%PDF-1.3 1 0 obj << /Type /Catalog /Outlines 2 0 R /Pages 3 0 R >> endobj 2 0 obj << /Type /Outlines /Count 0 >> endobj 3 0 obj << /Type /Pages /Kids [6 0 R 202 0 R 229 0 R 244 0 R 271 0 R 298 0 R 348 0 R 350 0 R ] /Count 8 /Resources << /ProcSet 4 0 R /Font << /F1 8 0 R /F2 9 0 R /F3 10 0 R /F4 11 0 R /F5 12 0 R /F6 13 0 R >> /XObject << /I1 14 0 R /I2 15 0 R /I3 212 0 R /I4 235 0 R /I5 254 0 R /I6 281 0 R >> >> /MediaBox [0.000 0.000 612.000 792.000] >> endobj 4 0 obj [/PDF /Text /ImageC ] endobj 5 0 obj << /Creator (DOMPDF) /CreationDate (D:20180722234702+00'00') /ModDate (D:20180722234702+00'00') /Title (Aggregation-Prone Proteins Modulate Huntingtin Inclusion Body Formation in Yeast PLOS Currents Huntington Disease) >> endobj 6 0 obj << /Type /Page /Parent 3 0 R /Annots [ 16 0 R 18 0 R 20 0 R 22 0 R 24 0 R 26 0 R 28 0 R 30 0 R 32 0 R 34 0 R 36 0 R 38 0 R 40 0 R 42 0 R 44 0 R 46 0 R 48 0 R 50 0 R 52 0 R 54 0 R 56 0 R 58 0 R 60 0 R 62 0 R 64 0 R 66 0 R 68 0 R 70 0 R 72 0 R 74 0 R 76 0 R 78 0 R 80 0 R 82 0 R 84 0 R 86 0 R 88 0 R 90 0 R 92 0 R 94 0 R 96 0 R 98 0 R 100 0 R 102 0 R 104 0 R 106 0 R 108 0 R 110 0 R 112 0 R 114 0 R 116 0 R 118 0 R 120 0 R 122 0 R 124 0 R 126 0 R 128 0 R 130 0 R 132 0 R 134 0 R 136 0 R 138 0 R 140 0 R 142 0 R 144 0 R 146 0 R 148 0 R 150 0 R 152 0 R 154 0 R 156 0 R 158 0 R 160 0 R 162 0 R 164 0 R 166 0 R 168 0 R 170 0 R 172 0 R 174 0 R 176 0 R 178 0 R 180 0 R 182 0 R 184 0 R 186 0 R 188 0 R 190 0 R 192 0 R 194 0 R 196 0 R 198 0 R 200 0 R ] /Contents 7 0 R >> endobj 7 0 obj << /Length 36170 >> stream q 375.000 0 0 39.000 222.000 738.000 cm /I2 Do Q q 15.000 684.354 577.500 53.646 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Aggregation-Prone Proteins Modulate Huntingtin Inclusion )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(Body Formation in Yeast)] TJ ET Q 0.271 0.267 0.267 rg BT 15.000 675.088 Td /F3 9.8 Tf [(April 23, 2014)] TJ ET BT 76.483 675.088 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 81.358 675.088 Td /F3 9.8 Tf [(Molecular/Cellular)] TJ ET BT 26.250 663.247 Td /F1 9.8 Tf [(Ralitsa B. Kantcheva)] TJ ET 0.271 0.267 0.267 rg BT 116.749 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 122.171 663.247 Td /F1 9.8 Tf [(Robert Mason)] TJ ET 0.271 0.267 0.267 rg BT 183.400 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 188.821 663.247 Td /F1 9.8 Tf [(Flaviano Giorgini)] TJ ET 0.271 0.267 0.267 rg BT 26.250 651.342 Td /F1 9.8 Tf [(Kantcheva RB, Mason R, Giorgini F. Aggregation-Prone Proteins Modulate Huntingtin Inclusion Body Formation in Yeast. )] TJ ET BT 551.892 651.342 Td /F1 9.8 Tf [(PLOS )] TJ ET BT 26.250 639.438 Td /F1 9.8 Tf [(Currents Huntington Disease. 2014 Apr 23 . Edition 1. doi: 10.1371/currents.hd.501008f3051342c9a5c0cd0f3a5bf3a4.)] TJ ET q 15.000 26.911 577.500 610.146 re W n 0.271 0.267 0.267 rg BT 26.250 610.335 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 590.381 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is a fatal neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin \(HTT\) )] TJ ET BT 26.250 578.476 Td /F1 9.8 Tf [(protein. The expression of mutant HTT in the bakers yeast Saccharomyces cerevisiae recapitulates many of the cellular )] TJ ET BT 26.250 566.571 Td /F1 9.8 Tf [(phenotypes observed in mammalian HD models. Mutant HTT aggregation and toxicity in yeast is influenced by the presence of )] TJ ET BT 26.250 554.667 Td /F1 9.8 Tf [(the Rnq1p and Sup35p prions, as well as other glutamine/asparagine-rich aggregation-prone proteins. Here we investigated the )] TJ ET BT 26.250 542.762 Td /F1 9.8 Tf [(ability of a subset of these proteins to modulate mutant HTT aggregation and to substitute for the prion form of Rnq1p. We find )] TJ ET BT 26.250 530.857 Td /F1 9.8 Tf [(that overexpression of either the putative prion Ybr016wp or the Sup35p prion restores aggregation of mutant HTT in yeast cells )] TJ ET BT 26.250 518.952 Td /F1 9.8 Tf [(lacking the Rnq1p prion. These results indicate that an interchangeable suite of aggregation-prone proteins regulates mutant )] TJ ET BT 26.250 507.048 Td /F1 9.8 Tf [(HTT aggregation dynamics in yeast, which may have implications for mutant HTT aggregation in human cells.)] TJ ET BT 26.250 470.445 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 450.491 Td /F1 9.8 Tf [(Flaviano Giorgini was supported by a MRC New Investigator award \(G0700090\). The authors have declared that no competing )] TJ ET BT 26.250 438.586 Td /F1 9.8 Tf [(interests exist.)] TJ ET BT 26.250 409.484 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 389.529 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is an autosomal dominant neurodegenerative disease )] TJ ET 0.267 0.267 0.267 rg BT 376.860 391.037 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 381.679 393.418 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 384.088 391.037 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 388.907 389.529 Td /F1 9.8 Tf [( caused by the expansion of a )] TJ ET BT 26.250 377.625 Td /F1 9.8 Tf [(polyglutamine \(polyQ\) tract in the huntingtin \(HTT\) protein, which leads to its misfolding and aggregation)] TJ ET 0.267 0.267 0.267 rg BT 474.389 379.132 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 479.208 377.625 Td /F1 9.8 Tf [( . Intracellular HTT )] TJ ET BT 26.250 365.720 Td /F1 9.8 Tf [(aggregates are a pathological hallmark of HD)] TJ ET 0.267 0.267 0.267 rg BT 221.874 367.227 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 226.693 369.608 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 229.102 367.227 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 233.921 365.720 Td /F1 9.8 Tf [(, with their morphology and localization influencing downstream cellular effects)] TJ ET 0.267 0.267 0.267 rg BT 572.051 367.227 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 576.869 369.608 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 26.250 355.322 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 31.069 353.815 Td /F1 9.8 Tf [( . Several studies suggest that macromolecular HTT aggregates which prevent further intermolecular interactions are )] TJ ET BT 26.250 341.910 Td /F1 9.8 Tf [(neuroprotective)] TJ ET 0.267 0.267 0.267 rg BT 93.447 343.418 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 98.266 341.910 Td /F1 9.8 Tf [(, while soluble oligomeric species of mutant HTT capable of sequestering other cellular proteins are neurotoxic)] TJ ET 0.267 0.267 0.267 rg BT 574.036 343.418 Td /F4 8.7 Tf [(9)] TJ ET 0.271 0.267 0.267 rg BT 26.250 330.006 Td /F1 9.8 Tf [(. HD has been modelled in a wide range of organisms)] TJ ET 0.267 0.267 0.267 rg BT 258.183 331.513 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 267.820 333.894 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 270.230 331.513 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 279.867 330.006 Td /F1 9.8 Tf [( including the bakers yeast, )] TJ ET BT 402.883 330.006 Td /F5 9.8 Tf [(Saccharomyces cerevisiae)] TJ ET 0.267 0.267 0.267 rg BT 518.830 331.513 Td /F6 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 528.467 330.006 Td /F1 9.8 Tf [(. Yeast )] TJ ET BT 26.250 318.101 Td /F1 9.8 Tf [(models recapitulate many of the cellular disease phenotypes seen in HD patients, including disturbances in mitochondrial )] TJ ET BT 26.250 306.196 Td /F1 9.8 Tf [(function, increased production of reactive oxygen species, disruption of the cytoskeleton, proteasome dysfunction and increased )] TJ ET BT 26.250 294.291 Td /F1 9.8 Tf [(flux through the kynurenine pathway of tryptophan degradation)] TJ ET 0.267 0.267 0.267 rg BT 296.695 295.799 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 306.333 294.291 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 274.887 Td /F1 9.8 Tf [(A number of yeast models have been developed to study the cellular effects of mutant HTT expression)] TJ ET 0.267 0.267 0.267 rg BT 468.461 276.394 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 473.280 278.775 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 475.689 276.394 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 485.327 278.775 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 487.736 276.394 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 497.373 274.887 Td /F1 9.8 Tf [( . These models )] TJ ET BT 26.250 262.982 Td /F1 9.8 Tf [(have shown that aggregation and cellular toxicity of mutant HTT in yeast is dependent upon the presence of the Rnq1p in its )] TJ ET BT 26.250 251.077 Td /F1 9.8 Tf [(prion [)] TJ ET BT 53.345 251.077 Td /F5 9.8 Tf [(PIN)] TJ ET BT 69.599 254.965 Td /F5 8.7 Tf [(+)] TJ ET BT 74.660 251.077 Td /F1 9.8 Tf [(] conformation)] TJ ET 0.267 0.267 0.267 rg BT 136.436 252.584 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 146.073 251.077 Td /F1 9.8 Tf [( , as deletion of )] TJ ET BT 214.908 251.077 Td /F5 9.8 Tf [(RNQ1)] TJ ET BT 241.994 251.077 Td /F1 9.8 Tf [( or curing of [)] TJ ET BT 298.895 251.077 Td /F5 9.8 Tf [(PIN)] TJ ET BT 315.148 254.965 Td /F5 8.7 Tf [(+)] TJ ET BT 320.209 251.077 Td /F1 9.8 Tf [(] ameliorates these phenotypes. Furthermore, a network of )] TJ ET BT 26.250 239.172 Td /F1 9.8 Tf [(prion-like glutamine-rich proteins modulates mutant HTT-dependent toxicity in yeast)] TJ ET 0.267 0.267 0.267 rg BT 387.127 240.680 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 396.764 239.172 Td /F1 9.8 Tf [( , with expression of these proteins as )] TJ ET BT 26.250 227.268 Td /F1 9.8 Tf [(well as Rnq1p having an additive effect on the overall level of toxicity observed. Although the cellular function of Rnq1p is )] TJ ET BT 26.250 215.363 Td /F1 9.8 Tf [(unclear, it is crucial for the )] TJ ET BT 142.207 215.363 Td /F5 9.8 Tf [(de novo)] TJ ET BT 176.897 215.363 Td /F1 9.8 Tf [( formation of [)] TJ ET BT 236.509 215.363 Td /F5 9.8 Tf [(PSI)] TJ ET BT 252.226 219.251 Td /F5 8.7 Tf [(+)] TJ ET BT 257.287 215.363 Td /F1 9.8 Tf [(], the prion form of Sup35p)] TJ ET 0.267 0.267 0.267 rg BT 372.727 216.870 Td /F4 8.7 Tf [(16)] TJ ET 0.271 0.267 0.267 rg BT 382.364 219.251 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 384.774 216.870 Td /F4 8.7 Tf [(17)] TJ ET 0.271 0.267 0.267 rg BT 394.411 215.363 Td /F1 9.8 Tf [( . [)] TJ ET BT 405.253 215.363 Td /F5 9.8 Tf [(PSI)] TJ ET BT 420.970 219.251 Td /F5 8.7 Tf [(+)] TJ ET BT 426.031 215.363 Td /F1 9.8 Tf [(] is one of the most extensively )] TJ ET BT 26.250 203.458 Td /F1 9.8 Tf [(studied yeast prions and is encoded by )] TJ ET BT 197.509 203.458 Td /F5 9.8 Tf [(SUP35)] TJ ET BT 228.397 203.458 Td /F1 9.8 Tf [(, with the non-prion form of the protein being crucial for the release of newly )] TJ ET BT 26.250 191.553 Td /F1 9.8 Tf [(synthesized polypeptides from the ribosomal complex through ATP hydrolysis)] TJ ET 0.267 0.267 0.267 rg BT 361.123 193.061 Td /F4 8.7 Tf [(18)] TJ ET 0.271 0.267 0.267 rg BT 370.761 191.553 Td /F1 9.8 Tf [( . A recent study found that Sup35p interacts )] TJ ET BT 26.250 179.649 Td /F1 9.8 Tf [(with mutant HTT through its Q/N-rich prion domain, and the presence of [)] TJ ET BT 341.087 179.649 Td /F5 9.8 Tf [(PSI)] TJ ET BT 356.804 183.537 Td /F5 8.7 Tf [(+)] TJ ET BT 361.866 179.649 Td /F1 9.8 Tf [(] is critical for full toxicity of mutant HTT )] TJ ET BT 26.250 167.744 Td /F1 9.8 Tf [(constructs with extended polyproline regions)] TJ ET 0.267 0.267 0.267 rg BT 218.081 169.251 Td /F4 8.7 Tf [(19)] TJ ET 0.271 0.267 0.267 rg BT 227.719 167.744 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 148.339 Td /F1 9.8 Tf [(We previously identified a number of putative yeast prions in a screen for gene deletion suppressors of HTT103Q-dependent )] TJ ET BT 26.250 136.434 Td /F1 9.8 Tf [(toxicity in yeast)] TJ ET 0.267 0.267 0.267 rg BT 92.355 137.942 Td /F4 8.7 Tf [(20)] TJ ET 0.271 0.267 0.267 rg BT 101.992 136.434 Td /F1 9.8 Tf [( . The proteins in question Def1p, Ybr016wp, Yir003wp and Ylr278cp all have glutamine/asparagine-rich )] TJ ET BT 26.250 124.530 Td /F1 9.8 Tf [(\(Q/N-rich\) domains and computational analyses suggests that they share primary sequence features with known prions)] TJ ET 0.267 0.267 0.267 rg BT 539.412 126.037 Td /F4 8.7 Tf [(21)] TJ ET 0.271 0.267 0.267 rg BT 549.049 124.530 Td /F1 9.8 Tf [( . )] TJ ET BT 26.250 112.625 Td /F1 9.8 Tf [(Subsequent work has found that Def1p, Yir003wp and Ylr278cp do not behave as typical yeast prion proteins, while Ybr016wp )] TJ ET BT 26.250 100.720 Td /F1 9.8 Tf [(has a large number of characteristics in common with established yeast prions such as Sup35p)] TJ ET 0.267 0.267 0.267 rg BT 436.462 102.227 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 446.099 100.720 Td /F1 9.8 Tf [( . Here we further investigate )] TJ ET BT 26.250 88.815 Td /F1 9.8 Tf [(the effect of Ybr016wp and Sup35p on mutant HTT aggregation dynamics in yeast. In order to unmask the effect these proteins )] TJ ET BT 26.250 76.911 Td /F1 9.8 Tf [(have on this protein misfolding process, we focused our attention on changes in HTT aggregation dynamics in a )] TJ ET BT 510.728 76.911 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 530.237 76.911 Td /F1 9.8 Tf [(? )] TJ ET BT 26.250 65.006 Td /F1 9.8 Tf [(background. We find that both Sup35p and Ybr016wp modulate the formation of mutant HTT aggregates in yeast and that these )] TJ ET BT 26.250 53.101 Td /F1 9.8 Tf [(effects are partially dependent on the presence of Rnq1p in the cell. In total, this work indicates that other aggregation-prone )] TJ ET BT 26.250 41.196 Td /F1 9.8 Tf [(proteins can substitute for Rnq1p in the context of HTT misfolding, and suggests that dynamic interplay between a suite of such )] TJ ET Q q 15.000 684.354 577.500 53.646 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Aggregation-Prone Proteins Modulate Huntingtin Inclusion )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(Body Formation in Yeast)] TJ ET Q 0.271 0.267 0.267 rg BT 15.000 675.088 Td /F3 9.8 Tf [(April 23, 2014)] TJ ET BT 76.483 675.088 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 81.358 675.088 Td /F3 9.8 Tf [(Molecular/Cellular)] TJ ET BT 26.250 663.247 Td /F1 9.8 Tf [(Ralitsa B. Kantcheva)] TJ ET 0.271 0.267 0.267 rg BT 116.749 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 122.171 663.247 Td /F1 9.8 Tf [(Robert Mason)] TJ ET 0.271 0.267 0.267 rg BT 183.400 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 188.821 663.247 Td /F1 9.8 Tf [(Flaviano Giorgini)] TJ ET 0.271 0.267 0.267 rg BT 26.250 651.342 Td /F1 9.8 Tf [(Kantcheva RB, Mason R, Giorgini F. Aggregation-Prone Proteins Modulate Huntingtin Inclusion Body Formation in Yeast. )] TJ ET BT 551.892 651.342 Td /F1 9.8 Tf [(PLOS )] TJ ET BT 26.250 639.438 Td /F1 9.8 Tf [(Currents Huntington Disease. 2014 Apr 23 . Edition 1. doi: 10.1371/currents.hd.501008f3051342c9a5c0cd0f3a5bf3a4.)] TJ ET q 15.000 26.911 577.500 610.146 re W n 0.271 0.267 0.267 rg BT 26.250 610.335 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 590.381 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is a fatal neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin \(HTT\) )] TJ ET BT 26.250 578.476 Td /F1 9.8 Tf [(protein. The expression of mutant HTT in the bakers yeast Saccharomyces cerevisiae recapitulates many of the cellular )] TJ ET BT 26.250 566.571 Td /F1 9.8 Tf [(phenotypes observed in mammalian HD models. Mutant HTT aggregation and toxicity in yeast is influenced by the presence of )] TJ ET BT 26.250 554.667 Td /F1 9.8 Tf [(the Rnq1p and Sup35p prions, as well as other glutamine/asparagine-rich aggregation-prone proteins. Here we investigated the )] TJ ET BT 26.250 542.762 Td /F1 9.8 Tf [(ability of a subset of these proteins to modulate mutant HTT aggregation and to substitute for the prion form of Rnq1p. We find )] TJ ET BT 26.250 530.857 Td /F1 9.8 Tf [(that overexpression of either the putative prion Ybr016wp or the Sup35p prion restores aggregation of mutant HTT in yeast cells )] TJ ET BT 26.250 518.952 Td /F1 9.8 Tf [(lacking the Rnq1p prion. These results indicate that an interchangeable suite of aggregation-prone proteins regulates mutant )] TJ ET BT 26.250 507.048 Td /F1 9.8 Tf [(HTT aggregation dynamics in yeast, which may have implications for mutant HTT aggregation in human cells.)] TJ ET BT 26.250 470.445 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 450.491 Td /F1 9.8 Tf [(Flaviano Giorgini was supported by a MRC New Investigator award \(G0700090\). The authors have declared that no competing )] TJ ET BT 26.250 438.586 Td /F1 9.8 Tf [(interests exist.)] TJ ET BT 26.250 409.484 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 389.529 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is an autosomal dominant neurodegenerative disease )] TJ ET 0.267 0.267 0.267 rg BT 376.860 391.037 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 381.679 393.418 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 384.088 391.037 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 388.907 389.529 Td /F1 9.8 Tf [( caused by the expansion of a )] TJ ET BT 26.250 377.625 Td /F1 9.8 Tf [(polyglutamine \(polyQ\) tract in the huntingtin \(HTT\) protein, which leads to its misfolding and aggregation)] TJ ET 0.267 0.267 0.267 rg BT 474.389 379.132 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 479.208 377.625 Td /F1 9.8 Tf [( . Intracellular HTT )] TJ ET BT 26.250 365.720 Td /F1 9.8 Tf [(aggregates are a pathological hallmark of HD)] TJ ET 0.267 0.267 0.267 rg BT 221.874 367.227 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 226.693 369.608 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 229.102 367.227 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 233.921 365.720 Td /F1 9.8 Tf [(, with their morphology and localization influencing downstream cellular effects)] TJ ET 0.267 0.267 0.267 rg BT 572.051 367.227 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 576.869 369.608 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 26.250 355.322 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 31.069 353.815 Td /F1 9.8 Tf [( . Several studies suggest that macromolecular HTT aggregates which prevent further intermolecular interactions are )] TJ ET BT 26.250 341.910 Td /F1 9.8 Tf [(neuroprotective)] TJ ET 0.267 0.267 0.267 rg BT 93.447 343.418 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 98.266 341.910 Td /F1 9.8 Tf [(, while soluble oligomeric species of mutant HTT capable of sequestering other cellular proteins are neurotoxic)] TJ ET 0.267 0.267 0.267 rg BT 574.036 343.418 Td /F4 8.7 Tf [(9)] TJ ET 0.271 0.267 0.267 rg BT 26.250 330.006 Td /F1 9.8 Tf [(. HD has been modelled in a wide range of organisms)] TJ ET 0.267 0.267 0.267 rg BT 258.183 331.513 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 267.820 333.894 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 270.230 331.513 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 279.867 330.006 Td /F1 9.8 Tf [( including the bakers yeast, )] TJ ET BT 402.883 330.006 Td /F5 9.8 Tf [(Saccharomyces cerevisiae)] TJ ET 0.267 0.267 0.267 rg BT 518.830 331.513 Td /F6 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 528.467 330.006 Td /F1 9.8 Tf [(. Yeast )] TJ ET BT 26.250 318.101 Td /F1 9.8 Tf [(models recapitulate many of the cellular disease phenotypes seen in HD patients, including disturbances in mitochondrial )] TJ ET BT 26.250 306.196 Td /F1 9.8 Tf [(function, increased production of reactive oxygen species, disruption of the cytoskeleton, proteasome dysfunction and increased )] TJ ET BT 26.250 294.291 Td /F1 9.8 Tf [(flux through the kynurenine pathway of tryptophan degradation)] TJ ET 0.267 0.267 0.267 rg BT 296.695 295.799 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 306.333 294.291 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 274.887 Td /F1 9.8 Tf [(A number of yeast models have been developed to study the cellular effects of mutant HTT expression)] TJ ET 0.267 0.267 0.267 rg BT 468.461 276.394 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 473.280 278.775 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 475.689 276.394 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 485.327 278.775 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 487.736 276.394 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 497.373 274.887 Td /F1 9.8 Tf [( . These models )] TJ ET BT 26.250 262.982 Td /F1 9.8 Tf [(have shown that aggregation and cellular toxicity of mutant HTT in yeast is dependent upon the presence of the Rnq1p in its )] TJ ET BT 26.250 251.077 Td /F1 9.8 Tf [(prion [)] TJ ET BT 53.345 251.077 Td /F5 9.8 Tf [(PIN)] TJ ET BT 69.599 254.965 Td /F5 8.7 Tf [(+)] TJ ET BT 74.660 251.077 Td /F1 9.8 Tf [(] conformation)] TJ ET 0.267 0.267 0.267 rg BT 136.436 252.584 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 146.073 251.077 Td /F1 9.8 Tf [( , as deletion of )] TJ ET BT 214.908 251.077 Td /F5 9.8 Tf [(RNQ1)] TJ ET BT 241.994 251.077 Td /F1 9.8 Tf [( or curing of [)] TJ ET BT 298.895 251.077 Td /F5 9.8 Tf [(PIN)] TJ ET BT 315.148 254.965 Td /F5 8.7 Tf [(+)] TJ ET BT 320.209 251.077 Td /F1 9.8 Tf [(] ameliorates these phenotypes. Furthermore, a network of )] TJ ET BT 26.250 239.172 Td /F1 9.8 Tf [(prion-like glutamine-rich proteins modulates mutant HTT-dependent toxicity in yeast)] TJ ET 0.267 0.267 0.267 rg BT 387.127 240.680 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 396.764 239.172 Td /F1 9.8 Tf [( , with expression of these proteins as )] TJ ET BT 26.250 227.268 Td /F1 9.8 Tf [(well as Rnq1p having an additive effect on the overall level of toxicity observed. Although the cellular function of Rnq1p is )] TJ ET BT 26.250 215.363 Td /F1 9.8 Tf [(unclear, it is crucial for the )] TJ ET BT 142.207 215.363 Td /F5 9.8 Tf [(de novo)] TJ ET BT 176.897 215.363 Td /F1 9.8 Tf [( formation of [)] TJ ET BT 236.509 215.363 Td /F5 9.8 Tf [(PSI)] TJ ET BT 252.226 219.251 Td /F5 8.7 Tf [(+)] TJ ET BT 257.287 215.363 Td /F1 9.8 Tf [(], the prion form of Sup35p)] TJ ET 0.267 0.267 0.267 rg BT 372.727 216.870 Td /F4 8.7 Tf [(16)] TJ ET 0.271 0.267 0.267 rg BT 382.364 219.251 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 384.774 216.870 Td /F4 8.7 Tf [(17)] TJ ET 0.271 0.267 0.267 rg BT 394.411 215.363 Td /F1 9.8 Tf [( . [)] TJ ET BT 405.253 215.363 Td /F5 9.8 Tf [(PSI)] TJ ET BT 420.970 219.251 Td /F5 8.7 Tf [(+)] TJ ET BT 426.031 215.363 Td /F1 9.8 Tf [(] is one of the most extensively )] TJ ET BT 26.250 203.458 Td /F1 9.8 Tf [(studied yeast prions and is encoded by )] TJ ET BT 197.509 203.458 Td /F5 9.8 Tf [(SUP35)] TJ ET BT 228.397 203.458 Td /F1 9.8 Tf [(, with the non-prion form of the protein being crucial for the release of newly )] TJ ET BT 26.250 191.553 Td /F1 9.8 Tf [(synthesized polypeptides from the ribosomal complex through ATP hydrolysis)] TJ ET 0.267 0.267 0.267 rg BT 361.123 193.061 Td /F4 8.7 Tf [(18)] TJ ET 0.271 0.267 0.267 rg BT 370.761 191.553 Td /F1 9.8 Tf [( . A recent study found that Sup35p interacts )] TJ ET BT 26.250 179.649 Td /F1 9.8 Tf [(with mutant HTT through its Q/N-rich prion domain, and the presence of [)] TJ ET BT 341.087 179.649 Td /F5 9.8 Tf [(PSI)] TJ ET BT 356.804 183.537 Td /F5 8.7 Tf [(+)] TJ ET BT 361.866 179.649 Td /F1 9.8 Tf [(] is critical for full toxicity of mutant HTT )] TJ ET BT 26.250 167.744 Td /F1 9.8 Tf [(constructs with extended polyproline regions)] TJ ET 0.267 0.267 0.267 rg BT 218.081 169.251 Td /F4 8.7 Tf [(19)] TJ ET 0.271 0.267 0.267 rg BT 227.719 167.744 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 148.339 Td /F1 9.8 Tf [(We previously identified a number of putative yeast prions in a screen for gene deletion suppressors of HTT103Q-dependent )] TJ ET BT 26.250 136.434 Td /F1 9.8 Tf [(toxicity in yeast)] TJ ET 0.267 0.267 0.267 rg BT 92.355 137.942 Td /F4 8.7 Tf [(20)] TJ ET 0.271 0.267 0.267 rg BT 101.992 136.434 Td /F1 9.8 Tf [( . The proteins in question Def1p, Ybr016wp, Yir003wp and Ylr278cp all have glutamine/asparagine-rich )] TJ ET BT 26.250 124.530 Td /F1 9.8 Tf [(\(Q/N-rich\) domains and computational analyses suggests that they share primary sequence features with known prions)] TJ ET 0.267 0.267 0.267 rg BT 539.412 126.037 Td /F4 8.7 Tf [(21)] TJ ET 0.271 0.267 0.267 rg BT 549.049 124.530 Td /F1 9.8 Tf [( . )] TJ ET BT 26.250 112.625 Td /F1 9.8 Tf [(Subsequent work has found that Def1p, Yir003wp and Ylr278cp do not behave as typical yeast prion proteins, while Ybr016wp )] TJ ET BT 26.250 100.720 Td /F1 9.8 Tf [(has a large number of characteristics in common with established yeast prions such as Sup35p)] TJ ET 0.267 0.267 0.267 rg BT 436.462 102.227 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 446.099 100.720 Td /F1 9.8 Tf [( . Here we further investigate )] TJ ET BT 26.250 88.815 Td /F1 9.8 Tf [(the effect of Ybr016wp and Sup35p on mutant HTT aggregation dynamics in yeast. In order to unmask the effect these proteins )] TJ ET BT 26.250 76.911 Td /F1 9.8 Tf [(have on this protein misfolding process, we focused our attention on changes in HTT aggregation dynamics in a )] TJ ET BT 510.728 76.911 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 530.237 76.911 Td /F1 9.8 Tf [(? )] TJ ET BT 26.250 65.006 Td /F1 9.8 Tf [(background. We find that both Sup35p and Ybr016wp modulate the formation of mutant HTT aggregates in yeast and that these )] TJ ET BT 26.250 53.101 Td /F1 9.8 Tf [(effects are partially dependent on the presence of Rnq1p in the cell. In total, this work indicates that other aggregation-prone )] TJ ET BT 26.250 41.196 Td /F1 9.8 Tf [(proteins can substitute for Rnq1p in the context of HTT misfolding, and suggests that dynamic interplay between a suite of such )] TJ ET Q q 15.000 684.354 577.500 53.646 re W n 0.267 0.267 0.267 rg BT 15.000 718.042 Td /F2 21.0 Tf [(Aggregation-Prone Proteins Modulate Huntingtin Inclusion )] TJ ET BT 15.000 693.094 Td /F2 21.0 Tf [(Body Formation in Yeast)] TJ ET Q 0.271 0.267 0.267 rg BT 15.000 675.088 Td /F3 9.8 Tf [(April 23, 2014)] TJ ET BT 76.483 675.088 Td /F3 9.8 Tf [()] TJ ET 0.267 0.267 0.267 rg BT 81.358 675.088 Td /F3 9.8 Tf [(Molecular/Cellular)] TJ ET BT 26.250 663.247 Td /F1 9.8 Tf [(Ralitsa B. Kantcheva)] TJ ET 0.271 0.267 0.267 rg BT 116.749 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 122.171 663.247 Td /F1 9.8 Tf [(Robert Mason)] TJ ET 0.271 0.267 0.267 rg BT 183.400 663.247 Td /F1 9.8 Tf [(, )] TJ ET 0.267 0.267 0.267 rg BT 188.821 663.247 Td /F1 9.8 Tf [(Flaviano Giorgini)] TJ ET 0.271 0.267 0.267 rg BT 26.250 651.342 Td /F1 9.8 Tf [(Kantcheva RB, Mason R, Giorgini F. Aggregation-Prone Proteins Modulate Huntingtin Inclusion Body Formation in Yeast. )] TJ ET BT 551.892 651.342 Td /F1 9.8 Tf [(PLOS )] TJ ET BT 26.250 639.438 Td /F1 9.8 Tf [(Currents Huntington Disease. 2014 Apr 23 . Edition 1. doi: 10.1371/currents.hd.501008f3051342c9a5c0cd0f3a5bf3a4.)] TJ ET q 15.000 26.911 577.500 610.146 re W n 0.271 0.267 0.267 rg BT 26.250 610.335 Td /F4 12.0 Tf [(Abstract)] TJ ET BT 26.250 590.381 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is a fatal neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin \(HTT\) )] TJ ET BT 26.250 578.476 Td /F1 9.8 Tf [(protein. The expression of mutant HTT in the bakers yeast Saccharomyces cerevisiae recapitulates many of the cellular )] TJ ET BT 26.250 566.571 Td /F1 9.8 Tf [(phenotypes observed in mammalian HD models. Mutant HTT aggregation and toxicity in yeast is influenced by the presence of )] TJ ET BT 26.250 554.667 Td /F1 9.8 Tf [(the Rnq1p and Sup35p prions, as well as other glutamine/asparagine-rich aggregation-prone proteins. Here we investigated the )] TJ ET BT 26.250 542.762 Td /F1 9.8 Tf [(ability of a subset of these proteins to modulate mutant HTT aggregation and to substitute for the prion form of Rnq1p. We find )] TJ ET BT 26.250 530.857 Td /F1 9.8 Tf [(that overexpression of either the putative prion Ybr016wp or the Sup35p prion restores aggregation of mutant HTT in yeast cells )] TJ ET BT 26.250 518.952 Td /F1 9.8 Tf [(lacking the Rnq1p prion. These results indicate that an interchangeable suite of aggregation-prone proteins regulates mutant )] TJ ET BT 26.250 507.048 Td /F1 9.8 Tf [(HTT aggregation dynamics in yeast, which may have implications for mutant HTT aggregation in human cells.)] TJ ET BT 26.250 470.445 Td /F4 12.0 Tf [(Funding Statement)] TJ ET BT 26.250 450.491 Td /F1 9.8 Tf [(Flaviano Giorgini was supported by a MRC New Investigator award \(G0700090\). The authors have declared that no competing )] TJ ET BT 26.250 438.586 Td /F1 9.8 Tf [(interests exist.)] TJ ET BT 26.250 409.484 Td /F4 12.0 Tf [(Introduction)] TJ ET BT 26.250 389.529 Td /F1 9.8 Tf [(Huntingtons disease \(HD\) is an autosomal dominant neurodegenerative disease )] TJ ET 0.267 0.267 0.267 rg BT 376.860 391.037 Td /F4 8.7 Tf [(1)] TJ ET 0.271 0.267 0.267 rg BT 381.679 393.418 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 384.088 391.037 Td /F4 8.7 Tf [(2)] TJ ET 0.271 0.267 0.267 rg BT 388.907 389.529 Td /F1 9.8 Tf [( caused by the expansion of a )] TJ ET BT 26.250 377.625 Td /F1 9.8 Tf [(polyglutamine \(polyQ\) tract in the huntingtin \(HTT\) protein, which leads to its misfolding and aggregation)] TJ ET 0.267 0.267 0.267 rg BT 474.389 379.132 Td /F4 8.7 Tf [(3)] TJ ET 0.271 0.267 0.267 rg BT 479.208 377.625 Td /F1 9.8 Tf [( . Intracellular HTT )] TJ ET BT 26.250 365.720 Td /F1 9.8 Tf [(aggregates are a pathological hallmark of HD)] TJ ET 0.267 0.267 0.267 rg BT 221.874 367.227 Td /F4 8.7 Tf [(4)] TJ ET 0.271 0.267 0.267 rg BT 226.693 369.608 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 229.102 367.227 Td /F4 8.7 Tf [(5)] TJ ET 0.271 0.267 0.267 rg BT 233.921 365.720 Td /F1 9.8 Tf [(, with their morphology and localization influencing downstream cellular effects)] TJ ET 0.267 0.267 0.267 rg BT 572.051 367.227 Td /F4 8.7 Tf [(6)] TJ ET 0.271 0.267 0.267 rg BT 576.869 369.608 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 26.250 355.322 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 31.069 353.815 Td /F1 9.8 Tf [( . Several studies suggest that macromolecular HTT aggregates which prevent further intermolecular interactions are )] TJ ET BT 26.250 341.910 Td /F1 9.8 Tf [(neuroprotective)] TJ ET 0.267 0.267 0.267 rg BT 93.447 343.418 Td /F4 8.7 Tf [(8)] TJ ET 0.271 0.267 0.267 rg BT 98.266 341.910 Td /F1 9.8 Tf [(, while soluble oligomeric species of mutant HTT capable of sequestering other cellular proteins are neurotoxic)] TJ ET 0.267 0.267 0.267 rg BT 574.036 343.418 Td /F4 8.7 Tf [(9)] TJ ET 0.271 0.267 0.267 rg BT 26.250 330.006 Td /F1 9.8 Tf [(. HD has been modelled in a wide range of organisms)] TJ ET 0.267 0.267 0.267 rg BT 258.183 331.513 Td /F4 8.7 Tf [(10)] TJ ET 0.271 0.267 0.267 rg BT 267.820 333.894 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 270.230 331.513 Td /F4 8.7 Tf [(11)] TJ ET 0.271 0.267 0.267 rg BT 279.867 330.006 Td /F1 9.8 Tf [( including the bakers yeast, )] TJ ET BT 402.883 330.006 Td /F5 9.8 Tf [(Saccharomyces cerevisiae)] TJ ET 0.267 0.267 0.267 rg BT 518.830 331.513 Td /F6 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 528.467 330.006 Td /F1 9.8 Tf [(. Yeast )] TJ ET BT 26.250 318.101 Td /F1 9.8 Tf [(models recapitulate many of the cellular disease phenotypes seen in HD patients, including disturbances in mitochondrial )] TJ ET BT 26.250 306.196 Td /F1 9.8 Tf [(function, increased production of reactive oxygen species, disruption of the cytoskeleton, proteasome dysfunction and increased )] TJ ET BT 26.250 294.291 Td /F1 9.8 Tf [(flux through the kynurenine pathway of tryptophan degradation)] TJ ET 0.267 0.267 0.267 rg BT 296.695 295.799 Td /F4 8.7 Tf [(13)] TJ ET 0.271 0.267 0.267 rg BT 306.333 294.291 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 274.887 Td /F1 9.8 Tf [(A number of yeast models have been developed to study the cellular effects of mutant HTT expression)] TJ ET 0.267 0.267 0.267 rg BT 468.461 276.394 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 473.280 278.775 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 475.689 276.394 Td /F4 8.7 Tf [(12)] TJ ET 0.271 0.267 0.267 rg BT 485.327 278.775 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 487.736 276.394 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 497.373 274.887 Td /F1 9.8 Tf [( . These models )] TJ ET BT 26.250 262.982 Td /F1 9.8 Tf [(have shown that aggregation and cellular toxicity of mutant HTT in yeast is dependent upon the presence of the Rnq1p in its )] TJ ET BT 26.250 251.077 Td /F1 9.8 Tf [(prion [)] TJ ET BT 53.345 251.077 Td /F5 9.8 Tf [(PIN)] TJ ET BT 69.599 254.965 Td /F5 8.7 Tf [(+)] TJ ET BT 74.660 251.077 Td /F1 9.8 Tf [(] conformation)] TJ ET 0.267 0.267 0.267 rg BT 136.436 252.584 Td /F4 8.7 Tf [(14)] TJ ET 0.271 0.267 0.267 rg BT 146.073 251.077 Td /F1 9.8 Tf [( , as deletion of )] TJ ET BT 214.908 251.077 Td /F5 9.8 Tf [(RNQ1)] TJ ET BT 241.994 251.077 Td /F1 9.8 Tf [( or curing of [)] TJ ET BT 298.895 251.077 Td /F5 9.8 Tf [(PIN)] TJ ET BT 315.148 254.965 Td /F5 8.7 Tf [(+)] TJ ET BT 320.209 251.077 Td /F1 9.8 Tf [(] ameliorates these phenotypes. Furthermore, a network of )] TJ ET BT 26.250 239.172 Td /F1 9.8 Tf [(prion-like glutamine-rich proteins modulates mutant HTT-dependent toxicity in yeast)] TJ ET 0.267 0.267 0.267 rg BT 387.127 240.680 Td /F4 8.7 Tf [(15)] TJ ET 0.271 0.267 0.267 rg BT 396.764 239.172 Td /F1 9.8 Tf [( , with expression of these proteins as )] TJ ET BT 26.250 227.268 Td /F1 9.8 Tf [(well as Rnq1p having an additive effect on the overall level of toxicity observed. Although the cellular function of Rnq1p is )] TJ ET BT 26.250 215.363 Td /F1 9.8 Tf [(unclear, it is crucial for the )] TJ ET BT 142.207 215.363 Td /F5 9.8 Tf [(de novo)] TJ ET BT 176.897 215.363 Td /F1 9.8 Tf [( formation of [)] TJ ET BT 236.509 215.363 Td /F5 9.8 Tf [(PSI)] TJ ET BT 252.226 219.251 Td /F5 8.7 Tf [(+)] TJ ET BT 257.287 215.363 Td /F1 9.8 Tf [(], the prion form of Sup35p)] TJ ET 0.267 0.267 0.267 rg BT 372.727 216.870 Td /F4 8.7 Tf [(16)] TJ ET 0.271 0.267 0.267 rg BT 382.364 219.251 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 384.774 216.870 Td /F4 8.7 Tf [(17)] TJ ET 0.271 0.267 0.267 rg BT 394.411 215.363 Td /F1 9.8 Tf [( . [)] TJ ET BT 405.253 215.363 Td /F5 9.8 Tf [(PSI)] TJ ET BT 420.970 219.251 Td /F5 8.7 Tf [(+)] TJ ET BT 426.031 215.363 Td /F1 9.8 Tf [(] is one of the most extensively )] TJ ET BT 26.250 203.458 Td /F1 9.8 Tf [(studied yeast prions and is encoded by )] TJ ET BT 197.509 203.458 Td /F5 9.8 Tf [(SUP35)] TJ ET BT 228.397 203.458 Td /F1 9.8 Tf [(, with the non-prion form of the protein being crucial for the release of newly )] TJ ET BT 26.250 191.553 Td /F1 9.8 Tf [(synthesized polypeptides from the ribosomal complex through ATP hydrolysis)] TJ ET 0.267 0.267 0.267 rg BT 361.123 193.061 Td /F4 8.7 Tf [(18)] TJ ET 0.271 0.267 0.267 rg BT 370.761 191.553 Td /F1 9.8 Tf [( . A recent study found that Sup35p interacts )] TJ ET BT 26.250 179.649 Td /F1 9.8 Tf [(with mutant HTT through its Q/N-rich prion domain, and the presence of [)] TJ ET BT 341.087 179.649 Td /F5 9.8 Tf [(PSI)] TJ ET BT 356.804 183.537 Td /F5 8.7 Tf [(+)] TJ ET BT 361.866 179.649 Td /F1 9.8 Tf [(] is critical for full toxicity of mutant HTT )] TJ ET BT 26.250 167.744 Td /F1 9.8 Tf [(constructs with extended polyproline regions)] TJ ET 0.267 0.267 0.267 rg BT 218.081 169.251 Td /F4 8.7 Tf [(19)] TJ ET 0.271 0.267 0.267 rg BT 227.719 167.744 Td /F1 9.8 Tf [( .)] TJ ET BT 26.250 148.339 Td /F1 9.8 Tf [(We previously identified a number of putative yeast prions in a screen for gene deletion suppressors of HTT103Q-dependent )] TJ ET BT 26.250 136.434 Td /F1 9.8 Tf [(toxicity in yeast)] TJ ET 0.267 0.267 0.267 rg BT 92.355 137.942 Td /F4 8.7 Tf [(20)] TJ ET 0.271 0.267 0.267 rg BT 101.992 136.434 Td /F1 9.8 Tf [( . The proteins in question Def1p, Ybr016wp, Yir003wp and Ylr278cp all have glutamine/asparagine-rich )] TJ ET BT 26.250 124.530 Td /F1 9.8 Tf [(\(Q/N-rich\) domains and computational analyses suggests that they share primary sequence features with known prions)] TJ ET 0.267 0.267 0.267 rg BT 539.412 126.037 Td /F4 8.7 Tf [(21)] TJ ET 0.271 0.267 0.267 rg BT 549.049 124.530 Td /F1 9.8 Tf [( . )] TJ ET BT 26.250 112.625 Td /F1 9.8 Tf [(Subsequent work has found that Def1p, Yir003wp and Ylr278cp do not behave as typical yeast prion proteins, while Ybr016wp )] TJ ET BT 26.250 100.720 Td /F1 9.8 Tf [(has a large number of characteristics in common with established yeast prions such as Sup35p)] TJ ET 0.267 0.267 0.267 rg BT 436.462 102.227 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 446.099 100.720 Td /F1 9.8 Tf [( . Here we further investigate )] TJ ET BT 26.250 88.815 Td /F1 9.8 Tf [(the effect of Ybr016wp and Sup35p on mutant HTT aggregation dynamics in yeast. In order to unmask the effect these proteins )] TJ ET BT 26.250 76.911 Td /F1 9.8 Tf [(have on this protein misfolding process, we focused our attention on changes in HTT aggregation dynamics in a )] TJ ET BT 510.728 76.911 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 530.237 76.911 Td /F1 9.8 Tf [(? )] TJ ET BT 26.250 65.006 Td /F1 9.8 Tf [(background. We find that both Sup35p and Ybr016wp modulate the formation of mutant HTT aggregates in yeast and that these )] TJ ET BT 26.250 53.101 Td /F1 9.8 Tf [(effects are partially dependent on the presence of Rnq1p in the cell. In total, this work indicates that other aggregation-prone )] TJ ET BT 26.250 41.196 Td /F1 9.8 Tf [(proteins can substitute for Rnq1p in the context of HTT misfolding, and suggests that dynamic interplay between a suite of such )] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(1)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 8 0 obj << /Type /Font /Subtype /Type1 /Name /F1 /BaseFont /Helvetica /Encoding /WinAnsiEncoding >> endobj 9 0 obj << /Type /Font /Subtype /Type1 /Name /F2 /BaseFont /Times-Bold /Encoding /WinAnsiEncoding >> endobj 10 0 obj << /Type /Font /Subtype /Type1 /Name /F3 /BaseFont /Times-Italic /Encoding /WinAnsiEncoding >> endobj 11 0 obj << /Type /Font /Subtype /Type1 /Name /F4 /BaseFont /Helvetica-Bold /Encoding /WinAnsiEncoding >> endobj 12 0 obj << /Type /Font /Subtype /Type1 /Name /F5 /BaseFont /Helvetica-Oblique /Encoding /WinAnsiEncoding >> endobj 13 0 obj << /Type /Font /Subtype /Type1 /Name /F6 /BaseFont /Helvetica-BoldOblique /Encoding /WinAnsiEncoding >> endobj 14 0 obj << /Type /XObject /Subtype /Image /Width 500 /Height 52 /Filter /FlateDecode /DecodeParms << /Predictor 15 /Colors 1 /Columns 500 /BitsPerComponent 8>> /ColorSpace /DeviceGray /BitsPerComponent 8 /Length 144>> stream x1 0 'ݲ؎"e{dzAdzAdzAdzAdzAdzAdzAdzAdzAdzAdzAdzAtlM0\ endstream endobj 15 0 obj << /Type /XObject /Subtype /Image /Width 500 /Height 52 /SMask 14 0 R /Filter /FlateDecode /DecodeParms << /Predictor 15 /Colors 3 /Columns 500 /BitsPerComponent 8>> /ColorSpace /DeviceRGB /BitsPerComponent 8 /Length 4223>> stream xە8=,8C#h!hGv#0=j$q1uaNĥT*J&a_Zaa0 a0 a0 H^UUUUX!0 3SY|^G0 ÌÔ{]wa_>"OeYaG!8e}a6̖{4M&d:"qʲ\QUUMS<ϛNld2l6Z a<=2B(2vK7M\.GW0 rrWWavߖe9C?Eq8Gqy~~}_qdϽ(neYʒGjK4EQNjͷm{X?|7m۶mqP:Ȳp8Kmu]lNzuZ,NTeY"jlUUQW\,aRiVbȗLkHkEsjd.L6=Iו P]1t7Sc2uIuMM; K5>A$Ǒ$I׋nY 0_Hat;aq䃰U$nR ,zO7bT mhhԫܻu1F*qm'P}ܙ<ϕALO6Lw3nB.T4co{߽.lV $laԀIu!gwChxJ]Ne (+M"d-Eqc=P ! 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/Type /Action >> endobj 202 0 obj << /Type /Page /Parent 3 0 R /Annots [ 204 0 R 206 0 R 208 0 R 210 0 R 213 0 R 215 0 R 217 0 R 219 0 R 221 0 R 223 0 R 225 0 R 227 0 R ] /Contents 203 0 R >> endobj 203 0 obj << /Length 28296 >> stream 0.271 0.267 0.267 rg q 15.000 -66.744 577.500 843.744 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(proteins modulates HTT aggregation in yeast, which may have implications for HTT aggregation in human cells.)] TJ ET BT 26.250 730.874 Td /F4 12.0 Tf [(Methods)] TJ ET BT 26.250 710.919 Td /F4 9.8 Tf [(Yeast strains and culturing: )] TJ ET BT 157.368 710.919 Td /F1 9.8 Tf [(The BY4741 and )] TJ ET BT 233.252 710.919 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 252.762 710.919 Td /F1 9.8 Tf [(? deletion strains \(MAT a)] TJ ET BT 361.133 710.919 Td /F5 9.8 Tf [(, )] TJ ET BT 366.554 710.919 Td /F5 9.8 Tf [(his3?)] TJ ET BT 389.857 710.919 Td /F5 9.8 Tf [(1,)] TJ ET BT 397.988 710.919 Td /F5 9.8 Tf [(leu2-3,112, trp1-1, ura3-1, ade2-1)] TJ ET BT 544.316 710.919 Td /F1 9.8 Tf [(\) )] TJ ET BT 26.250 699.015 Td /F1 9.8 Tf [(employed carry integrated constructs encoding a human HTT fragment consisting of the first 17 N-terminal amino acids followed )] TJ ET BT 26.250 687.110 Td /F1 9.8 Tf [(by either 25 or 103 glutamines)] TJ ET 0.267 0.267 0.267 rg BT 157.397 688.617 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 162.216 687.110 Td /F1 9.8 Tf [( . Yeast strains were typically grown on YPD \(1 % yeast extract, 2 % peptone, 2 % glucose, 2.5 )] TJ ET BT 26.250 675.205 Td /F1 9.8 Tf [(% agar\). Recombinant strains were selected on synthetic medium lacking relevant amino acids \(0.68 % yeast nitrogen base )] TJ ET BT 26.250 663.300 Td /F1 9.8 Tf [(without amino acids, 2.5 % agar, 2 % glucose, 0.6 % leucine, 0.3 % lysine, 0.09 % dropout powder lacking the respective amino )] TJ ET BT 26.250 651.396 Td /F1 9.8 Tf [(acids)] TJ ET 0.267 0.267 0.267 rg BT 49.007 652.903 Td /F4 8.7 Tf [(23)] TJ ET 0.271 0.267 0.267 rg BT 58.644 651.396 Td /F1 9.8 Tf [( \).)] TJ ET BT 26.250 631.991 Td /F4 9.8 Tf [(Yeast growth assays: )] TJ ET BT 128.664 631.991 Td /F1 9.8 Tf [(Yeast colonies were inoculated into 100 l of medium in 96-well plates and incubated at 30 C for 16 to )] TJ ET BT 26.250 620.086 Td /F1 9.8 Tf [(18 hours. Cultures were serially diluted by 5 or 10-fold in distilled water and spotted \(5 ?l\) onto selective plates containing either )] TJ ET BT 26.250 608.181 Td /F1 9.8 Tf [(2 % galactose or 2 % glucose as a carbon source and incubated for 60 to 72 hours at 30 C.)] TJ ET BT 26.250 588.777 Td /F4 9.8 Tf [(Fluorescence microscopy)] TJ ET BT 146.546 588.777 Td /F4 9.8 Tf [(: )] TJ ET BT 152.503 588.777 Td /F1 9.8 Tf [(Colonies were inoculated in 5 ml of glucose containing selective medium and incubated at 30 C )] TJ ET BT 26.250 576.872 Td /F1 9.8 Tf [(with shaking for 24 hours. Cultures were washed once with water and diluted to an OD)] TJ ET BT 399.080 574.808 Td /F1 8.7 Tf [(600 )] TJ ET BT 415.946 576.872 Td /F1 9.8 Tf [(of 0.2 in 5 ml selective media )] TJ ET BT 26.250 564.967 Td /F1 9.8 Tf [(containing 2 % raffinose. After a further 6 hours at 30 C with shaking, galactose was added to a final concentration of 2 % to )] TJ ET BT 26.250 553.062 Td /F1 9.8 Tf [(induce expression of constructs under the control of )] TJ ET BT 252.236 553.062 Td /F5 9.8 Tf [(GAL1 )] TJ ET BT 279.877 553.062 Td /F1 9.8 Tf [(promoters. After a further 12 hours the number of cells containing )] TJ ET BT 26.250 541.158 Td /F1 9.8 Tf [(aggregates was scored using a Zeiss Axioscope 2 fluorescent microscope with a Zeiss 100 X Plan-NEOFLUAR \(1.30/oil, )] TJ ET BT 26.250 529.253 Td /F1 9.8 Tf [(?/0.17\) objective and a chromomycin filter.)] TJ ET BT 26.250 509.848 Td /F4 9.8 Tf [(Determination of yeast )] TJ ET BT 134.065 509.848 Td /F6 9.8 Tf [([PIN])] TJ ET BT 156.812 509.848 Td /F4 9.8 Tf [( and )] TJ ET BT 179.569 509.848 Td /F6 9.8 Tf [([PSI])] TJ ET BT 201.779 509.848 Td /F4 9.8 Tf [( status: )] TJ ET BT 239.161 509.848 Td /F1 9.8 Tf [(The presence of the [)] TJ ET BT 331.298 509.848 Td /F5 9.8 Tf [(PIN)] TJ ET BT 347.551 513.736 Td /F5 8.7 Tf [(+)] TJ ET BT 352.613 509.848 Td /F1 9.8 Tf [(] prion was detected by a previously described )] TJ ET BT 26.250 497.943 Td /F1 9.8 Tf [(protocol)] TJ ET 0.267 0.267 0.267 rg BT 60.931 499.451 Td /F4 8.7 Tf [(24)] TJ ET 0.271 0.267 0.267 rg BT 70.568 497.943 Td /F1 9.8 Tf [( using a primary antibody against Rnq1p \(Santa Cruz Biotechnology Inc., 1:10,000 dilution\). Samples for the )] TJ ET BT 26.250 486.039 Td /F1 9.8 Tf [(determination of [)] TJ ET BT 101.579 486.039 Td /F5 9.8 Tf [(PSI)] TJ ET BT 117.296 486.039 Td /F1 9.8 Tf [(] status were grown the same way as for the [)] TJ ET BT 313.466 486.039 Td /F5 9.8 Tf [(PIN)] TJ ET BT 329.719 486.039 Td /F1 9.8 Tf [(] status assay, and 4 OD units harvested by )] TJ ET BT 26.250 474.134 Td /F1 9.8 Tf [(centrifugation and suspended in 100 l of ice-cold lysis buffer [1X PBS without Ca)] TJ ET BT 378.508 478.022 Td /F1 8.7 Tf [(2+)] TJ ET BT 388.388 474.134 Td /F1 9.8 Tf [(and Mg)] TJ ET BT 420.904 478.022 Td /F1 8.7 Tf [(2+)] TJ ET BT 430.784 474.134 Td /F1 9.8 Tf [(\(PAA Laboratories GmbH, )] TJ ET BT 26.250 462.229 Td /F1 9.8 Tf [(Austria\), 100 mM NaCl, 2 mM PMSF and 1x EDTA-free protease inhibitors \(Roche\)]. Samples were lysed with 100 l of acid )] TJ ET BT 26.250 450.324 Td /F1 9.8 Tf [(washed glass beads \(425-600 ?m, Sigma-Aldrich, USA\) in a bead-beater for 1 minute at maximum speed. After adding an )] TJ ET BT 26.250 438.420 Td /F1 9.8 Tf [(additional 100 l of ice-cold lysis buffer, samples were left on ice for 1 minute to allow the beads to sediment and the )] TJ ET BT 26.250 426.515 Td /F1 9.8 Tf [(supernatant was removed for further analysis. The amount of protein was quantified using a NanoPhotometer Pearl \(IMPLEN )] TJ ET BT 26.250 414.610 Td /F1 9.8 Tf [(GmbH, Germany\) and 10 ?g of protein in 50 ?l of lysis buffer were spun at 50,000 rpm at 4 C for 15 minutes in a BECKMAN TL-)] TJ ET BT 26.250 402.705 Td /F1 9.8 Tf [(100 ultracentrifuge \(Beckman, USA\). The supernatant was removed and used as the Soluble fraction, while the pellet was )] TJ ET BT 26.250 390.801 Td /F1 9.8 Tf [(resuspended in 50 ?l of lysis buffer and used as the Pellet fraction. Protein from the total, soluble and pellet fractions were )] TJ ET BT 26.250 378.896 Td /F1 9.8 Tf [(mixed with 2 l of 5 X protein loading dye [50 mM Tris-HCl \(pH 6.8\), 2 % SDS, 10 % glycerol, 1 % ?-mercaptoethanol, 12.5 mM )] TJ ET BT 26.250 366.991 Td /F1 9.8 Tf [(EDTA, and 0.02 % bromophenol blue] to give a final volume of 10 ?l. Samples were denatured at 95 C for 5 minutes, )] TJ ET BT 26.250 355.086 Td /F1 9.8 Tf [(separated by SDS-PAGE, and transferred to PVDF membranes. Sup35p was detected with a primary antibody against yeast )] TJ ET BT 26.250 343.182 Td /F1 9.8 Tf [(Sup35p \(1:10,000 dilution\), a generous gift from Mick Tuite \(University of Kent, UK\).)] TJ ET BT 26.250 323.777 Td /F4 9.8 Tf [(Statistical Analyses: )] TJ ET BT 123.789 323.777 Td /F1 9.8 Tf [(Data was analysed by unpaired, two-tailed Mann-Whitney tests using Prism 6 \(GraphPad Software\).)] TJ ET BT 26.250 287.174 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 267.220 Td /F4 9.8 Tf [(HD model yeast exhibit HTT aggregation independent of toxicity in a [)] TJ ET BT 348.604 267.220 Td /F6 9.8 Tf [(PIN)] TJ ET BT 364.858 271.108 Td /F6 8.7 Tf [(+)] TJ ET BT 369.919 267.220 Td /F4 9.8 Tf [(] and [)] TJ ET BT 399.169 267.220 Td /F6 9.8 Tf [(PSI)] TJ ET BT 414.886 271.108 Td /F6 8.7 Tf [(+)] TJ ET BT 419.947 267.220 Td /F4 9.8 Tf [(] background: )] TJ ET BT 487.661 267.220 Td /F1 9.8 Tf [(To avoid )] TJ ET BT 26.250 255.315 Td /F1 9.8 Tf [(confounding issues due to variable plasmid copy number we generated yeast strains containing either HTT25Q or HTT103Q )] TJ ET BT 26.250 243.411 Td /F1 9.8 Tf [(constructs integrated at the )] TJ ET BT 146.565 243.411 Td /F5 9.8 Tf [(HIS3 )] TJ ET BT 170.950 243.411 Td /F1 9.8 Tf [(locus in parental BY4741 yeast, as well as in a )] TJ ET BT 374.715 243.411 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 394.225 243.411 Td /F1 9.8 Tf [(? strain in the same background. The )] TJ ET BT 26.250 231.506 Td /F1 9.8 Tf [(Rnq1p-deficient strain was generated by deleting )] TJ ET BT 240.311 231.506 Td /F5 9.8 Tf [(RNQ1 )] TJ ET BT 270.107 231.506 Td /F1 9.8 Tf [(using homologous recombination, which was verified by PCR )] TJ ET BT 26.250 219.601 Td /F1 9.8 Tf [(genotyping and immunoblotting \(Figure 1b; data not shown\). These constructs encode the first 17 amino acids of HTT followed )] TJ ET BT 26.250 207.696 Td /F1 9.8 Tf [(by the respective polyQ length under the control of an inducible )] TJ ET BT 301.561 207.696 Td /F5 9.8 Tf [(GAL1 )] TJ ET BT 329.202 207.696 Td /F1 9.8 Tf [(promoter, and have an N-terminal FLAG epitope tag and )] TJ ET BT 26.250 195.792 Td /F1 9.8 Tf [(a C-terminal CFP fusion. Integration of the HTT constructs was achieved by employing the integrative vector pRS303, and )] TJ ET BT 26.250 183.887 Td /F1 9.8 Tf [(successful integration events were confirmed by PCR and sequencing of the )] TJ ET BT 358.442 183.887 Td /F5 9.8 Tf [(HIS3 )] TJ ET BT 382.827 183.887 Td /F1 9.8 Tf [(locus \(data not shown\).)] TJ ET 0.965 0.965 0.965 rg 26.250 -66.744 555.000 240.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 174.006 m 581.250 174.006 l 581.250 173.256 l 26.250 173.256 l f q 450.000 0 0 225.000 35.250 -60.744 cm /I3 Do Q q 35.250 -66.744 537.000 0.000 re W n Q Q q 15.000 -66.744 577.500 843.744 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(proteins modulates HTT aggregation in yeast, which may have implications for HTT aggregation in human cells.)] TJ ET BT 26.250 730.874 Td /F4 12.0 Tf [(Methods)] TJ ET BT 26.250 710.919 Td /F4 9.8 Tf [(Yeast strains and culturing: )] TJ ET BT 157.368 710.919 Td /F1 9.8 Tf [(The BY4741 and )] TJ ET BT 233.252 710.919 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 252.762 710.919 Td /F1 9.8 Tf [(? deletion strains \(MAT a)] TJ ET BT 361.133 710.919 Td /F5 9.8 Tf [(, )] TJ ET BT 366.554 710.919 Td /F5 9.8 Tf [(his3?)] TJ ET BT 389.857 710.919 Td /F5 9.8 Tf [(1,)] TJ ET BT 397.988 710.919 Td /F5 9.8 Tf [(leu2-3,112, trp1-1, ura3-1, ade2-1)] TJ ET BT 544.316 710.919 Td /F1 9.8 Tf [(\) )] TJ ET BT 26.250 699.015 Td /F1 9.8 Tf [(employed carry integrated constructs encoding a human HTT fragment consisting of the first 17 N-terminal amino acids followed )] TJ ET BT 26.250 687.110 Td /F1 9.8 Tf [(by either 25 or 103 glutamines)] TJ ET 0.267 0.267 0.267 rg BT 157.397 688.617 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 162.216 687.110 Td /F1 9.8 Tf [( . Yeast strains were typically grown on YPD \(1 % yeast extract, 2 % peptone, 2 % glucose, 2.5 )] TJ ET BT 26.250 675.205 Td /F1 9.8 Tf [(% agar\). Recombinant strains were selected on synthetic medium lacking relevant amino acids \(0.68 % yeast nitrogen base )] TJ ET BT 26.250 663.300 Td /F1 9.8 Tf [(without amino acids, 2.5 % agar, 2 % glucose, 0.6 % leucine, 0.3 % lysine, 0.09 % dropout powder lacking the respective amino )] TJ ET BT 26.250 651.396 Td /F1 9.8 Tf [(acids)] TJ ET 0.267 0.267 0.267 rg BT 49.007 652.903 Td /F4 8.7 Tf [(23)] TJ ET 0.271 0.267 0.267 rg BT 58.644 651.396 Td /F1 9.8 Tf [( \).)] TJ ET BT 26.250 631.991 Td /F4 9.8 Tf [(Yeast growth assays: )] TJ ET BT 128.664 631.991 Td /F1 9.8 Tf [(Yeast colonies were inoculated into 100 l of medium in 96-well plates and incubated at 30 C for 16 to )] TJ ET BT 26.250 620.086 Td /F1 9.8 Tf [(18 hours. Cultures were serially diluted by 5 or 10-fold in distilled water and spotted \(5 ?l\) onto selective plates containing either )] TJ ET BT 26.250 608.181 Td /F1 9.8 Tf [(2 % galactose or 2 % glucose as a carbon source and incubated for 60 to 72 hours at 30 C.)] TJ ET BT 26.250 588.777 Td /F4 9.8 Tf [(Fluorescence microscopy)] TJ ET BT 146.546 588.777 Td /F4 9.8 Tf [(: )] TJ ET BT 152.503 588.777 Td /F1 9.8 Tf [(Colonies were inoculated in 5 ml of glucose containing selective medium and incubated at 30 C )] TJ ET BT 26.250 576.872 Td /F1 9.8 Tf [(with shaking for 24 hours. Cultures were washed once with water and diluted to an OD)] TJ ET BT 399.080 574.808 Td /F1 8.7 Tf [(600 )] TJ ET BT 415.946 576.872 Td /F1 9.8 Tf [(of 0.2 in 5 ml selective media )] TJ ET BT 26.250 564.967 Td /F1 9.8 Tf [(containing 2 % raffinose. After a further 6 hours at 30 C with shaking, galactose was added to a final concentration of 2 % to )] TJ ET BT 26.250 553.062 Td /F1 9.8 Tf [(induce expression of constructs under the control of )] TJ ET BT 252.236 553.062 Td /F5 9.8 Tf [(GAL1 )] TJ ET BT 279.877 553.062 Td /F1 9.8 Tf [(promoters. After a further 12 hours the number of cells containing )] TJ ET BT 26.250 541.158 Td /F1 9.8 Tf [(aggregates was scored using a Zeiss Axioscope 2 fluorescent microscope with a Zeiss 100 X Plan-NEOFLUAR \(1.30/oil, )] TJ ET BT 26.250 529.253 Td /F1 9.8 Tf [(?/0.17\) objective and a chromomycin filter.)] TJ ET BT 26.250 509.848 Td /F4 9.8 Tf [(Determination of yeast )] TJ ET BT 134.065 509.848 Td /F6 9.8 Tf [([PIN])] TJ ET BT 156.812 509.848 Td /F4 9.8 Tf [( and )] TJ ET BT 179.569 509.848 Td /F6 9.8 Tf [([PSI])] TJ ET BT 201.779 509.848 Td /F4 9.8 Tf [( status: )] TJ ET BT 239.161 509.848 Td /F1 9.8 Tf [(The presence of the [)] TJ ET BT 331.298 509.848 Td /F5 9.8 Tf [(PIN)] TJ ET BT 347.551 513.736 Td /F5 8.7 Tf [(+)] TJ ET BT 352.613 509.848 Td /F1 9.8 Tf [(] prion was detected by a previously described )] TJ ET BT 26.250 497.943 Td /F1 9.8 Tf [(protocol)] TJ ET 0.267 0.267 0.267 rg BT 60.931 499.451 Td /F4 8.7 Tf [(24)] TJ ET 0.271 0.267 0.267 rg BT 70.568 497.943 Td /F1 9.8 Tf [( using a primary antibody against Rnq1p \(Santa Cruz Biotechnology Inc., 1:10,000 dilution\). Samples for the )] TJ ET BT 26.250 486.039 Td /F1 9.8 Tf [(determination of [)] TJ ET BT 101.579 486.039 Td /F5 9.8 Tf [(PSI)] TJ ET BT 117.296 486.039 Td /F1 9.8 Tf [(] status were grown the same way as for the [)] TJ ET BT 313.466 486.039 Td /F5 9.8 Tf [(PIN)] TJ ET BT 329.719 486.039 Td /F1 9.8 Tf [(] status assay, and 4 OD units harvested by )] TJ ET BT 26.250 474.134 Td /F1 9.8 Tf [(centrifugation and suspended in 100 l of ice-cold lysis buffer [1X PBS without Ca)] TJ ET BT 378.508 478.022 Td /F1 8.7 Tf [(2+)] TJ ET BT 388.388 474.134 Td /F1 9.8 Tf [(and Mg)] TJ ET BT 420.904 478.022 Td /F1 8.7 Tf [(2+)] TJ ET BT 430.784 474.134 Td /F1 9.8 Tf [(\(PAA Laboratories GmbH, )] TJ ET BT 26.250 462.229 Td /F1 9.8 Tf [(Austria\), 100 mM NaCl, 2 mM PMSF and 1x EDTA-free protease inhibitors \(Roche\)]. Samples were lysed with 100 l of acid )] TJ ET BT 26.250 450.324 Td /F1 9.8 Tf [(washed glass beads \(425-600 ?m, Sigma-Aldrich, USA\) in a bead-beater for 1 minute at maximum speed. After adding an )] TJ ET BT 26.250 438.420 Td /F1 9.8 Tf [(additional 100 l of ice-cold lysis buffer, samples were left on ice for 1 minute to allow the beads to sediment and the )] TJ ET BT 26.250 426.515 Td /F1 9.8 Tf [(supernatant was removed for further analysis. The amount of protein was quantified using a NanoPhotometer Pearl \(IMPLEN )] TJ ET BT 26.250 414.610 Td /F1 9.8 Tf [(GmbH, Germany\) and 10 ?g of protein in 50 ?l of lysis buffer were spun at 50,000 rpm at 4 C for 15 minutes in a BECKMAN TL-)] TJ ET BT 26.250 402.705 Td /F1 9.8 Tf [(100 ultracentrifuge \(Beckman, USA\). The supernatant was removed and used as the Soluble fraction, while the pellet was )] TJ ET BT 26.250 390.801 Td /F1 9.8 Tf [(resuspended in 50 ?l of lysis buffer and used as the Pellet fraction. Protein from the total, soluble and pellet fractions were )] TJ ET BT 26.250 378.896 Td /F1 9.8 Tf [(mixed with 2 l of 5 X protein loading dye [50 mM Tris-HCl \(pH 6.8\), 2 % SDS, 10 % glycerol, 1 % ?-mercaptoethanol, 12.5 mM )] TJ ET BT 26.250 366.991 Td /F1 9.8 Tf [(EDTA, and 0.02 % bromophenol blue] to give a final volume of 10 ?l. Samples were denatured at 95 C for 5 minutes, )] TJ ET BT 26.250 355.086 Td /F1 9.8 Tf [(separated by SDS-PAGE, and transferred to PVDF membranes. Sup35p was detected with a primary antibody against yeast )] TJ ET BT 26.250 343.182 Td /F1 9.8 Tf [(Sup35p \(1:10,000 dilution\), a generous gift from Mick Tuite \(University of Kent, UK\).)] TJ ET BT 26.250 323.777 Td /F4 9.8 Tf [(Statistical Analyses: )] TJ ET BT 123.789 323.777 Td /F1 9.8 Tf [(Data was analysed by unpaired, two-tailed Mann-Whitney tests using Prism 6 \(GraphPad Software\).)] TJ ET BT 26.250 287.174 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 267.220 Td /F4 9.8 Tf [(HD model yeast exhibit HTT aggregation independent of toxicity in a [)] TJ ET BT 348.604 267.220 Td /F6 9.8 Tf [(PIN)] TJ ET BT 364.858 271.108 Td /F6 8.7 Tf [(+)] TJ ET BT 369.919 267.220 Td /F4 9.8 Tf [(] and [)] TJ ET BT 399.169 267.220 Td /F6 9.8 Tf [(PSI)] TJ ET BT 414.886 271.108 Td /F6 8.7 Tf [(+)] TJ ET BT 419.947 267.220 Td /F4 9.8 Tf [(] background: )] TJ ET BT 487.661 267.220 Td /F1 9.8 Tf [(To avoid )] TJ ET BT 26.250 255.315 Td /F1 9.8 Tf [(confounding issues due to variable plasmid copy number we generated yeast strains containing either HTT25Q or HTT103Q )] TJ ET BT 26.250 243.411 Td /F1 9.8 Tf [(constructs integrated at the )] TJ ET BT 146.565 243.411 Td /F5 9.8 Tf [(HIS3 )] TJ ET BT 170.950 243.411 Td /F1 9.8 Tf [(locus in parental BY4741 yeast, as well as in a )] TJ ET BT 374.715 243.411 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 394.225 243.411 Td /F1 9.8 Tf [(? strain in the same background. The )] TJ ET BT 26.250 231.506 Td /F1 9.8 Tf [(Rnq1p-deficient strain was generated by deleting )] TJ ET BT 240.311 231.506 Td /F5 9.8 Tf [(RNQ1 )] TJ ET BT 270.107 231.506 Td /F1 9.8 Tf [(using homologous recombination, which was verified by PCR )] TJ ET BT 26.250 219.601 Td /F1 9.8 Tf [(genotyping and immunoblotting \(Figure 1b; data not shown\). These constructs encode the first 17 amino acids of HTT followed )] TJ ET BT 26.250 207.696 Td /F1 9.8 Tf [(by the respective polyQ length under the control of an inducible )] TJ ET BT 301.561 207.696 Td /F5 9.8 Tf [(GAL1 )] TJ ET BT 329.202 207.696 Td /F1 9.8 Tf [(promoter, and have an N-terminal FLAG epitope tag and )] TJ ET BT 26.250 195.792 Td /F1 9.8 Tf [(a C-terminal CFP fusion. Integration of the HTT constructs was achieved by employing the integrative vector pRS303, and )] TJ ET BT 26.250 183.887 Td /F1 9.8 Tf [(successful integration events were confirmed by PCR and sequencing of the )] TJ ET BT 358.442 183.887 Td /F5 9.8 Tf [(HIS3 )] TJ ET BT 382.827 183.887 Td /F1 9.8 Tf [(locus \(data not shown\).)] TJ ET 0.965 0.965 0.965 rg 26.250 -66.744 555.000 240.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 174.006 m 581.250 174.006 l 581.250 173.256 l 26.250 173.256 l f q 450.000 0 0 225.000 35.250 -60.744 cm /I3 Do Q q 35.250 -66.744 537.000 0.000 re W n Q Q q 15.000 -66.744 577.500 843.744 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(proteins modulates HTT aggregation in yeast, which may have implications for HTT aggregation in human cells.)] TJ ET BT 26.250 730.874 Td /F4 12.0 Tf [(Methods)] TJ ET BT 26.250 710.919 Td /F4 9.8 Tf [(Yeast strains and culturing: )] TJ ET BT 157.368 710.919 Td /F1 9.8 Tf [(The BY4741 and )] TJ ET BT 233.252 710.919 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 252.762 710.919 Td /F1 9.8 Tf [(? deletion strains \(MAT a)] TJ ET BT 361.133 710.919 Td /F5 9.8 Tf [(, )] TJ ET BT 366.554 710.919 Td /F5 9.8 Tf [(his3?)] TJ ET BT 389.857 710.919 Td /F5 9.8 Tf [(1,)] TJ ET BT 397.988 710.919 Td /F5 9.8 Tf [(leu2-3,112, trp1-1, ura3-1, ade2-1)] TJ ET BT 544.316 710.919 Td /F1 9.8 Tf [(\) )] TJ ET BT 26.250 699.015 Td /F1 9.8 Tf [(employed carry integrated constructs encoding a human HTT fragment consisting of the first 17 N-terminal amino acids followed )] TJ ET BT 26.250 687.110 Td /F1 9.8 Tf [(by either 25 or 103 glutamines)] TJ ET 0.267 0.267 0.267 rg BT 157.397 688.617 Td /F4 8.7 Tf [(7)] TJ ET 0.271 0.267 0.267 rg BT 162.216 687.110 Td /F1 9.8 Tf [( . Yeast strains were typically grown on YPD \(1 % yeast extract, 2 % peptone, 2 % glucose, 2.5 )] TJ ET BT 26.250 675.205 Td /F1 9.8 Tf [(% agar\). Recombinant strains were selected on synthetic medium lacking relevant amino acids \(0.68 % yeast nitrogen base )] TJ ET BT 26.250 663.300 Td /F1 9.8 Tf [(without amino acids, 2.5 % agar, 2 % glucose, 0.6 % leucine, 0.3 % lysine, 0.09 % dropout powder lacking the respective amino )] TJ ET BT 26.250 651.396 Td /F1 9.8 Tf [(acids)] TJ ET 0.267 0.267 0.267 rg BT 49.007 652.903 Td /F4 8.7 Tf [(23)] TJ ET 0.271 0.267 0.267 rg BT 58.644 651.396 Td /F1 9.8 Tf [( \).)] TJ ET BT 26.250 631.991 Td /F4 9.8 Tf [(Yeast growth assays: )] TJ ET BT 128.664 631.991 Td /F1 9.8 Tf [(Yeast colonies were inoculated into 100 l of medium in 96-well plates and incubated at 30 C for 16 to )] TJ ET BT 26.250 620.086 Td /F1 9.8 Tf [(18 hours. Cultures were serially diluted by 5 or 10-fold in distilled water and spotted \(5 ?l\) onto selective plates containing either )] TJ ET BT 26.250 608.181 Td /F1 9.8 Tf [(2 % galactose or 2 % glucose as a carbon source and incubated for 60 to 72 hours at 30 C.)] TJ ET BT 26.250 588.777 Td /F4 9.8 Tf [(Fluorescence microscopy)] TJ ET BT 146.546 588.777 Td /F4 9.8 Tf [(: )] TJ ET BT 152.503 588.777 Td /F1 9.8 Tf [(Colonies were inoculated in 5 ml of glucose containing selective medium and incubated at 30 C )] TJ ET BT 26.250 576.872 Td /F1 9.8 Tf [(with shaking for 24 hours. Cultures were washed once with water and diluted to an OD)] TJ ET BT 399.080 574.808 Td /F1 8.7 Tf [(600 )] TJ ET BT 415.946 576.872 Td /F1 9.8 Tf [(of 0.2 in 5 ml selective media )] TJ ET BT 26.250 564.967 Td /F1 9.8 Tf [(containing 2 % raffinose. After a further 6 hours at 30 C with shaking, galactose was added to a final concentration of 2 % to )] TJ ET BT 26.250 553.062 Td /F1 9.8 Tf [(induce expression of constructs under the control of )] TJ ET BT 252.236 553.062 Td /F5 9.8 Tf [(GAL1 )] TJ ET BT 279.877 553.062 Td /F1 9.8 Tf [(promoters. After a further 12 hours the number of cells containing )] TJ ET BT 26.250 541.158 Td /F1 9.8 Tf [(aggregates was scored using a Zeiss Axioscope 2 fluorescent microscope with a Zeiss 100 X Plan-NEOFLUAR \(1.30/oil, )] TJ ET BT 26.250 529.253 Td /F1 9.8 Tf [(?/0.17\) objective and a chromomycin filter.)] TJ ET BT 26.250 509.848 Td /F4 9.8 Tf [(Determination of yeast )] TJ ET BT 134.065 509.848 Td /F6 9.8 Tf [([PIN])] TJ ET BT 156.812 509.848 Td /F4 9.8 Tf [( and )] TJ ET BT 179.569 509.848 Td /F6 9.8 Tf [([PSI])] TJ ET BT 201.779 509.848 Td /F4 9.8 Tf [( status: )] TJ ET BT 239.161 509.848 Td /F1 9.8 Tf [(The presence of the [)] TJ ET BT 331.298 509.848 Td /F5 9.8 Tf [(PIN)] TJ ET BT 347.551 513.736 Td /F5 8.7 Tf [(+)] TJ ET BT 352.613 509.848 Td /F1 9.8 Tf [(] prion was detected by a previously described )] TJ ET BT 26.250 497.943 Td /F1 9.8 Tf [(protocol)] TJ ET 0.267 0.267 0.267 rg BT 60.931 499.451 Td /F4 8.7 Tf [(24)] TJ ET 0.271 0.267 0.267 rg BT 70.568 497.943 Td /F1 9.8 Tf [( using a primary antibody against Rnq1p \(Santa Cruz Biotechnology Inc., 1:10,000 dilution\). Samples for the )] TJ ET BT 26.250 486.039 Td /F1 9.8 Tf [(determination of [)] TJ ET BT 101.579 486.039 Td /F5 9.8 Tf [(PSI)] TJ ET BT 117.296 486.039 Td /F1 9.8 Tf [(] status were grown the same way as for the [)] TJ ET BT 313.466 486.039 Td /F5 9.8 Tf [(PIN)] TJ ET BT 329.719 486.039 Td /F1 9.8 Tf [(] status assay, and 4 OD units harvested by )] TJ ET BT 26.250 474.134 Td /F1 9.8 Tf [(centrifugation and suspended in 100 l of ice-cold lysis buffer [1X PBS without Ca)] TJ ET BT 378.508 478.022 Td /F1 8.7 Tf [(2+)] TJ ET BT 388.388 474.134 Td /F1 9.8 Tf [(and Mg)] TJ ET BT 420.904 478.022 Td /F1 8.7 Tf [(2+)] TJ ET BT 430.784 474.134 Td /F1 9.8 Tf [(\(PAA Laboratories GmbH, )] TJ ET BT 26.250 462.229 Td /F1 9.8 Tf [(Austria\), 100 mM NaCl, 2 mM PMSF and 1x EDTA-free protease inhibitors \(Roche\)]. Samples were lysed with 100 l of acid )] TJ ET BT 26.250 450.324 Td /F1 9.8 Tf [(washed glass beads \(425-600 ?m, Sigma-Aldrich, USA\) in a bead-beater for 1 minute at maximum speed. After adding an )] TJ ET BT 26.250 438.420 Td /F1 9.8 Tf [(additional 100 l of ice-cold lysis buffer, samples were left on ice for 1 minute to allow the beads to sediment and the )] TJ ET BT 26.250 426.515 Td /F1 9.8 Tf [(supernatant was removed for further analysis. The amount of protein was quantified using a NanoPhotometer Pearl \(IMPLEN )] TJ ET BT 26.250 414.610 Td /F1 9.8 Tf [(GmbH, Germany\) and 10 ?g of protein in 50 ?l of lysis buffer were spun at 50,000 rpm at 4 C for 15 minutes in a BECKMAN TL-)] TJ ET BT 26.250 402.705 Td /F1 9.8 Tf [(100 ultracentrifuge \(Beckman, USA\). The supernatant was removed and used as the Soluble fraction, while the pellet was )] TJ ET BT 26.250 390.801 Td /F1 9.8 Tf [(resuspended in 50 ?l of lysis buffer and used as the Pellet fraction. Protein from the total, soluble and pellet fractions were )] TJ ET BT 26.250 378.896 Td /F1 9.8 Tf [(mixed with 2 l of 5 X protein loading dye [50 mM Tris-HCl \(pH 6.8\), 2 % SDS, 10 % glycerol, 1 % ?-mercaptoethanol, 12.5 mM )] TJ ET BT 26.250 366.991 Td /F1 9.8 Tf [(EDTA, and 0.02 % bromophenol blue] to give a final volume of 10 ?l. Samples were denatured at 95 C for 5 minutes, )] TJ ET BT 26.250 355.086 Td /F1 9.8 Tf [(separated by SDS-PAGE, and transferred to PVDF membranes. Sup35p was detected with a primary antibody against yeast )] TJ ET BT 26.250 343.182 Td /F1 9.8 Tf [(Sup35p \(1:10,000 dilution\), a generous gift from Mick Tuite \(University of Kent, UK\).)] TJ ET BT 26.250 323.777 Td /F4 9.8 Tf [(Statistical Analyses: )] TJ ET BT 123.789 323.777 Td /F1 9.8 Tf [(Data was analysed by unpaired, two-tailed Mann-Whitney tests using Prism 6 \(GraphPad Software\).)] TJ ET BT 26.250 287.174 Td /F4 12.0 Tf [(Results)] TJ ET BT 26.250 267.220 Td /F4 9.8 Tf [(HD model yeast exhibit HTT aggregation independent of toxicity in a [)] TJ ET BT 348.604 267.220 Td /F6 9.8 Tf [(PIN)] TJ ET BT 364.858 271.108 Td /F6 8.7 Tf [(+)] TJ ET BT 369.919 267.220 Td /F4 9.8 Tf [(] and [)] TJ ET BT 399.169 267.220 Td /F6 9.8 Tf [(PSI)] TJ ET BT 414.886 271.108 Td /F6 8.7 Tf [(+)] TJ ET BT 419.947 267.220 Td /F4 9.8 Tf [(] background: )] TJ ET BT 487.661 267.220 Td /F1 9.8 Tf [(To avoid )] TJ ET BT 26.250 255.315 Td /F1 9.8 Tf [(confounding issues due to variable plasmid copy number we generated yeast strains containing either HTT25Q or HTT103Q )] TJ ET BT 26.250 243.411 Td /F1 9.8 Tf [(constructs integrated at the )] TJ ET BT 146.565 243.411 Td /F5 9.8 Tf [(HIS3 )] TJ ET BT 170.950 243.411 Td /F1 9.8 Tf [(locus in parental BY4741 yeast, as well as in a )] TJ ET BT 374.715 243.411 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 394.225 243.411 Td /F1 9.8 Tf [(? strain in the same background. The )] TJ ET BT 26.250 231.506 Td /F1 9.8 Tf [(Rnq1p-deficient strain was generated by deleting )] TJ ET BT 240.311 231.506 Td /F5 9.8 Tf [(RNQ1 )] TJ ET BT 270.107 231.506 Td /F1 9.8 Tf [(using homologous recombination, which was verified by PCR )] TJ ET BT 26.250 219.601 Td /F1 9.8 Tf [(genotyping and immunoblotting \(Figure 1b; data not shown\). These constructs encode the first 17 amino acids of HTT followed )] TJ ET BT 26.250 207.696 Td /F1 9.8 Tf [(by the respective polyQ length under the control of an inducible )] TJ ET BT 301.561 207.696 Td /F5 9.8 Tf [(GAL1 )] TJ ET BT 329.202 207.696 Td /F1 9.8 Tf [(promoter, and have an N-terminal FLAG epitope tag and )] TJ ET BT 26.250 195.792 Td /F1 9.8 Tf [(a C-terminal CFP fusion. Integration of the HTT constructs was achieved by employing the integrative vector pRS303, and )] TJ ET BT 26.250 183.887 Td /F1 9.8 Tf [(successful integration events were confirmed by PCR and sequencing of the )] TJ ET BT 358.442 183.887 Td /F5 9.8 Tf [(HIS3 )] TJ ET BT 382.827 183.887 Td /F1 9.8 Tf [(locus \(data not shown\).)] TJ ET 0.965 0.965 0.965 rg 26.250 -66.744 555.000 240.750 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 174.006 m 581.250 174.006 l 581.250 173.256 l 26.250 173.256 l f q 450.000 0 0 225.000 35.250 -60.744 cm /I3 Do Q q 35.250 -66.744 537.000 0.000 re W n Q Q q 450.000 0 0 225.000 35.250 -60.744 cm /I3 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(2)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 204 0 obj << /Type /Annot /Subtype /Link /A 205 0 R /Border [0 0 0] /H /I /Rect [ 157.3973 687.8154 162.2159 696.6337 ] >> endobj 205 0 obj << /Type /Action >> endobj 206 0 obj << /Type /Annot /Subtype /Link /A 207 0 R /Border [0 0 0] /H /I /Rect [ 49.0065 652.1012 58.6438 660.9195 ] >> endobj 207 0 obj << /Type /Action >> endobj 208 0 obj << /Type /Annot /Subtype /Link /A 209 0 R /Border [0 0 0] /H /I /Rect [ 60.9308 498.6489 70.5681 507.4672 ] >> endobj 209 0 obj << /Type /Action >> endobj 210 0 obj << /Type /Annot /Subtype /Link /A 211 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 -60.7440 485.2500 164.2560 ] >> endobj 211 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2014/03/Fig-1-prp-article-Mar-v2C.jpg) >> endobj 212 0 obj << /Type /XObject /Subtype /Image /Width 600 /Height 300 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 35858>> stream JFIF;CREATOR: gd-jpeg v1.0 (using IJG JPEG v62), quality = 90 C     C   ,X" }!1AQa"q2#BR$3br %&'()*456789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz w!1AQaq"2B #3Rbr $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?R&(((qF)? ? \QO€R~~.(((qF'G@ 1IR`QO€R~~QIQPF'G@ .R~~qIQPQIQPF'G@ .R~~qIQPQ'F=xL{Qj\ 8ǵ)1F=xcڀ8ǵ)1F=p(cڀ8ǵ L{Qj^(cڀ8ǵL{Qj^(cڀ0)1F=xL{Qj^(cڀL{Q@^ۣoC}=[>coz֖W7PZ K4Q4hAA{vCiW[4,T;ៈ<}wX?7EWQkr򉤛HVᙱP:xFմ5-2I0ʺ ǭ|~bi'< ~}'GKqG^U^_ךeX[ۍRb%޸0QGx?GOLj3D^bn Us[>`fhkg#F7|m$l*K{n*ű jhk߅gցxg]љf;ifBW_yW>#Ϋ1/,|OǢJ4$.I";Fc-rTz4/4s@ G4o CBZ,s]_].o $^[S[tMkÞ*6X5n$1C!9F|/g/) o֚ b_α!8䞀I$Q|A֕i%޻_OyͺP2Z5Xc ƏưuHkgw$3.Xml[s\H29T.@'O 7B[C>-,VXSQ4 amh53Z}Ċ[J%e]](hk~-xOFVkb&:Ui$Ep՘ '5$/q+]Qzzuӣ\#ی44~5a/T=+OLXZ(c7L-{h@5[+T-.#v4' G\k?m@iFX pH* g/:|j+~5 1Zo-Ĭ|p:OƏƪiŖZe}k5մd2;'GKF~cE={n5(DDQLjԹ>U'u|BρՅm·Mƣg{u 0qi3K 2 Fd#tR#vrV3Y﩮b5;mYmpK*2r@nxwzwtkm[HO4y e  PE/4PJ<k,?an&-&^\&';śH`=`vhqß <SzoOM$ 4͍8J_Əƀ8i?*|4wۛ]^-&_ qqfg! x9O $&u]u}$v x81Ufff'x:o}kYVSc Q+29SҸEn =1J B5]3RoI MlnI o~Ϻ77gȼ\xHV3|Z6s!Ӿ@wqпFj|4?=O%P~%> o ɡXj3Ie ƅ@`o ԊֹG ko4<6?g2y$o.6nk3iC\U_Ok⏁Guougu}:|-ݣ̡nn,$b# Q'7ɼn>zOxsBO8mE n9=O9cGԾw[%o<]=g#-)Gw  Ҹ_1K\[h8G⏳ŇJw/\ kY4]𧊀:KEM|jC6o٭ig,ݝƘ%(,ħhR|DSZiI^YmLp'7b+Q?ڏPwᧉ]f5ux`PhT |b?|d Aɡj OkE ?Q?]𧊀:WYiKee{;l2F/.8=O{o&a%Ӿɫi`w5W(ܸ '= <3~ OdoG xS?UGMM4DnZ6ȷ;*\b־uOO/!#~kg-W.(8w&L S*doG x>5{Qž*d?Wxxɠ'7$w&d3lfU~RȻPeҼ{ OjxoSouv>&v5E4\ۑzQǥ`ZCV'.&qqE;8 lOa]@ ǥzR4~4x ]wo OiO#[oy;IټpY? |+N  KHf oƏƀJ8hh8J_ƏƀJc㗀xLPk 4$,>ey0E*$ 3a\:̶EZiҪnмu!\$ .N+7KRG}]t˛MB >m5V?0Ib'~kN!,ٝ.)K m,`GNN]?P|?:+k&u˝!-,fKM&5 & ( !I }R0pGC~,>|7C3dtNڮ{q_zIu(lG$ON 5kaaojsisuĮGTͷ#㑃񏅮#m ^Y^޼k"tgh|+CjK ֎6ۧM>(˱y#tKbHa_q.o zUy[_7FApwm,Dd){}psp?%xuOMQVht?JO#o@x r~k~8ċ+84MY=Gp'u5MA? \?'Pg^ҡ״=#ñA=,`]+sllV_cznD^P<?:<៌/ONuSYoOa \/M&^ -uodִ[[ sELSZ"$ .-nBC,70+ڿ$׿x#uT/-.o^5MGMui./mRY7KI2b%nHukP<?:I5(l@{=[;o@{j:XjS8&2RJLqa j-uf?z팺~8֞H ,0[kn*BF?P|?:/֍(sG~@ŸO-wzu;Ge9 F?1RyJIGh-:>Zqsq iV;{V7_3a= eT%ߏ|s.4˯ Y%΢gY%hPʲ#0a$׍i 'xwO5 -QmG.fu67ѻϴ4vDUԜ׶K@G dCMqkuy|?x᭛TڷG.mhYXjdǴ}<1w+xkZۚ?u}!{xKBoi n*WQgj,:+Gʣ<}SKA? _j-HKi Gݿ!rsqֹoj3xOHhw\#ۛ$3 2*Ov %uĺ'Pxg! 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Equal numbers of cells were serially diluted threefold and )] TJ ET BT 35.250 708.729 Td /F1 9.8 Tf [(plated on medium containing glucose to assess cell numbers and medium containing galactose to induce the expression of )] TJ ET BT 35.250 694.993 Td /F1 9.8 Tf [(mutant HTT. b\) [)] TJ ET BT 106.230 694.993 Td /F5 9.8 Tf [(PIN)] TJ ET BT 122.483 700.712 Td /F1 8.7 Tf [(+)] TJ ET BT 127.545 694.993 Td /F1 9.8 Tf [(] is present in the integrated BY4741 strains and is absent in the )] TJ ET BT 406.658 694.993 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 426.168 694.993 Td /F1 9.8 Tf [(? background. \(T=total protein, )] TJ ET BT 35.250 681.256 Td /F1 9.8 Tf [(S=soluble protein, and P=insoluble protein\). The [PIN] status of the cells was determined by using standard immunoblot )] TJ ET BT 35.250 667.520 Td /F1 9.8 Tf [(techniques and an antibody raised against yeast Rnq1p. c\) [)] TJ ET BT 294.308 667.520 Td /F5 9.8 Tf [(PSI)] TJ ET BT 310.024 673.240 Td /F1 8.7 Tf [(+)] TJ ET BT 315.086 667.520 Td /F1 9.8 Tf [(] is present in both the integrated BY4741 strains and the )] TJ ET BT 35.250 653.784 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 54.760 653.784 Td /F1 9.8 Tf [(? background. The presence of [)] TJ ET BT 195.130 653.784 Td /F5 9.8 Tf [(PSI)] TJ ET BT 210.847 659.504 Td /F1 8.7 Tf [(+)] TJ ET BT 215.909 653.784 Td /F1 9.8 Tf [(] was assessed using an antibody raised against yeast Sup35p.)] TJ ET Q BT 26.250 615.263 Td /F1 9.8 Tf [(The effect of expressing the integrated HTT constructs was assessed via growth assays and fluorescence microscopy. )] TJ ET BT 26.250 603.358 Td /F1 9.8 Tf [(Surprisingly, we observed that expression of HTT103Q in the integrated strain did not impair growth in multiple integrants )] TJ ET BT 26.250 591.453 Td /F1 9.8 Tf [(generated from independent transformations \(Figure 1a\). As the presence of the [)] TJ ET BT 377.425 591.453 Td /F5 9.8 Tf [(PIN)] TJ ET BT 393.679 595.342 Td /F5 8.7 Tf [(+)] TJ ET BT 398.740 591.453 Td /F1 9.8 Tf [(] and [)] TJ ET BT 425.845 591.453 Td /F5 9.8 Tf [(PSI)] TJ ET BT 441.562 595.342 Td /F5 8.7 Tf [(+)] TJ ET BT 446.623 591.453 Td /F1 9.8 Tf [(] prions modulates mutant )] TJ ET BT 26.250 579.549 Td /F1 9.8 Tf [(HTT aggregation and toxicity in bakers yeast)] TJ ET 0.267 0.267 0.267 rg BT 221.328 581.056 Td /F4 8.7 Tf [(25)] TJ ET 0.271 0.267 0.267 rg BT 230.965 579.549 Td /F1 9.8 Tf [( , we assessed their status in the integrated strains. We observed that Rnq1p is )] TJ ET BT 26.250 567.644 Td /F1 9.8 Tf [(predominantly found as [)] TJ ET BT 133.012 567.644 Td /F5 9.8 Tf [(PIN)] TJ ET BT 149.266 571.532 Td /F5 8.7 Tf [(+)] TJ ET BT 154.327 567.644 Td /F1 9.8 Tf [(] in the BY4741 integrated strains, and as expected is not present in the )] TJ ET BT 465.966 567.644 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 485.476 567.644 Td /F1 9.8 Tf [(? strains \(Figure 1b\). )] TJ ET BT 26.250 555.739 Td /F1 9.8 Tf [(Sup35p \(Figure 1c\) was found as [)] TJ ET BT 175.279 555.739 Td /F5 9.8 Tf [(PSI)] TJ ET BT 190.996 559.627 Td /F5 8.7 Tf [(+)] TJ ET BT 196.057 555.739 Td /F1 9.8 Tf [(] in both the BY4741 and )] TJ ET BT 306.096 555.739 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 325.605 555.739 Td /F1 9.8 Tf [(? integrated strains. These data confirm that lack of [)] TJ ET BT 552.673 555.739 Td /F5 9.8 Tf [(PIN)] TJ ET BT 568.926 559.627 Td /F5 8.7 Tf [(+)] TJ ET BT 573.988 555.739 Td /F1 9.8 Tf [(] )] TJ ET BT 26.250 543.834 Td /F1 9.8 Tf [(or [)] TJ ET BT 40.339 543.834 Td /F5 9.8 Tf [(PSI)] TJ ET BT 56.056 547.723 Td /F5 8.7 Tf [(+)] TJ ET BT 61.117 543.834 Td /F1 9.8 Tf [(] is not responsible for the absence of HTT103Q toxicity in the generated strains.)] TJ ET BT 26.250 524.430 Td /F1 9.8 Tf [(Despite the lack of HTT103Q-dependent toxicity in the newly created integrated strains \(Figure 1a\), we observed a high level of )] TJ ET BT 26.250 512.525 Td /F1 9.8 Tf [(inclusion body formation in these cells \(Figure 2\). Indeed, ~90 % of the cells in the integrated HTT103Q strain contained mutant )] TJ ET BT 26.250 500.620 Td /F1 9.8 Tf [(HTT inclusion bodies 12 hours post induction \(Figure 2\), while in the )] TJ ET BT 322.660 500.620 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 342.169 500.620 Td /F1 9.8 Tf [(? strain the number of cells containing inclusions is )] TJ ET BT 26.250 488.715 Td /F1 9.8 Tf [(reduced to ~7 %. Reintroduction of a plasmid expressing Rnq1p into the )] TJ ET BT 339.761 488.715 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 359.271 488.715 Td /F1 9.8 Tf [(? strain restored inclusion body formation to )] TJ ET BT 26.250 476.811 Td /F1 9.8 Tf [(control levels. Thus, this novel integrated yeast model of HD dissociates the toxicity caused by HTT103Q expression from its )] TJ ET BT 26.250 464.906 Td /F1 9.8 Tf [(aggregation and allowed us to further dissect the relationship between prion-like yeast proteins and the dynamics of mutant HTT )] TJ ET BT 26.250 453.001 Td /F1 9.8 Tf [(aggregation without the confounding effects of toxicity.)] TJ ET 0.965 0.965 0.965 rg 26.250 80.965 555.000 362.155 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 443.120 m 581.250 443.120 l 581.250 442.370 l 26.250 442.370 l f q 450.000 0 0 328.500 35.250 104.870 cm /I4 Do Q q 35.250 80.965 537.000 17.905 re W n Q Q q 15.000 80.965 577.500 696.035 re W n 0.965 0.965 0.965 rg 26.250 632.287 555.000 144.713 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 632.287 m 581.250 632.287 l 581.250 633.037 l 26.250 633.037 l f q 35.250 643.537 537.000 133.463 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 1: An integrated HD yeast model exhibits a reduced level of mutant HTT toxicity in the cell independent of the )] TJ ET BT 35.250 755.571 Td /F4 9.8 Tf [(presence of [PIN+] and [PSI+])] TJ ET BT 35.250 736.201 Td /F1 9.8 Tf [(a\) Expression of the integrated HTT103Q construct in BY4741 yielded no toxicity or cell death, even when Rnq1p was )] TJ ET BT 35.250 722.465 Td /F1 9.8 Tf [(expressed on a high copy number plasmid, labelled here as 2. Equal numbers of cells were serially diluted threefold and )] TJ ET BT 35.250 708.729 Td /F1 9.8 Tf [(plated on medium containing glucose to assess cell numbers and medium containing galactose to induce the expression of )] TJ ET BT 35.250 694.993 Td /F1 9.8 Tf [(mutant HTT. b\) [)] TJ ET BT 106.230 694.993 Td /F5 9.8 Tf [(PIN)] TJ ET BT 122.483 700.712 Td /F1 8.7 Tf [(+)] TJ ET BT 127.545 694.993 Td /F1 9.8 Tf [(] is present in the integrated BY4741 strains and is absent in the )] TJ ET BT 406.658 694.993 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 426.168 694.993 Td /F1 9.8 Tf [(? background. \(T=total protein, )] TJ ET BT 35.250 681.256 Td /F1 9.8 Tf [(S=soluble protein, and P=insoluble protein\). The [PIN] status of the cells was determined by using standard immunoblot )] TJ ET BT 35.250 667.520 Td /F1 9.8 Tf [(techniques and an antibody raised against yeast Rnq1p. c\) [)] TJ ET BT 294.308 667.520 Td /F5 9.8 Tf [(PSI)] TJ ET BT 310.024 673.240 Td /F1 8.7 Tf [(+)] TJ ET BT 315.086 667.520 Td /F1 9.8 Tf [(] is present in both the integrated BY4741 strains and the )] TJ ET BT 35.250 653.784 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 54.760 653.784 Td /F1 9.8 Tf [(? background. The presence of [)] TJ ET BT 195.130 653.784 Td /F5 9.8 Tf [(PSI)] TJ ET BT 210.847 659.504 Td /F1 8.7 Tf [(+)] TJ ET BT 215.909 653.784 Td /F1 9.8 Tf [(] was assessed using an antibody raised against yeast Sup35p.)] TJ ET Q BT 26.250 615.263 Td /F1 9.8 Tf [(The effect of expressing the integrated HTT constructs was assessed via growth assays and fluorescence microscopy. )] TJ ET BT 26.250 603.358 Td /F1 9.8 Tf [(Surprisingly, we observed that expression of HTT103Q in the integrated strain did not impair growth in multiple integrants )] TJ ET BT 26.250 591.453 Td /F1 9.8 Tf [(generated from independent transformations \(Figure 1a\). As the presence of the [)] TJ ET BT 377.425 591.453 Td /F5 9.8 Tf [(PIN)] TJ ET BT 393.679 595.342 Td /F5 8.7 Tf [(+)] TJ ET BT 398.740 591.453 Td /F1 9.8 Tf [(] and [)] TJ ET BT 425.845 591.453 Td /F5 9.8 Tf [(PSI)] TJ ET BT 441.562 595.342 Td /F5 8.7 Tf [(+)] TJ ET BT 446.623 591.453 Td /F1 9.8 Tf [(] prions modulates mutant )] TJ ET BT 26.250 579.549 Td /F1 9.8 Tf [(HTT aggregation and toxicity in bakers yeast)] TJ ET 0.267 0.267 0.267 rg BT 221.328 581.056 Td /F4 8.7 Tf [(25)] TJ ET 0.271 0.267 0.267 rg BT 230.965 579.549 Td /F1 9.8 Tf [( , we assessed their status in the integrated strains. We observed that Rnq1p is )] TJ ET BT 26.250 567.644 Td /F1 9.8 Tf [(predominantly found as [)] TJ ET BT 133.012 567.644 Td /F5 9.8 Tf [(PIN)] TJ ET BT 149.266 571.532 Td /F5 8.7 Tf [(+)] TJ ET BT 154.327 567.644 Td /F1 9.8 Tf [(] in the BY4741 integrated strains, and as expected is not present in the )] TJ ET BT 465.966 567.644 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 485.476 567.644 Td /F1 9.8 Tf [(? strains \(Figure 1b\). )] TJ ET BT 26.250 555.739 Td /F1 9.8 Tf [(Sup35p \(Figure 1c\) was found as [)] TJ ET BT 175.279 555.739 Td /F5 9.8 Tf [(PSI)] TJ ET BT 190.996 559.627 Td /F5 8.7 Tf [(+)] TJ ET BT 196.057 555.739 Td /F1 9.8 Tf [(] in both the BY4741 and )] TJ ET BT 306.096 555.739 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 325.605 555.739 Td /F1 9.8 Tf [(? integrated strains. These data confirm that lack of [)] TJ ET BT 552.673 555.739 Td /F5 9.8 Tf [(PIN)] TJ ET BT 568.926 559.627 Td /F5 8.7 Tf [(+)] TJ ET BT 573.988 555.739 Td /F1 9.8 Tf [(] )] TJ ET BT 26.250 543.834 Td /F1 9.8 Tf [(or [)] TJ ET BT 40.339 543.834 Td /F5 9.8 Tf [(PSI)] TJ ET BT 56.056 547.723 Td /F5 8.7 Tf [(+)] TJ ET BT 61.117 543.834 Td /F1 9.8 Tf [(] is not responsible for the absence of HTT103Q toxicity in the generated strains.)] TJ ET BT 26.250 524.430 Td /F1 9.8 Tf [(Despite the lack of HTT103Q-dependent toxicity in the newly created integrated strains \(Figure 1a\), we observed a high level of )] TJ ET BT 26.250 512.525 Td /F1 9.8 Tf [(inclusion body formation in these cells \(Figure 2\). Indeed, ~90 % of the cells in the integrated HTT103Q strain contained mutant )] TJ ET BT 26.250 500.620 Td /F1 9.8 Tf [(HTT inclusion bodies 12 hours post induction \(Figure 2\), while in the )] TJ ET BT 322.660 500.620 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 342.169 500.620 Td /F1 9.8 Tf [(? strain the number of cells containing inclusions is )] TJ ET BT 26.250 488.715 Td /F1 9.8 Tf [(reduced to ~7 %. Reintroduction of a plasmid expressing Rnq1p into the )] TJ ET BT 339.761 488.715 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 359.271 488.715 Td /F1 9.8 Tf [(? strain restored inclusion body formation to )] TJ ET BT 26.250 476.811 Td /F1 9.8 Tf [(control levels. Thus, this novel integrated yeast model of HD dissociates the toxicity caused by HTT103Q expression from its )] TJ ET BT 26.250 464.906 Td /F1 9.8 Tf [(aggregation and allowed us to further dissect the relationship between prion-like yeast proteins and the dynamics of mutant HTT )] TJ ET BT 26.250 453.001 Td /F1 9.8 Tf [(aggregation without the confounding effects of toxicity.)] TJ ET 0.965 0.965 0.965 rg 26.250 80.965 555.000 362.155 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 443.120 m 581.250 443.120 l 581.250 442.370 l 26.250 442.370 l f q 450.000 0 0 328.500 35.250 104.870 cm /I4 Do Q q 35.250 80.965 537.000 17.905 re W n Q Q q 15.000 80.965 577.500 696.035 re W n 0.965 0.965 0.965 rg 26.250 632.287 555.000 144.713 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 632.287 m 581.250 632.287 l 581.250 633.037 l 26.250 633.037 l f q 35.250 643.537 537.000 133.463 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 1: An integrated HD yeast model exhibits a reduced level of mutant HTT toxicity in the cell independent of the )] TJ ET BT 35.250 755.571 Td /F4 9.8 Tf [(presence of [PIN+] and [PSI+])] TJ ET BT 35.250 736.201 Td /F1 9.8 Tf [(a\) Expression of the integrated HTT103Q construct in BY4741 yielded no toxicity or cell death, even when Rnq1p was )] TJ ET BT 35.250 722.465 Td /F1 9.8 Tf [(expressed on a high copy number plasmid, labelled here as 2. Equal numbers of cells were serially diluted threefold and )] TJ ET BT 35.250 708.729 Td /F1 9.8 Tf [(plated on medium containing glucose to assess cell numbers and medium containing galactose to induce the expression of )] TJ ET BT 35.250 694.993 Td /F1 9.8 Tf [(mutant HTT. b\) [)] TJ ET BT 106.230 694.993 Td /F5 9.8 Tf [(PIN)] TJ ET BT 122.483 700.712 Td /F1 8.7 Tf [(+)] TJ ET BT 127.545 694.993 Td /F1 9.8 Tf [(] is present in the integrated BY4741 strains and is absent in the )] TJ ET BT 406.658 694.993 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 426.168 694.993 Td /F1 9.8 Tf [(? background. \(T=total protein, )] TJ ET BT 35.250 681.256 Td /F1 9.8 Tf [(S=soluble protein, and P=insoluble protein\). The [PIN] status of the cells was determined by using standard immunoblot )] TJ ET BT 35.250 667.520 Td /F1 9.8 Tf [(techniques and an antibody raised against yeast Rnq1p. c\) [)] TJ ET BT 294.308 667.520 Td /F5 9.8 Tf [(PSI)] TJ ET BT 310.024 673.240 Td /F1 8.7 Tf [(+)] TJ ET BT 315.086 667.520 Td /F1 9.8 Tf [(] is present in both the integrated BY4741 strains and the )] TJ ET BT 35.250 653.784 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 54.760 653.784 Td /F1 9.8 Tf [(? background. The presence of [)] TJ ET BT 195.130 653.784 Td /F5 9.8 Tf [(PSI)] TJ ET BT 210.847 659.504 Td /F1 8.7 Tf [(+)] TJ ET BT 215.909 653.784 Td /F1 9.8 Tf [(] was assessed using an antibody raised against yeast Sup35p.)] TJ ET Q BT 26.250 615.263 Td /F1 9.8 Tf [(The effect of expressing the integrated HTT constructs was assessed via growth assays and fluorescence microscopy. )] TJ ET BT 26.250 603.358 Td /F1 9.8 Tf [(Surprisingly, we observed that expression of HTT103Q in the integrated strain did not impair growth in multiple integrants )] TJ ET BT 26.250 591.453 Td /F1 9.8 Tf [(generated from independent transformations \(Figure 1a\). As the presence of the [)] TJ ET BT 377.425 591.453 Td /F5 9.8 Tf [(PIN)] TJ ET BT 393.679 595.342 Td /F5 8.7 Tf [(+)] TJ ET BT 398.740 591.453 Td /F1 9.8 Tf [(] and [)] TJ ET BT 425.845 591.453 Td /F5 9.8 Tf [(PSI)] TJ ET BT 441.562 595.342 Td /F5 8.7 Tf [(+)] TJ ET BT 446.623 591.453 Td /F1 9.8 Tf [(] prions modulates mutant )] TJ ET BT 26.250 579.549 Td /F1 9.8 Tf [(HTT aggregation and toxicity in bakers yeast)] TJ ET 0.267 0.267 0.267 rg BT 221.328 581.056 Td /F4 8.7 Tf [(25)] TJ ET 0.271 0.267 0.267 rg BT 230.965 579.549 Td /F1 9.8 Tf [( , we assessed their status in the integrated strains. We observed that Rnq1p is )] TJ ET BT 26.250 567.644 Td /F1 9.8 Tf [(predominantly found as [)] TJ ET BT 133.012 567.644 Td /F5 9.8 Tf [(PIN)] TJ ET BT 149.266 571.532 Td /F5 8.7 Tf [(+)] TJ ET BT 154.327 567.644 Td /F1 9.8 Tf [(] in the BY4741 integrated strains, and as expected is not present in the )] TJ ET BT 465.966 567.644 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 485.476 567.644 Td /F1 9.8 Tf [(? strains \(Figure 1b\). )] TJ ET BT 26.250 555.739 Td /F1 9.8 Tf [(Sup35p \(Figure 1c\) was found as [)] TJ ET BT 175.279 555.739 Td /F5 9.8 Tf [(PSI)] TJ ET BT 190.996 559.627 Td /F5 8.7 Tf [(+)] TJ ET BT 196.057 555.739 Td /F1 9.8 Tf [(] in both the BY4741 and )] TJ ET BT 306.096 555.739 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 325.605 555.739 Td /F1 9.8 Tf [(? integrated strains. These data confirm that lack of [)] TJ ET BT 552.673 555.739 Td /F5 9.8 Tf [(PIN)] TJ ET BT 568.926 559.627 Td /F5 8.7 Tf [(+)] TJ ET BT 573.988 555.739 Td /F1 9.8 Tf [(] )] TJ ET BT 26.250 543.834 Td /F1 9.8 Tf [(or [)] TJ ET BT 40.339 543.834 Td /F5 9.8 Tf [(PSI)] TJ ET BT 56.056 547.723 Td /F5 8.7 Tf [(+)] TJ ET BT 61.117 543.834 Td /F1 9.8 Tf [(] is not responsible for the absence of HTT103Q toxicity in the generated strains.)] TJ ET BT 26.250 524.430 Td /F1 9.8 Tf [(Despite the lack of HTT103Q-dependent toxicity in the newly created integrated strains \(Figure 1a\), we observed a high level of )] TJ ET BT 26.250 512.525 Td /F1 9.8 Tf [(inclusion body formation in these cells \(Figure 2\). Indeed, ~90 % of the cells in the integrated HTT103Q strain contained mutant )] TJ ET BT 26.250 500.620 Td /F1 9.8 Tf [(HTT inclusion bodies 12 hours post induction \(Figure 2\), while in the )] TJ ET BT 322.660 500.620 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 342.169 500.620 Td /F1 9.8 Tf [(? strain the number of cells containing inclusions is )] TJ ET BT 26.250 488.715 Td /F1 9.8 Tf [(reduced to ~7 %. Reintroduction of a plasmid expressing Rnq1p into the )] TJ ET BT 339.761 488.715 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 359.271 488.715 Td /F1 9.8 Tf [(? strain restored inclusion body formation to )] TJ ET BT 26.250 476.811 Td /F1 9.8 Tf [(control levels. Thus, this novel integrated yeast model of HD dissociates the toxicity caused by HTT103Q expression from its )] TJ ET BT 26.250 464.906 Td /F1 9.8 Tf [(aggregation and allowed us to further dissect the relationship between prion-like yeast proteins and the dynamics of mutant HTT )] TJ ET BT 26.250 453.001 Td /F1 9.8 Tf [(aggregation without the confounding effects of toxicity.)] TJ ET 0.965 0.965 0.965 rg 26.250 80.965 555.000 362.155 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 443.120 m 581.250 443.120 l 581.250 442.370 l 26.250 442.370 l f q 450.000 0 0 328.500 35.250 104.870 cm /I4 Do Q q 35.250 80.965 537.000 17.905 re W n Q Q q 450.000 0 0 328.500 35.250 104.870 cm /I4 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(3)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 231 0 obj << /Type /Annot /Subtype /Link /A 232 0 R /Border [0 0 0] /H /I /Rect [ 221.3280 580.2542 230.9653 589.0725 ] >> endobj 232 0 obj << /Type /Action >> endobj 233 0 obj << /Type /Annot /Subtype /Link /A 234 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 104.8702 485.2500 433.3702 ] >> endobj 234 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2014/02/Figure-2.3.jpg) >> endobj 235 0 obj << /Type /XObject /Subtype /Image /Width 600 /Height 438 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 27296>> stream JFIF;CREATOR: gd-jpeg v1.0 (using IJG JPEG v62), quality = 90 C     C   X" }!1AQa"q2#BR$3br %&'()*456789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz w!1AQaq"2B #3Rbr $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?Rh1K0hb9.(1F(P1G4s@ \R`bs9ъ91@1IҐh&(.h4PbQ@(PLRP1K Q&(|tk:zVEt"}EvP\FaF9"|mgw{ GZZfĔ 661w8x¿OC kM5:ub/KnƂU$`fLR1@)1K1@ Q\LPLRъLQ\Q.)1@)qI\PbR9.(bQ0h1K0h&(.(&(. 4(2)x8(/QPqGsGQJ^0((ڗ8' QJ1@ (◌QRqJH~PqE|-}|~*^G:cM-7!^@Ñ}kGqW8+ѱxwqVʾjF/ivil^2Ɲh^=jF/ivil^2Ɲh^)x+ѱxwqVʾjF/ivil^2Ɲh^1GW46/NOWb4;@JQ| ?__8ӿؼe;O|#kwjZF?DaXȗ )%yQ5Oߴ_ğڗ !̓nI|G9`Uf1`澩F/ivil^2Ɲhoً׶>h<]⯈ WDLJn5;B㜩W?ؼe; ?__8ӿ}+W46/NOWb4;@KRW_ؼe; ?__8ӿ}+W_ؼe; ?__8ӿ}+W_ؼe; ?__8ӿ}+W_ؼe; ?__8ӿ}+W_ؼe; ?__8ӿ}+E| ?__8ӿؼe;ҴW_ؼe;ץ_?9OA^xvEӮ4G hBA{PqGt4Q/q@ R~TRI@qGG@qKGQ/w)r(")x)hbj?*\{у@GF=*1F(G)qG@GGF(?*_ʌ{яz?*?*1F(ڃF=/J]ok^+ҧ< 2DA<2< 2Ipݑ@T-:Mn'.Ub26pg5_p?+4hcrdh0Sݸ }kom9A$rԌpA$ڗZ;qGn/F=/E1@ RTcޏƀǽҊSE'KQ1@ QRƀʓ.=;-yR"RK! dO@zG++4"Y! 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JSzWlmy| jwt/FEx_|i|j&F}[^lt_ %Ē#9›Wnʯ8&4~6~&(xsAƅkw*)*ٲ4-5?]hѷ|!wFNWl+M#/ :m*!OvEbGo]+3[>w6oiNILB2Hx]Nr5gQƝFZxjLZqr\[x 娕>\8""'!c۽qdge͕,w"'88&.EKFE%-4.hȠ"O4dPsG4dPsG4挚\7 L)w (h Q(F((Q(юh PP3F((4Q@(4Q@(Ph81F( Q(cEqA1F( Q(c(b(1F( Q(b(1F91F( Qh Q(b(1F( Q(b(1F( sEP endstream endobj 236 0 obj << /Type /Annot /Subtype /Link /A 237 0 R /Border [0 0 0] /H /I /Rect [ 221.3280 580.2542 230.9653 589.0725 ] >> endobj 237 0 obj << /Type /Action >> endobj 238 0 obj << /Type /Annot /Subtype /Link /A 239 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 104.8702 485.2500 433.3702 ] >> endobj 239 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2014/02/Figure-2.3.jpg) >> endobj 240 0 obj << /Type /Annot /Subtype /Link /A 241 0 R /Border [0 0 0] /H /I /Rect [ 221.3280 580.2542 230.9653 589.0725 ] >> endobj 241 0 obj << /Type /Action >> endobj 242 0 obj << /Type /Annot /Subtype /Link /A 243 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 104.8702 485.2500 433.3702 ] >> endobj 243 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2014/02/Figure-2.3.jpg) >> endobj 244 0 obj << /Type /Page /Parent 3 0 R /Annots [ 246 0 R 248 0 R 250 0 R 252 0 R 255 0 R 257 0 R 259 0 R 261 0 R 263 0 R 265 0 R 267 0 R 269 0 R ] /Contents 245 0 R >> endobj 245 0 obj << /Length 18149 >> stream q 15.000 66.069 577.500 710.931 re W n 0.965 0.965 0.965 rg 26.250 638.759 555.000 138.241 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 638.759 m 581.250 638.759 l 581.250 639.509 l 26.250 639.509 l f q 35.250 650.009 537.000 126.991 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 2: Overexpression of Rnq1p increases the aggregation of mutant huntingtin in yeast)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(Expression of HTT was induced in overnight cultures and the number of cells containing inclusion bodies was assessed by )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(fluorescence microscopy. Overexpression of Rnq1p in a )] TJ ET BT 279.634 734.370 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 299.144 734.370 Td /F1 9.8 Tf [(? background restores inclusion body formation in cells )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(expressing HTT103Q. Counts were converted to the percentage of cells containing inclusion bodies and statistical analyses )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(were performed using unpaired, two-way Mann-Whitney tests \(**)] TJ ET BT 313.759 706.897 Td /F5 9.8 Tf [(P)] TJ ET BT 320.262 706.897 Td /F1 9.8 Tf [(< 0.01, ***)] TJ ET BT 364.439 706.897 Td /F5 9.8 Tf [(P)] TJ ET BT 370.942 706.897 Td /F1 9.8 Tf [(< 0.001\), NS = not significant. All data are )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(shown as the mean SEM; n ? 5 per genotype.)] TJ ET Q BT 26.250 621.735 Td /F1 9.8 Tf [(Interestingly, we found that deletion of the endogenous )] TJ ET BT 265.817 621.735 Td /F5 9.8 Tf [(RNQ1)] TJ ET BT 292.903 621.735 Td /F1 9.8 Tf [( gene leads to the formation of inclusion bodies in a small number )] TJ ET BT 26.250 609.831 Td /F1 9.8 Tf [(of cells expressing the HTT25Q construct, which does not normally form these structures \(Figure 2; )] TJ ET BT 456.488 609.831 Td /F5 9.8 Tf [(P)] TJ ET BT 462.991 609.831 Td /F1 9.8 Tf [(=0.0002\). It has recently )] TJ ET BT 26.250 597.926 Td /F1 9.8 Tf [(been observed that a network of proteins in yeast modify the formation of mutant HTT aggregates)] TJ ET 0.267 0.267 0.267 rg BT 446.787 599.433 Td /F4 8.7 Tf [(26)] TJ ET 0.271 0.267 0.267 rg BT 456.424 597.926 Td /F1 9.8 Tf [( , and it is thus possible that )] TJ ET BT 26.250 586.021 Td /F1 9.8 Tf [(deletion of the endogenous )] TJ ET BT 146.584 586.021 Td /F5 9.8 Tf [(RNQ1)] TJ ET BT 173.670 586.021 Td /F1 9.8 Tf [( disrupts the balance between other yeast genes that govern HTT aggregation, leading to the )] TJ ET BT 26.250 574.116 Td /F1 9.8 Tf [(aggregation of the HTT25Q construct, which is highly enriched for glutamines in comparison to most endogenous yeast proteins.)] TJ ET BT 26.250 554.712 Td /F4 9.8 Tf [(Ybr016wp and Sup35p modulate HTT aggregation independently of Rnq1p: )] TJ ET BT 376.226 554.712 Td /F1 9.8 Tf [(We next focused on characterizing the role of )] TJ ET BT 26.250 542.807 Td /F1 9.8 Tf [(Ybr016wp and Sup35p in the aggregation dynamics of mutant HTT in yeast. We initially examined the ability of Ybr016wp to )] TJ ET BT 26.250 530.902 Td /F1 9.8 Tf [(modulate the number of cells containing inclusion bodies when overexpressed in the presence and absence of Rnq1p \(Figure )] TJ ET BT 26.250 518.997 Td /F1 9.8 Tf [(3\). Interestingly, overexpression of Ybr016wp causes a small but significant increase in the formation of inclusion bodies in )] TJ ET BT 26.250 507.093 Td /F1 9.8 Tf [(HTT25Q-expressing BY4741 \(~ 12 %; )] TJ ET BT 193.423 507.093 Td /F5 9.8 Tf [(P)] TJ ET BT 199.927 507.093 Td /F1 9.8 Tf [(=0.0022\) and )] TJ ET BT 260.367 507.093 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 279.877 507.093 Td /F1 9.8 Tf [(? \(~ 7 %; )] TJ ET BT 321.880 507.093 Td /F5 9.8 Tf [(P)] TJ ET BT 328.383 507.093 Td /F1 9.8 Tf [(=0.0007\) cells \(Figure 3\). A possible explanation for this )] TJ ET BT 26.250 495.188 Td /F1 9.8 Tf [(is the high level of Ybr016wp overexpression, which contains five consecutive perfect repeats \(GYNQQ\) that are enriched for )] TJ ET BT 26.250 483.283 Td /F1 9.8 Tf [(asparagine, glutamine, tyrosine and glycine. Overexpression of such sequences induces aggregation of other proteins, and )] TJ ET BT 26.250 471.378 Td /F1 9.8 Tf [(could seed formation of inclusion bodies by polyQ-rich sequences)] TJ ET 0.267 0.267 0.267 rg BT 309.663 472.886 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 319.300 475.267 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 321.710 472.886 Td /F4 8.7 Tf [(27)] TJ ET 0.271 0.267 0.267 rg BT 331.347 471.378 Td /F1 9.8 Tf [( . Analysis of the HTT103Q integrated strain revealed )] TJ ET BT 26.250 459.474 Td /F1 9.8 Tf [(that Ybr016wp overexpression yileds a small but significant increase in the number of BY4741 cells exhibiting inclusion bodies \()] TJ ET BT 26.250 447.569 Td /F5 9.8 Tf [(P)] TJ ET BT 32.753 447.569 Td /F1 9.8 Tf [(=0.008\). Ybr016wp overexpression in the )] TJ ET BT 213.489 447.569 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 232.999 447.569 Td /F1 9.8 Tf [(? background also significantly increased the number of )] TJ ET BT 476.320 447.569 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 495.829 447.569 Td /F1 9.8 Tf [(? cells forming )] TJ ET BT 26.250 435.664 Td /F1 9.8 Tf [(HTT103Q inclusions, from ~ 7 % to ~ 16 % \()] TJ ET BT 217.535 435.664 Td /F5 9.8 Tf [(P)] TJ ET BT 224.038 435.664 Td /F1 9.8 Tf [(=0.0293\). These data indicate that Ybr016wp is able to partially compensate for )] TJ ET BT 26.250 423.759 Td /F1 9.8 Tf [(the loss of Rnq1p in the )] TJ ET BT 131.394 423.759 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 150.904 423.759 Td /F1 9.8 Tf [(? background with respect to mutant HTT aggregation, and that this aggregation is not polyQ-)] TJ ET BT 26.250 411.855 Td /F1 9.8 Tf [(length dependent.)] TJ ET 0.965 0.965 0.965 rg 26.250 66.069 555.000 335.905 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 401.974 m 581.250 401.974 l 581.250 401.224 l 26.250 401.224 l f q 450.000 0 0 302.250 35.250 89.974 cm /I5 Do Q q 35.250 66.069 537.000 17.905 re W n Q Q q 15.000 66.069 577.500 710.931 re W n 0.965 0.965 0.965 rg 26.250 638.759 555.000 138.241 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 638.759 m 581.250 638.759 l 581.250 639.509 l 26.250 639.509 l f q 35.250 650.009 537.000 126.991 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 2: Overexpression of Rnq1p increases the aggregation of mutant huntingtin in yeast)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(Expression of HTT was induced in overnight cultures and the number of cells containing inclusion bodies was assessed by )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(fluorescence microscopy. Overexpression of Rnq1p in a )] TJ ET BT 279.634 734.370 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 299.144 734.370 Td /F1 9.8 Tf [(? background restores inclusion body formation in cells )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(expressing HTT103Q. Counts were converted to the percentage of cells containing inclusion bodies and statistical analyses )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(were performed using unpaired, two-way Mann-Whitney tests \(**)] TJ ET BT 313.759 706.897 Td /F5 9.8 Tf [(P)] TJ ET BT 320.262 706.897 Td /F1 9.8 Tf [(< 0.01, ***)] TJ ET BT 364.439 706.897 Td /F5 9.8 Tf [(P)] TJ ET BT 370.942 706.897 Td /F1 9.8 Tf [(< 0.001\), NS = not significant. All data are )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(shown as the mean SEM; n ? 5 per genotype.)] TJ ET Q BT 26.250 621.735 Td /F1 9.8 Tf [(Interestingly, we found that deletion of the endogenous )] TJ ET BT 265.817 621.735 Td /F5 9.8 Tf [(RNQ1)] TJ ET BT 292.903 621.735 Td /F1 9.8 Tf [( gene leads to the formation of inclusion bodies in a small number )] TJ ET BT 26.250 609.831 Td /F1 9.8 Tf [(of cells expressing the HTT25Q construct, which does not normally form these structures \(Figure 2; )] TJ ET BT 456.488 609.831 Td /F5 9.8 Tf [(P)] TJ ET BT 462.991 609.831 Td /F1 9.8 Tf [(=0.0002\). It has recently )] TJ ET BT 26.250 597.926 Td /F1 9.8 Tf [(been observed that a network of proteins in yeast modify the formation of mutant HTT aggregates)] TJ ET 0.267 0.267 0.267 rg BT 446.787 599.433 Td /F4 8.7 Tf [(26)] TJ ET 0.271 0.267 0.267 rg BT 456.424 597.926 Td /F1 9.8 Tf [( , and it is thus possible that )] TJ ET BT 26.250 586.021 Td /F1 9.8 Tf [(deletion of the endogenous )] TJ ET BT 146.584 586.021 Td /F5 9.8 Tf [(RNQ1)] TJ ET BT 173.670 586.021 Td /F1 9.8 Tf [( disrupts the balance between other yeast genes that govern HTT aggregation, leading to the )] TJ ET BT 26.250 574.116 Td /F1 9.8 Tf [(aggregation of the HTT25Q construct, which is highly enriched for glutamines in comparison to most endogenous yeast proteins.)] TJ ET BT 26.250 554.712 Td /F4 9.8 Tf [(Ybr016wp and Sup35p modulate HTT aggregation independently of Rnq1p: )] TJ ET BT 376.226 554.712 Td /F1 9.8 Tf [(We next focused on characterizing the role of )] TJ ET BT 26.250 542.807 Td /F1 9.8 Tf [(Ybr016wp and Sup35p in the aggregation dynamics of mutant HTT in yeast. We initially examined the ability of Ybr016wp to )] TJ ET BT 26.250 530.902 Td /F1 9.8 Tf [(modulate the number of cells containing inclusion bodies when overexpressed in the presence and absence of Rnq1p \(Figure )] TJ ET BT 26.250 518.997 Td /F1 9.8 Tf [(3\). Interestingly, overexpression of Ybr016wp causes a small but significant increase in the formation of inclusion bodies in )] TJ ET BT 26.250 507.093 Td /F1 9.8 Tf [(HTT25Q-expressing BY4741 \(~ 12 %; )] TJ ET BT 193.423 507.093 Td /F5 9.8 Tf [(P)] TJ ET BT 199.927 507.093 Td /F1 9.8 Tf [(=0.0022\) and )] TJ ET BT 260.367 507.093 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 279.877 507.093 Td /F1 9.8 Tf [(? \(~ 7 %; )] TJ ET BT 321.880 507.093 Td /F5 9.8 Tf [(P)] TJ ET BT 328.383 507.093 Td /F1 9.8 Tf [(=0.0007\) cells \(Figure 3\). A possible explanation for this )] TJ ET BT 26.250 495.188 Td /F1 9.8 Tf [(is the high level of Ybr016wp overexpression, which contains five consecutive perfect repeats \(GYNQQ\) that are enriched for )] TJ ET BT 26.250 483.283 Td /F1 9.8 Tf [(asparagine, glutamine, tyrosine and glycine. Overexpression of such sequences induces aggregation of other proteins, and )] TJ ET BT 26.250 471.378 Td /F1 9.8 Tf [(could seed formation of inclusion bodies by polyQ-rich sequences)] TJ ET 0.267 0.267 0.267 rg BT 309.663 472.886 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 319.300 475.267 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 321.710 472.886 Td /F4 8.7 Tf [(27)] TJ ET 0.271 0.267 0.267 rg BT 331.347 471.378 Td /F1 9.8 Tf [( . Analysis of the HTT103Q integrated strain revealed )] TJ ET BT 26.250 459.474 Td /F1 9.8 Tf [(that Ybr016wp overexpression yileds a small but significant increase in the number of BY4741 cells exhibiting inclusion bodies \()] TJ ET BT 26.250 447.569 Td /F5 9.8 Tf [(P)] TJ ET BT 32.753 447.569 Td /F1 9.8 Tf [(=0.008\). Ybr016wp overexpression in the )] TJ ET BT 213.489 447.569 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 232.999 447.569 Td /F1 9.8 Tf [(? background also significantly increased the number of )] TJ ET BT 476.320 447.569 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 495.829 447.569 Td /F1 9.8 Tf [(? cells forming )] TJ ET BT 26.250 435.664 Td /F1 9.8 Tf [(HTT103Q inclusions, from ~ 7 % to ~ 16 % \()] TJ ET BT 217.535 435.664 Td /F5 9.8 Tf [(P)] TJ ET BT 224.038 435.664 Td /F1 9.8 Tf [(=0.0293\). These data indicate that Ybr016wp is able to partially compensate for )] TJ ET BT 26.250 423.759 Td /F1 9.8 Tf [(the loss of Rnq1p in the )] TJ ET BT 131.394 423.759 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 150.904 423.759 Td /F1 9.8 Tf [(? background with respect to mutant HTT aggregation, and that this aggregation is not polyQ-)] TJ ET BT 26.250 411.855 Td /F1 9.8 Tf [(length dependent.)] TJ ET 0.965 0.965 0.965 rg 26.250 66.069 555.000 335.905 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 401.974 m 581.250 401.974 l 581.250 401.224 l 26.250 401.224 l f q 450.000 0 0 302.250 35.250 89.974 cm /I5 Do Q q 35.250 66.069 537.000 17.905 re W n Q Q q 15.000 66.069 577.500 710.931 re W n 0.965 0.965 0.965 rg 26.250 638.759 555.000 138.241 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 638.759 m 581.250 638.759 l 581.250 639.509 l 26.250 639.509 l f q 35.250 650.009 537.000 126.991 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 2: Overexpression of Rnq1p increases the aggregation of mutant huntingtin in yeast)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(Expression of HTT was induced in overnight cultures and the number of cells containing inclusion bodies was assessed by )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(fluorescence microscopy. Overexpression of Rnq1p in a )] TJ ET BT 279.634 734.370 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 299.144 734.370 Td /F1 9.8 Tf [(? background restores inclusion body formation in cells )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(expressing HTT103Q. Counts were converted to the percentage of cells containing inclusion bodies and statistical analyses )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(were performed using unpaired, two-way Mann-Whitney tests \(**)] TJ ET BT 313.759 706.897 Td /F5 9.8 Tf [(P)] TJ ET BT 320.262 706.897 Td /F1 9.8 Tf [(< 0.01, ***)] TJ ET BT 364.439 706.897 Td /F5 9.8 Tf [(P)] TJ ET BT 370.942 706.897 Td /F1 9.8 Tf [(< 0.001\), NS = not significant. All data are )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(shown as the mean SEM; n ? 5 per genotype.)] TJ ET Q BT 26.250 621.735 Td /F1 9.8 Tf [(Interestingly, we found that deletion of the endogenous )] TJ ET BT 265.817 621.735 Td /F5 9.8 Tf [(RNQ1)] TJ ET BT 292.903 621.735 Td /F1 9.8 Tf [( gene leads to the formation of inclusion bodies in a small number )] TJ ET BT 26.250 609.831 Td /F1 9.8 Tf [(of cells expressing the HTT25Q construct, which does not normally form these structures \(Figure 2; )] TJ ET BT 456.488 609.831 Td /F5 9.8 Tf [(P)] TJ ET BT 462.991 609.831 Td /F1 9.8 Tf [(=0.0002\). It has recently )] TJ ET BT 26.250 597.926 Td /F1 9.8 Tf [(been observed that a network of proteins in yeast modify the formation of mutant HTT aggregates)] TJ ET 0.267 0.267 0.267 rg BT 446.787 599.433 Td /F4 8.7 Tf [(26)] TJ ET 0.271 0.267 0.267 rg BT 456.424 597.926 Td /F1 9.8 Tf [( , and it is thus possible that )] TJ ET BT 26.250 586.021 Td /F1 9.8 Tf [(deletion of the endogenous )] TJ ET BT 146.584 586.021 Td /F5 9.8 Tf [(RNQ1)] TJ ET BT 173.670 586.021 Td /F1 9.8 Tf [( disrupts the balance between other yeast genes that govern HTT aggregation, leading to the )] TJ ET BT 26.250 574.116 Td /F1 9.8 Tf [(aggregation of the HTT25Q construct, which is highly enriched for glutamines in comparison to most endogenous yeast proteins.)] TJ ET BT 26.250 554.712 Td /F4 9.8 Tf [(Ybr016wp and Sup35p modulate HTT aggregation independently of Rnq1p: )] TJ ET BT 376.226 554.712 Td /F1 9.8 Tf [(We next focused on characterizing the role of )] TJ ET BT 26.250 542.807 Td /F1 9.8 Tf [(Ybr016wp and Sup35p in the aggregation dynamics of mutant HTT in yeast. We initially examined the ability of Ybr016wp to )] TJ ET BT 26.250 530.902 Td /F1 9.8 Tf [(modulate the number of cells containing inclusion bodies when overexpressed in the presence and absence of Rnq1p \(Figure )] TJ ET BT 26.250 518.997 Td /F1 9.8 Tf [(3\). Interestingly, overexpression of Ybr016wp causes a small but significant increase in the formation of inclusion bodies in )] TJ ET BT 26.250 507.093 Td /F1 9.8 Tf [(HTT25Q-expressing BY4741 \(~ 12 %; )] TJ ET BT 193.423 507.093 Td /F5 9.8 Tf [(P)] TJ ET BT 199.927 507.093 Td /F1 9.8 Tf [(=0.0022\) and )] TJ ET BT 260.367 507.093 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 279.877 507.093 Td /F1 9.8 Tf [(? \(~ 7 %; )] TJ ET BT 321.880 507.093 Td /F5 9.8 Tf [(P)] TJ ET BT 328.383 507.093 Td /F1 9.8 Tf [(=0.0007\) cells \(Figure 3\). A possible explanation for this )] TJ ET BT 26.250 495.188 Td /F1 9.8 Tf [(is the high level of Ybr016wp overexpression, which contains five consecutive perfect repeats \(GYNQQ\) that are enriched for )] TJ ET BT 26.250 483.283 Td /F1 9.8 Tf [(asparagine, glutamine, tyrosine and glycine. Overexpression of such sequences induces aggregation of other proteins, and )] TJ ET BT 26.250 471.378 Td /F1 9.8 Tf [(could seed formation of inclusion bodies by polyQ-rich sequences)] TJ ET 0.267 0.267 0.267 rg BT 309.663 472.886 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 319.300 475.267 Td /F1 8.7 Tf [(,)] TJ ET 0.267 0.267 0.267 rg BT 321.710 472.886 Td /F4 8.7 Tf [(27)] TJ ET 0.271 0.267 0.267 rg BT 331.347 471.378 Td /F1 9.8 Tf [( . Analysis of the HTT103Q integrated strain revealed )] TJ ET BT 26.250 459.474 Td /F1 9.8 Tf [(that Ybr016wp overexpression yileds a small but significant increase in the number of BY4741 cells exhibiting inclusion bodies \()] TJ ET BT 26.250 447.569 Td /F5 9.8 Tf [(P)] TJ ET BT 32.753 447.569 Td /F1 9.8 Tf [(=0.008\). Ybr016wp overexpression in the )] TJ ET BT 213.489 447.569 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 232.999 447.569 Td /F1 9.8 Tf [(? background also significantly increased the number of )] TJ ET BT 476.320 447.569 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 495.829 447.569 Td /F1 9.8 Tf [(? cells forming )] TJ ET BT 26.250 435.664 Td /F1 9.8 Tf [(HTT103Q inclusions, from ~ 7 % to ~ 16 % \()] TJ ET BT 217.535 435.664 Td /F5 9.8 Tf [(P)] TJ ET BT 224.038 435.664 Td /F1 9.8 Tf [(=0.0293\). These data indicate that Ybr016wp is able to partially compensate for )] TJ ET BT 26.250 423.759 Td /F1 9.8 Tf [(the loss of Rnq1p in the )] TJ ET BT 131.394 423.759 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 150.904 423.759 Td /F1 9.8 Tf [(? background with respect to mutant HTT aggregation, and that this aggregation is not polyQ-)] TJ ET BT 26.250 411.855 Td /F1 9.8 Tf [(length dependent.)] TJ ET 0.965 0.965 0.965 rg 26.250 66.069 555.000 335.905 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 401.974 m 581.250 401.974 l 581.250 401.224 l 26.250 401.224 l f q 450.000 0 0 302.250 35.250 89.974 cm /I5 Do Q q 35.250 66.069 537.000 17.905 re W n Q Q q 450.000 0 0 302.250 35.250 89.974 cm /I5 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(4)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 246 0 obj << /Type /Annot /Subtype /Link /A 247 0 R /Border [0 0 0] /H /I /Rect [ 446.7870 598.6314 456.4243 607.4497 ] >> endobj 247 0 obj << /Type /Action >> endobj 248 0 obj << /Type /Annot /Subtype /Link /A 249 0 R /Border [0 0 0] /H /I /Rect [ 309.6630 472.0839 319.3003 480.9022 ] >> endobj 249 0 obj << /Type /Action >> endobj 250 0 obj << /Type /Annot /Subtype /Link /A 251 0 R /Border [0 0 0] /H /I /Rect [ 321.7097 472.0839 331.3470 480.9022 ] >> endobj 251 0 obj << /Type /Action >> endobj 252 0 obj << /Type /Annot /Subtype /Link /A 253 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 89.9737 485.2500 392.2237 ] >> endobj 253 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2014/03/Figure-3.1-modified.jpg) >> endobj 254 0 obj << /Type /XObject /Subtype /Image /Width 600 /Height 403 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 31140>> stream JFIF;CREATOR: gd-jpeg v1.0 (using IJG JPEG v62), quality = 90 C     C   X" }!1AQa"q2#BR$3br %&'()*456789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz w!1AQaq"2B #3Rbr $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?R/I@ IF(/Iڊ(i(֏Ɗ(wQ?ZJ(}hQE/IE~4Q@b(hƊ(ii(4f((Q(?(Qz>%@xY^~T¨'#'$+fQYkoitm,6gq(9k(ϽPGGEQ@h(E(h 3E-%R4PIF(i{~4b4~4~4wOƊ_ʏʌQ@IQQPTRgPP ?*((~T()?G@EQPRQPQG@ R~TP _ʏʓ ZOʏʀʏʏʏʀʏʊ(ڱ<_ >Fi^ #y Fqx5^X>K;m~mnЬ%Ep$E<?᩾ ^_vjoW-Q?[E? /E{R?᩾ ^_vG8o[qD&(#y E?ŏH%$) J1:#_q"7ºQ?[E? /E{R?᩾ ^_vG8o[qD&(Mei᩾ ^_vG8o[qD&(Kek.h-A$SDE#! ^+O?k>aGY孤@C+;w#Fnz4r%+)? *?*?*3(ڏʏʏʀʃGG@GGG@GGEQEʗϵ/IQPRTgPRT(f_“? ? 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Overexpression of Ybr016wp in a)] TJ ET BT 292.094 734.370 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 311.604 734.370 Td /F1 9.8 Tf [(? background increases inclusion body formation in both )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(HTT25Q and HTT103Q expressing cells. Counts were converted to the percentage of cells containing inclusion bodies and )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(statistical analyses were performed using unpaired, two-way Mann-Whitney tests \(*)] TJ ET BT 393.952 706.897 Td /F5 9.8 Tf [(P)] TJ ET BT 400.456 706.897 Td /F1 9.8 Tf [(< 0.05, **)] TJ ET BT 440.840 706.897 Td /F5 9.8 Tf [(P)] TJ ET BT 447.343 706.897 Td /F1 9.8 Tf [(< 0.01, ***)] TJ ET BT 491.521 706.897 Td /F5 9.8 Tf [(P)] TJ ET BT 498.024 706.897 Td /F1 9.8 Tf [(< 0.001\). All )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(data are shown as the mean SEM; n ? 6 per genotype.)] TJ ET Q BT 26.250 621.735 Td /F1 9.8 Tf [(As the presence of Rnq1p is crucial for the )] TJ ET BT 212.670 621.735 Td /F5 9.8 Tf [(de novo)] TJ ET BT 247.361 621.735 Td /F1 9.8 Tf [( formation of [)] TJ ET BT 306.972 621.735 Td /F5 9.8 Tf [(PSI)] TJ ET BT 322.689 625.624 Td /F5 8.7 Tf [(+)] TJ ET BT 327.750 621.735 Td /F1 9.8 Tf [(] as well as the formation of mutant HTT aggregates in )] TJ ET BT 26.250 609.831 Td /F1 9.8 Tf [(yeast, we next investigated the connection between Sup35p expression and mutant HTT aggregation by overexpressing )] TJ ET BT 26.250 597.926 Td /F1 9.8 Tf [(Sup35p. As with Ybr016wp, overexpression of Sup35p leads to a small but significant increase in the formation of HTT25Q )] TJ ET BT 26.250 586.021 Td /F1 9.8 Tf [(inclusion bodies in the BY4741 background \(Figure 4; )] TJ ET BT 260.367 586.021 Td /F5 9.8 Tf [(P)] TJ ET BT 266.870 586.021 Td /F1 9.8 Tf [( < 0.0001\). It is possible that these inclusion bodies form due to the )] TJ ET BT 26.250 574.116 Td /F1 9.8 Tf [(presence of [)] TJ ET BT 82.615 574.116 Td /F5 9.8 Tf [(PSI)] TJ ET BT 98.332 578.005 Td /F5 8.7 Tf [(+)] TJ ET BT 103.393 574.116 Td /F1 9.8 Tf [(] in the cells, as formation of [)] TJ ET BT 230.202 574.116 Td /F5 9.8 Tf [(PSI)] TJ ET BT 245.919 578.005 Td /F5 8.7 Tf [(+)] TJ ET BT 250.980 574.116 Td /F1 9.8 Tf [(] can be triggered by overexpression of Sup35p)] TJ ET 0.267 0.267 0.267 rg BT 455.837 575.624 Td /F4 8.7 Tf [(28)] TJ ET 0.271 0.267 0.267 rg BT 465.475 574.116 Td /F1 9.8 Tf [( , which is able to cause )] TJ ET BT 26.250 562.212 Td /F1 9.8 Tf [(aggregation of heterogeneous polyQ-rich sequences)] TJ ET 0.267 0.267 0.267 rg BT 253.327 563.719 Td /F4 8.7 Tf [(29)] TJ ET 0.271 0.267 0.267 rg BT 262.965 562.212 Td /F1 9.8 Tf [( . Interestingly, in the )] TJ ET BT 355.639 562.212 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 375.148 562.212 Td /F1 9.8 Tf [(? strain overexpressing Sup35p the formation )] TJ ET BT 26.250 550.307 Td /F1 9.8 Tf [(of HTT25Q inclusion bodies is dramatically increased \(~ 68 %\) compared with the parental BY4741 strain \(~ 12 %; )] TJ ET BT 522.077 550.307 Td /F5 9.8 Tf [(P)] TJ ET BT 528.580 550.307 Td /F1 9.8 Tf [(=0.0264\). )] TJ ET BT 26.250 538.402 Td /F1 9.8 Tf [(As mentioned earlier a network of proteins is able to modify aggregation of mutant HTT in yeast)] TJ ET 0.267 0.267 0.267 rg BT 437.554 539.909 Td /F4 8.7 Tf [(25)] TJ ET 0.271 0.267 0.267 rg BT 447.191 538.402 Td /F1 9.8 Tf [( and has also been found to )] TJ ET BT 26.250 526.497 Td /F1 9.8 Tf [(trigger the formation of [)] TJ ET BT 129.756 526.497 Td /F5 9.8 Tf [(PSI)] TJ ET BT 145.473 530.386 Td /F5 8.7 Tf [(+)] TJ ET BT 150.534 526.497 Td /F1 9.8 Tf [(]. It is therefore possible that this network is activated by the absence of endogenous Rnq1p in the )] TJ ET BT 26.250 514.593 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 45.760 514.593 Td /F1 9.8 Tf [(? strain, and is further stimulated by the presence of [)] TJ ET BT 275.538 514.593 Td /F5 9.8 Tf [(PSI)] TJ ET BT 291.255 518.481 Td /F5 8.7 Tf [(+)] TJ ET BT 296.316 514.593 Td /F1 9.8 Tf [(]. Interestingly, the HTT25Q and HTT103Q strains exhibit a )] TJ ET BT 26.250 502.688 Td /F1 9.8 Tf [(similar number of cells with aggregates in the )] TJ ET BT 224.029 502.688 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 243.538 502.688 Td /F1 9.8 Tf [(? background with Sup35p expression, indicating that this substitution is not )] TJ ET BT 26.250 490.783 Td /F1 9.8 Tf [(polyQ-length dependent, and potentiates the aggregation of shorter polyQ stretches.)] TJ ET 0.965 0.965 0.965 rg 26.250 53.816 555.000 427.086 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 480.902 m 581.250 480.902 l 581.250 480.152 l 26.250 480.152 l f 26.250 53.816 m 581.250 53.816 l 581.250 54.566 l 26.250 54.566 l f q 450.000 0 0 306.000 35.250 165.152 cm /I6 Do Q q 35.250 65.066 537.000 94.086 re W n 0.271 0.267 0.267 rg BT 35.250 149.628 Td /F4 9.8 Tf [(Fig. 4: Overexpression of Sup35p increases the aggregation of mutant huntingtin in yeast)] TJ ET BT 35.250 130.258 Td /F1 9.8 Tf [(Expression of HTT was induced in overnight cultures and the number of cells containing inclusion bodies was assessed by )] TJ ET BT 35.250 116.522 Td /F1 9.8 Tf [(fluorescence microscopy. Overexpression of Sup35p in a )] TJ ET BT 284.519 116.522 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 304.028 116.522 Td /F1 9.8 Tf [(? background greatly enhances inclusion body formation in a )] TJ ET BT 35.250 102.786 Td /F1 9.8 Tf [(non-polyglutamine dependent manner. Counts were converted to the percentage of cells containing inclusion bodies and )] TJ ET BT 35.250 89.050 Td /F1 9.8 Tf [(statistical analyses were performed using unpaired, two-way Mann-Whitney tests \(*** )] TJ ET BT 404.248 89.050 Td /F5 9.8 Tf [(P)] TJ ET BT 410.752 89.050 Td /F1 9.8 Tf [(< 0.001, ****)] TJ ET BT 464.143 89.050 Td /F5 9.8 Tf [(P)] TJ ET BT 470.646 89.050 Td /F1 9.8 Tf [(< 0.0001\), NS = not )] TJ ET BT 35.250 75.313 Td /F1 9.8 Tf [(significant. All data are shown as the mean SEM; n ? 7 per genotype.)] TJ ET Q Q q 15.000 46.316 577.500 730.684 re W n 0.965 0.965 0.965 rg 26.250 638.759 555.000 138.241 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 638.759 m 581.250 638.759 l 581.250 639.509 l 26.250 639.509 l f q 35.250 650.009 537.000 126.991 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 3: Overexpression of Ybr016wp increases the aggregation of mutant huntingtin in yeast)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(Expression of HTT was induced in overnight cultures and the number of cells containing inclusion bodies was assessed by )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(fluorescence microscopy. Overexpression of Ybr016wp in a)] TJ ET BT 292.094 734.370 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 311.604 734.370 Td /F1 9.8 Tf [(? background increases inclusion body formation in both )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(HTT25Q and HTT103Q expressing cells. Counts were converted to the percentage of cells containing inclusion bodies and )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(statistical analyses were performed using unpaired, two-way Mann-Whitney tests \(*)] TJ ET BT 393.952 706.897 Td /F5 9.8 Tf [(P)] TJ ET BT 400.456 706.897 Td /F1 9.8 Tf [(< 0.05, **)] TJ ET BT 440.840 706.897 Td /F5 9.8 Tf [(P)] TJ ET BT 447.343 706.897 Td /F1 9.8 Tf [(< 0.01, ***)] TJ ET BT 491.521 706.897 Td /F5 9.8 Tf [(P)] TJ ET BT 498.024 706.897 Td /F1 9.8 Tf [(< 0.001\). All )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(data are shown as the mean SEM; n ? 6 per genotype.)] TJ ET Q BT 26.250 621.735 Td /F1 9.8 Tf [(As the presence of Rnq1p is crucial for the )] TJ ET BT 212.670 621.735 Td /F5 9.8 Tf [(de novo)] TJ ET BT 247.361 621.735 Td /F1 9.8 Tf [( formation of [)] TJ ET BT 306.972 621.735 Td /F5 9.8 Tf [(PSI)] TJ ET BT 322.689 625.624 Td /F5 8.7 Tf [(+)] TJ ET BT 327.750 621.735 Td /F1 9.8 Tf [(] as well as the formation of mutant HTT aggregates in )] TJ ET BT 26.250 609.831 Td /F1 9.8 Tf [(yeast, we next investigated the connection between Sup35p expression and mutant HTT aggregation by overexpressing )] TJ ET BT 26.250 597.926 Td /F1 9.8 Tf [(Sup35p. As with Ybr016wp, overexpression of Sup35p leads to a small but significant increase in the formation of HTT25Q )] TJ ET BT 26.250 586.021 Td /F1 9.8 Tf [(inclusion bodies in the BY4741 background \(Figure 4; )] TJ ET BT 260.367 586.021 Td /F5 9.8 Tf [(P)] TJ ET BT 266.870 586.021 Td /F1 9.8 Tf [( < 0.0001\). It is possible that these inclusion bodies form due to the )] TJ ET BT 26.250 574.116 Td /F1 9.8 Tf [(presence of [)] TJ ET BT 82.615 574.116 Td /F5 9.8 Tf [(PSI)] TJ ET BT 98.332 578.005 Td /F5 8.7 Tf [(+)] TJ ET BT 103.393 574.116 Td /F1 9.8 Tf [(] in the cells, as formation of [)] TJ ET BT 230.202 574.116 Td /F5 9.8 Tf [(PSI)] TJ ET BT 245.919 578.005 Td /F5 8.7 Tf [(+)] TJ ET BT 250.980 574.116 Td /F1 9.8 Tf [(] can be triggered by overexpression of Sup35p)] TJ ET 0.267 0.267 0.267 rg BT 455.837 575.624 Td /F4 8.7 Tf [(28)] TJ ET 0.271 0.267 0.267 rg BT 465.475 574.116 Td /F1 9.8 Tf [( , which is able to cause )] TJ ET BT 26.250 562.212 Td /F1 9.8 Tf [(aggregation of heterogeneous polyQ-rich sequences)] TJ ET 0.267 0.267 0.267 rg BT 253.327 563.719 Td /F4 8.7 Tf [(29)] TJ ET 0.271 0.267 0.267 rg BT 262.965 562.212 Td /F1 9.8 Tf [( . Interestingly, in the )] TJ ET BT 355.639 562.212 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 375.148 562.212 Td /F1 9.8 Tf [(? strain overexpressing Sup35p the formation )] TJ ET BT 26.250 550.307 Td /F1 9.8 Tf [(of HTT25Q inclusion bodies is dramatically increased \(~ 68 %\) compared with the parental BY4741 strain \(~ 12 %; )] TJ ET BT 522.077 550.307 Td /F5 9.8 Tf [(P)] TJ ET BT 528.580 550.307 Td /F1 9.8 Tf [(=0.0264\). )] TJ ET BT 26.250 538.402 Td /F1 9.8 Tf [(As mentioned earlier a network of proteins is able to modify aggregation of mutant HTT in yeast)] TJ ET 0.267 0.267 0.267 rg BT 437.554 539.909 Td /F4 8.7 Tf [(25)] TJ ET 0.271 0.267 0.267 rg BT 447.191 538.402 Td /F1 9.8 Tf [( and has also been found to )] TJ ET BT 26.250 526.497 Td /F1 9.8 Tf [(trigger the formation of [)] TJ ET BT 129.756 526.497 Td /F5 9.8 Tf [(PSI)] TJ ET BT 145.473 530.386 Td /F5 8.7 Tf [(+)] TJ ET BT 150.534 526.497 Td /F1 9.8 Tf [(]. It is therefore possible that this network is activated by the absence of endogenous Rnq1p in the )] TJ ET BT 26.250 514.593 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 45.760 514.593 Td /F1 9.8 Tf [(? strain, and is further stimulated by the presence of [)] TJ ET BT 275.538 514.593 Td /F5 9.8 Tf [(PSI)] TJ ET BT 291.255 518.481 Td /F5 8.7 Tf [(+)] TJ ET BT 296.316 514.593 Td /F1 9.8 Tf [(]. Interestingly, the HTT25Q and HTT103Q strains exhibit a )] TJ ET BT 26.250 502.688 Td /F1 9.8 Tf [(similar number of cells with aggregates in the )] TJ ET BT 224.029 502.688 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 243.538 502.688 Td /F1 9.8 Tf [(? background with Sup35p expression, indicating that this substitution is not )] TJ ET BT 26.250 490.783 Td /F1 9.8 Tf [(polyQ-length dependent, and potentiates the aggregation of shorter polyQ stretches.)] TJ ET 0.965 0.965 0.965 rg 26.250 53.816 555.000 427.086 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 480.902 m 581.250 480.902 l 581.250 480.152 l 26.250 480.152 l f 26.250 53.816 m 581.250 53.816 l 581.250 54.566 l 26.250 54.566 l f q 450.000 0 0 306.000 35.250 165.152 cm /I6 Do Q q 35.250 65.066 537.000 94.086 re W n 0.271 0.267 0.267 rg BT 35.250 149.628 Td /F4 9.8 Tf [(Fig. 4: Overexpression of Sup35p increases the aggregation of mutant huntingtin in yeast)] TJ ET BT 35.250 130.258 Td /F1 9.8 Tf [(Expression of HTT was induced in overnight cultures and the number of cells containing inclusion bodies was assessed by )] TJ ET BT 35.250 116.522 Td /F1 9.8 Tf [(fluorescence microscopy. Overexpression of Sup35p in a )] TJ ET BT 284.519 116.522 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 304.028 116.522 Td /F1 9.8 Tf [(? background greatly enhances inclusion body formation in a )] TJ ET BT 35.250 102.786 Td /F1 9.8 Tf [(non-polyglutamine dependent manner. Counts were converted to the percentage of cells containing inclusion bodies and )] TJ ET BT 35.250 89.050 Td /F1 9.8 Tf [(statistical analyses were performed using unpaired, two-way Mann-Whitney tests \(*** )] TJ ET BT 404.248 89.050 Td /F5 9.8 Tf [(P)] TJ ET BT 410.752 89.050 Td /F1 9.8 Tf [(< 0.001, ****)] TJ ET BT 464.143 89.050 Td /F5 9.8 Tf [(P)] TJ ET BT 470.646 89.050 Td /F1 9.8 Tf [(< 0.0001\), NS = not )] TJ ET BT 35.250 75.313 Td /F1 9.8 Tf [(significant. All data are shown as the mean SEM; n ? 7 per genotype.)] TJ ET Q Q q 15.000 46.316 577.500 730.684 re W n 0.965 0.965 0.965 rg 26.250 638.759 555.000 138.241 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 638.759 m 581.250 638.759 l 581.250 639.509 l 26.250 639.509 l f q 35.250 650.009 537.000 126.991 re W n 0.271 0.267 0.267 rg BT 35.250 767.476 Td /F4 9.8 Tf [(Fig. 3: Overexpression of Ybr016wp increases the aggregation of mutant huntingtin in yeast)] TJ ET BT 35.250 748.106 Td /F1 9.8 Tf [(Expression of HTT was induced in overnight cultures and the number of cells containing inclusion bodies was assessed by )] TJ ET BT 35.250 734.370 Td /F1 9.8 Tf [(fluorescence microscopy. Overexpression of Ybr016wp in a)] TJ ET BT 292.094 734.370 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 311.604 734.370 Td /F1 9.8 Tf [(? background increases inclusion body formation in both )] TJ ET BT 35.250 720.634 Td /F1 9.8 Tf [(HTT25Q and HTT103Q expressing cells. Counts were converted to the percentage of cells containing inclusion bodies and )] TJ ET BT 35.250 706.897 Td /F1 9.8 Tf [(statistical analyses were performed using unpaired, two-way Mann-Whitney tests \(*)] TJ ET BT 393.952 706.897 Td /F5 9.8 Tf [(P)] TJ ET BT 400.456 706.897 Td /F1 9.8 Tf [(< 0.05, **)] TJ ET BT 440.840 706.897 Td /F5 9.8 Tf [(P)] TJ ET BT 447.343 706.897 Td /F1 9.8 Tf [(< 0.01, ***)] TJ ET BT 491.521 706.897 Td /F5 9.8 Tf [(P)] TJ ET BT 498.024 706.897 Td /F1 9.8 Tf [(< 0.001\). All )] TJ ET BT 35.250 693.161 Td /F1 9.8 Tf [(data are shown as the mean SEM; n ? 6 per genotype.)] TJ ET Q BT 26.250 621.735 Td /F1 9.8 Tf [(As the presence of Rnq1p is crucial for the )] TJ ET BT 212.670 621.735 Td /F5 9.8 Tf [(de novo)] TJ ET BT 247.361 621.735 Td /F1 9.8 Tf [( formation of [)] TJ ET BT 306.972 621.735 Td /F5 9.8 Tf [(PSI)] TJ ET BT 322.689 625.624 Td /F5 8.7 Tf [(+)] TJ ET BT 327.750 621.735 Td /F1 9.8 Tf [(] as well as the formation of mutant HTT aggregates in )] TJ ET BT 26.250 609.831 Td /F1 9.8 Tf [(yeast, we next investigated the connection between Sup35p expression and mutant HTT aggregation by overexpressing )] TJ ET BT 26.250 597.926 Td /F1 9.8 Tf [(Sup35p. As with Ybr016wp, overexpression of Sup35p leads to a small but significant increase in the formation of HTT25Q )] TJ ET BT 26.250 586.021 Td /F1 9.8 Tf [(inclusion bodies in the BY4741 background \(Figure 4; )] TJ ET BT 260.367 586.021 Td /F5 9.8 Tf [(P)] TJ ET BT 266.870 586.021 Td /F1 9.8 Tf [( < 0.0001\). It is possible that these inclusion bodies form due to the )] TJ ET BT 26.250 574.116 Td /F1 9.8 Tf [(presence of [)] TJ ET BT 82.615 574.116 Td /F5 9.8 Tf [(PSI)] TJ ET BT 98.332 578.005 Td /F5 8.7 Tf [(+)] TJ ET BT 103.393 574.116 Td /F1 9.8 Tf [(] in the cells, as formation of [)] TJ ET BT 230.202 574.116 Td /F5 9.8 Tf [(PSI)] TJ ET BT 245.919 578.005 Td /F5 8.7 Tf [(+)] TJ ET BT 250.980 574.116 Td /F1 9.8 Tf [(] can be triggered by overexpression of Sup35p)] TJ ET 0.267 0.267 0.267 rg BT 455.837 575.624 Td /F4 8.7 Tf [(28)] TJ ET 0.271 0.267 0.267 rg BT 465.475 574.116 Td /F1 9.8 Tf [( , which is able to cause )] TJ ET BT 26.250 562.212 Td /F1 9.8 Tf [(aggregation of heterogeneous polyQ-rich sequences)] TJ ET 0.267 0.267 0.267 rg BT 253.327 563.719 Td /F4 8.7 Tf [(29)] TJ ET 0.271 0.267 0.267 rg BT 262.965 562.212 Td /F1 9.8 Tf [( . Interestingly, in the )] TJ ET BT 355.639 562.212 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 375.148 562.212 Td /F1 9.8 Tf [(? strain overexpressing Sup35p the formation )] TJ ET BT 26.250 550.307 Td /F1 9.8 Tf [(of HTT25Q inclusion bodies is dramatically increased \(~ 68 %\) compared with the parental BY4741 strain \(~ 12 %; )] TJ ET BT 522.077 550.307 Td /F5 9.8 Tf [(P)] TJ ET BT 528.580 550.307 Td /F1 9.8 Tf [(=0.0264\). )] TJ ET BT 26.250 538.402 Td /F1 9.8 Tf [(As mentioned earlier a network of proteins is able to modify aggregation of mutant HTT in yeast)] TJ ET 0.267 0.267 0.267 rg BT 437.554 539.909 Td /F4 8.7 Tf [(25)] TJ ET 0.271 0.267 0.267 rg BT 447.191 538.402 Td /F1 9.8 Tf [( and has also been found to )] TJ ET BT 26.250 526.497 Td /F1 9.8 Tf [(trigger the formation of [)] TJ ET BT 129.756 526.497 Td /F5 9.8 Tf [(PSI)] TJ ET BT 145.473 530.386 Td /F5 8.7 Tf [(+)] TJ ET BT 150.534 526.497 Td /F1 9.8 Tf [(]. It is therefore possible that this network is activated by the absence of endogenous Rnq1p in the )] TJ ET BT 26.250 514.593 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 45.760 514.593 Td /F1 9.8 Tf [(? strain, and is further stimulated by the presence of [)] TJ ET BT 275.538 514.593 Td /F5 9.8 Tf [(PSI)] TJ ET BT 291.255 518.481 Td /F5 8.7 Tf [(+)] TJ ET BT 296.316 514.593 Td /F1 9.8 Tf [(]. Interestingly, the HTT25Q and HTT103Q strains exhibit a )] TJ ET BT 26.250 502.688 Td /F1 9.8 Tf [(similar number of cells with aggregates in the )] TJ ET BT 224.029 502.688 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 243.538 502.688 Td /F1 9.8 Tf [(? background with Sup35p expression, indicating that this substitution is not )] TJ ET BT 26.250 490.783 Td /F1 9.8 Tf [(polyQ-length dependent, and potentiates the aggregation of shorter polyQ stretches.)] TJ ET 0.965 0.965 0.965 rg 26.250 53.816 555.000 427.086 re f 0.267 0.267 0.267 rg 0.267 0.267 0.267 RG 26.250 480.902 m 581.250 480.902 l 581.250 480.152 l 26.250 480.152 l f 26.250 53.816 m 581.250 53.816 l 581.250 54.566 l 26.250 54.566 l f q 450.000 0 0 306.000 35.250 165.152 cm /I6 Do Q q 35.250 65.066 537.000 94.086 re W n 0.271 0.267 0.267 rg BT 35.250 149.628 Td /F4 9.8 Tf [(Fig. 4: Overexpression of Sup35p increases the aggregation of mutant huntingtin in yeast)] TJ ET BT 35.250 130.258 Td /F1 9.8 Tf [(Expression of HTT was induced in overnight cultures and the number of cells containing inclusion bodies was assessed by )] TJ ET BT 35.250 116.522 Td /F1 9.8 Tf [(fluorescence microscopy. Overexpression of Sup35p in a )] TJ ET BT 284.519 116.522 Td /F5 9.8 Tf [(rnq1)] TJ ET BT 304.028 116.522 Td /F1 9.8 Tf [(? background greatly enhances inclusion body formation in a )] TJ ET BT 35.250 102.786 Td /F1 9.8 Tf [(non-polyglutamine dependent manner. Counts were converted to the percentage of cells containing inclusion bodies and )] TJ ET BT 35.250 89.050 Td /F1 9.8 Tf [(statistical analyses were performed using unpaired, two-way Mann-Whitney tests \(*** )] TJ ET BT 404.248 89.050 Td /F5 9.8 Tf [(P)] TJ ET BT 410.752 89.050 Td /F1 9.8 Tf [(< 0.001, ****)] TJ ET BT 464.143 89.050 Td /F5 9.8 Tf [(P)] TJ ET BT 470.646 89.050 Td /F1 9.8 Tf [(< 0.0001\), NS = not )] TJ ET BT 35.250 75.313 Td /F1 9.8 Tf [(significant. All data are shown as the mean SEM; n ? 7 per genotype.)] TJ ET Q Q q 450.000 0 0 306.000 35.250 165.152 cm /I6 Do Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(5)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 273 0 obj << /Type /Annot /Subtype /Link /A 274 0 R /Border [0 0 0] /H /I /Rect [ 455.8372 574.8219 465.4745 583.6402 ] >> endobj 274 0 obj << /Type /Action >> endobj 275 0 obj << /Type /Annot /Subtype /Link /A 276 0 R /Border [0 0 0] /H /I /Rect [ 253.3275 562.9172 262.9648 571.7355 ] >> endobj 276 0 obj << /Type /Action >> endobj 277 0 obj << /Type /Annot /Subtype /Link /A 278 0 R /Border [0 0 0] /H /I /Rect [ 437.5537 539.1077 447.1911 547.9260 ] >> endobj 278 0 obj << /Type /Action >> endobj 279 0 obj << /Type /Annot /Subtype /Link /A 280 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 165.1522 485.2500 471.1522 ] >> endobj 280 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2014/02/figure-4.1.jpg) >> endobj 281 0 obj << /Type /XObject /Subtype /Image /Width 600 /Height 408 /ColorSpace /DeviceRGB /Filter /DCTDecode /BitsPerComponent 8 /Length 31623>> stream JFIF;CREATOR: gd-jpeg v1.0 (using IJG JPEG v62), quality = 90 C     C   X" }!1AQa"q2#BR$3br %&'()*456789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz w!1AQaq"2B #3Rbr $4%&'()*56789:CDEFGHIJSTUVWXYZcdefghijstuvwxyz ?RJ8PъLQ^h&(.(PޓE&(4\4Phǽ&(. 4Q@ 4Q@ .=8N(( Q0}iqI@4"io>O;ۭ'Ri$UfY V$Oo!5Ld1ʗ߹!ms`shp}iq^[oF:WӵxjfKeunm6W#׏Q@= \{`ъ8֗\{`ъ8ǽ&=PKIG`Pޓb( (ǽ>b \{яz@h4qF.(ǽ `PF=0(cގ)0(h֊L (qE'PP~bhPE~4sG4Q@?9(h?*^hhh(4@4GG@ G4Ⓘ'))|7O 7ֵZus{u 2f5P(76?z;~{ /};~{ /_4E_4ýg'4s_;ýg'K?@Ds@+wD/_?ؿi}g7haceci74gWup J?߅74Oi_T|3K#X\7 $?u00=?=~wD/_?d_ $]Ng:h<i)5J%fa@ג@W_4ýg'ϵ|K?G;~{ /}'#Ҿw{Ob?=~>旚?=~wD/_#9ؿi}{Ob;v x?zxod^XO{#:B̌f @_Y|:i_vvv̄-.s9/G@ R4~4sI/G@ 4'4sK—j??Nh9~4QsG4~4PF=hh(ǽ~~RQQ(җ.()? \RPR'KRb/~4/IF /IF((?J1A'F(4bŽ(RIs@G\_Ï <m;?ْnpydkp~mmѶ](((~~bҏŒ0h((ŒQ@KRh? ? 1F 4`QQF QQF 4`Œ0h((~4`QQF 4bŒ1/IRPQQF(((~~bPQQ1@E~s@9QFMQE&(E/4@ KގhwC@~&w:m:rywVrRǙkd8j0w$Pq_;|0iE ?ƟZj@DQ_;|0iE ?ƟZj@DQ_;|0iE ?ƟZj@DQ| ~|i|0iE}+`O-5_H; MW(<ro/|cޕnd 8ჾύ?"`O-5_H(ჾύ?"`O-5_H(ჾύ?"`O-5_H(wa?>4U?ჾύ?">=hჾύ?"`O-5_H8; MW+%|/O~(sG>QQPQQϥQG>~QG>s@qGns@Qϥ/>Qϥ{PG~s@QR@ QϥqGG>qG~>RqKE@qG4s@QG4qE@sE'GA=h?)h=h(iZJ?(hh@ EexZW|=}kwiUofF{r@u>L-<\^|‡W뿍vߴÛ{Ś:/|,eyQ`I8>Ii*:>PJ˜؂;PG4QG*,Uq Efz@GF(|zoï^ /5׼`&exN]BrEsZoC5חULD nsyyG€#gw{?G\G/~۽_Ò^(Mԭ$ix&@SFvPE~4bj'Q]zzfmGKq p3uyğG_ >;o#{T ǀ*o {ZS[էnب`~9R;Ƙ)BPR;OhǵƏƏh1~4~5C^t hzuhYfl$h:޼_ ^T*G0BR?bQ 2=Isf r:}w3ث|\0=%WuPGF(ǵ~~~4cڌ{P~@GF=?Z??? 1@GG~~4gޖJ?Z(3GKj?ZOƌzPgފ\{Q@b_Ɗ1I@ I1@ ъLP4_״-?-ZEiеͬ9aVIsA VLF'-̍c^Q YG0_OgEz|$coNGk}b_$e=X|s߇|5xGIKl-6'㴵$h]j,p8'/G"`/U#kOTOٷ>2=xSFtk9Si1?֌}hրkڃÓ|A>*>"4ZWx{WNyl"vlgֶᇼC.u9@m4^~>A5ψ-xm'Pcls)H u A85^ '- 5K@Yzc XQB*nj>:~:7-^(ƒgWcoq XiDL%+ƾv:Oƾm-{[u-n.K}qٲŰ=X_/GI}h?LQY>(m!L4֒9Z!$LЕ<2ϥ|Y! 9x/^+n^ _q \JIŽt? =<_)h#R Lh$GEH,8sV+ "R@l)LiCq!grŤs?Jo`/V?zx =[(Əƾv Q,_H9o |׾$V[{;Ȧ]9C2Sӵ}4яZ1i?~img([F|[Y݄i;ݣH%UG>Ҁ>4=I57R؂Er|uiL[Kn*vfy#^ŝF1W(z$Q Y@gzSmC;1\jʅbRHqm9k`/V?zx =W(Ɠzx =W*_Q<-ޣppwVH+@ª} ۭ&>c@Aя4я/H>cF>~4&>c@4я/GI4~4Z_ƏƓbhi? 1hi1F()194P^MO>gԔw(ϵ%/z3GzJ^>ԔP%>ih> )ouUi-R>Yi?vNW<4?ٓ~.KV:M-#iM~c|W< Qҥ+i-[H."K"܆G`rt &ߠݧ"(fRiKvL)SI;io>n mr-*W-14JOtWR <1k_ToA <]kڄ:evVڛN[q_y_G(Z1jWSzKay֌x#W j3IE/F}(ϵ%sF}9>ԔPڌRQ@ j? 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" oh UH#I.?_]?Y imޛ!mpo3. I#88ȠԁI_#.jUx {5@xj]_oª |y,o"@Ob<)BW O3@_WO מYWտ+z?0ԿhO7_ÿ%vFyx |eu#KFsvJJ2ƞ.GݨV k5G+_Ky?z?T'G?(k~7~_4o]LX<#+%V,y?m Е?=S9Zu)IJ.ڭ~iTluIwNگD{KBCso w) Ȯ r ^񂒗Q%-@Q@PPh@ EQK@ E-@-%-UMgMื,lYOH >x/BM+K7\I&%vm*v@t (SW>&th<=g_Yt묨#o p=(Gzc//o?Ꮄ*ߨ:|_K .=?Z_O^tiy .=c+/o?ᎴT@F+Ꮄ*:ҿ|_~׀iU>/{ uT@Ex1֗ES]ZWO^tv:|_J .=Wi_U>/{ uT@(x1֗ES]^e7杪'įes_{y0`͇C2$ (bׇ>-k eX?^鰕 Gs3+(7 _{1|CU?< C?._?@@(7 _"b WQb W Qo!uS^ Qo!uEZ~(7 _"b W(ߍ?Z1|CUﴽ< C?._H1|CUW/]j~b W(߻Q^ Qo!u?< C?._H} ?g? |*:FۧkskF T"VP(c#S^ڀ QEQE((P( ((( ( )i(((iCƊ()JZ?J? (w@F}(){) J _MVxZx[zZ]^ LrW j+yI4W:?MoKpۦ {r‘T?/xG8uau[";<^UEmL8N*ğWӿi_|1X|#kw5֥zm]<[S̲̐q}K֭?x嶾񟈼]6eݥL]]s`19ȴۯZ\Y-|s.H襍5K䡉Zeā6A'*Dx^⏈}g^ŦˈSH32ڀ>h߲[o ƚ^>,/Ϗ91\Pxoޟx\I&{/]Ilk7t;S#0|g;ωږcmkkWY2# F/^2goUmýÚz^Kt,k Ĩ,KIcWw彟⳽,~Pњ? (~4QPQPFh((€ƏƏ€ƌG@(h3E'K@GIRR4Q@E'K@EPQ@Q@ KER (Q@-PRJ;EQ@ AZJ(Q@(QEQE(EQEPh ( ( ( ( ( ( ( ( PEP((EQE(((Q@Q@ endstream endobj 282 0 obj << /Type /Annot /Subtype /Link /A 283 0 R /Border [0 0 0] /H /I /Rect [ 455.8372 574.8219 465.4745 583.6402 ] >> endobj 283 0 obj << /Type /Action >> endobj 284 0 obj << /Type /Annot /Subtype /Link /A 285 0 R /Border [0 0 0] /H /I /Rect [ 253.3275 562.9172 262.9648 571.7355 ] >> endobj 285 0 obj << /Type /Action >> endobj 286 0 obj << /Type /Annot /Subtype /Link /A 287 0 R /Border [0 0 0] /H /I /Rect [ 437.5537 539.1077 447.1911 547.9260 ] >> endobj 287 0 obj << /Type /Action >> endobj 288 0 obj << /Type /Annot /Subtype /Link /A 289 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 165.1522 485.2500 471.1522 ] >> endobj 289 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2014/02/figure-4.1.jpg) >> endobj 290 0 obj << /Type /Annot /Subtype /Link /A 291 0 R /Border [0 0 0] /H /I /Rect [ 455.8372 574.8219 465.4745 583.6402 ] >> endobj 291 0 obj << /Type /Action >> endobj 292 0 obj << /Type /Annot /Subtype /Link /A 293 0 R /Border [0 0 0] /H /I /Rect [ 253.3275 562.9172 262.9648 571.7355 ] >> endobj 293 0 obj << /Type /Action >> endobj 294 0 obj << /Type /Annot /Subtype /Link /A 295 0 R /Border [0 0 0] /H /I /Rect [ 437.5537 539.1077 447.1911 547.9260 ] >> endobj 295 0 obj << /Type /Action >> endobj 296 0 obj << /Type /Annot /Subtype /Link /A 297 0 R /Border [0 0 0] /H /I /Rect [ 35.2500 165.1522 485.2500 471.1522 ] >> endobj 297 0 obj << /Type /Action /S /URI /URI (https://currents.plos.org/hd/files/2014/02/figure-4.1.jpg) >> endobj 298 0 obj << /Type /Page /Parent 3 0 R /Annots [ 300 0 R 302 0 R 304 0 R 306 0 R 308 0 R 310 0 R 312 0 R 314 0 R 316 0 R 318 0 R 320 0 R 322 0 R 324 0 R 326 0 R 328 0 R 330 0 R 332 0 R 334 0 R 336 0 R 338 0 R 340 0 R 342 0 R 344 0 R 346 0 R ] /Contents 299 0 R >> endobj 299 0 obj << /Length 27193 >> stream 0.271 0.267 0.267 rg q 15.000 35.425 577.500 741.575 re W n 0.271 0.267 0.267 rg BT 26.250 750.278 Td /F4 12.0 Tf [(Discussion)] TJ ET BT 26.250 730.324 Td /F1 9.8 Tf [(Our studies indicate that overexpression of Rnq1p, Sup35p and Ybr016wp can modulate the formation of mutant HTT )] TJ ET BT 26.250 718.419 Td /F1 9.8 Tf [(aggregates in yeast. Furthermore, the induction of mutant HTT inclusion body formation by both Sup35p and Ybr016wp is in )] TJ ET BT 26.250 706.515 Td /F1 9.8 Tf [(part dependent upon the presence of Rnq1p in the cell. The mechanism\(s\) by which these two aggregation-prone proteins )] TJ ET BT 26.250 694.610 Td /F1 9.8 Tf [(modulate mutant HTT aggregation is not clear, nor is it known if they are acting in a similar manner. In the case of Sup35p it has )] TJ ET BT 26.250 682.705 Td /F1 9.8 Tf [(been proposed that there are two groups of factors that govern both the formation of mutant HTT aggregates and the Sup35p to )] TJ ET BT 26.250 670.800 Td /F1 9.8 Tf [([)] TJ ET BT 28.960 670.800 Td /F5 9.8 Tf [(PSI)] TJ ET BT 44.678 674.689 Td /F5 8.7 Tf [(+)] TJ ET BT 49.739 670.800 Td /F1 9.8 Tf [(] switch in yeast)] TJ ET 0.267 0.267 0.267 rg BT 118.554 672.308 Td /F4 8.7 Tf [(26)] TJ ET 0.271 0.267 0.267 rg BT 128.192 670.800 Td /F1 9.8 Tf [( . The first group is involved in the formation of large mutant HTT aggregates and the switch between )] TJ ET BT 26.250 658.896 Td /F1 9.8 Tf [(Sup35p and [)] TJ ET BT 84.253 658.896 Td /F5 9.8 Tf [(PSI)] TJ ET BT 99.970 662.784 Td /F5 8.7 Tf [(+)] TJ ET BT 105.031 658.896 Td /F1 9.8 Tf [(], while the factors in the second group are crucial for the formation of soluble oligomeric species of mutant )] TJ ET BT 26.250 646.991 Td /F1 9.8 Tf [(HTT, as well as the formation of diffuse [)] TJ ET BT 200.736 646.991 Td /F5 9.8 Tf [(PSI)] TJ ET BT 216.453 650.879 Td /F5 8.7 Tf [(+)] TJ ET BT 221.514 646.991 Td /F1 9.8 Tf [(] aggregates. These findings further support the hypothesis that yeast prions and )] TJ ET BT 26.250 635.086 Td /F1 9.8 Tf [(glutamine-rich amyloidogenic proteins interact with similar molecular partners upon forming larger amyloid structures in yeast)] TJ ET 0.267 0.267 0.267 rg BT 564.284 636.593 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 26.250 623.181 Td /F1 9.8 Tf [(. Based upon this observation it is possible that mutant HTT interacts directly with Sup35p. Indeed, a recent investigation )] TJ ET BT 26.250 611.277 Td /F1 9.8 Tf [(indicates that mutant HTT can sequester Sup35p in [)] TJ ET BT 253.864 611.277 Td /F5 9.8 Tf [(PSI)] TJ ET BT 269.581 615.165 Td /F5 8.7 Tf [(+)] TJ ET BT 274.642 611.277 Td /F1 9.8 Tf [(] cells)] TJ ET 0.267 0.267 0.267 rg BT 299.563 612.784 Td /F4 8.7 Tf [(27)] TJ ET 0.271 0.267 0.267 rg BT 309.200 611.277 Td /F1 9.8 Tf [( . As most established yeast prions have a Q/N-rich sequence )] TJ ET BT 26.250 599.372 Td /F1 9.8 Tf [(and mutant HTT can sequester Q-rich sequences in yeast)] TJ ET 0.267 0.267 0.267 rg BT 276.065 600.879 Td /F4 8.7 Tf [(28)] TJ ET 0.271 0.267 0.267 rg BT 285.702 599.372 Td /F1 9.8 Tf [( , it is possible that mutant HTT can sequester other yeast prions. )] TJ ET BT 26.250 587.467 Td /F1 9.8 Tf [(Ybr016wp is poorly characterised, and the relationship between this protein and mutant HTT described here is novel. Although )] TJ ET BT 26.250 575.562 Td /F1 9.8 Tf [(Ybr016wp has an unknown cellular function it contains a CYSTM domain that is characteristic of eukaryotic proteins involved in )] TJ ET BT 26.250 563.658 Td /F1 9.8 Tf [(stress tolerance)] TJ ET 0.267 0.267 0.267 rg BT 95.066 565.165 Td /F4 8.7 Tf [(30)] TJ ET 0.271 0.267 0.267 rg BT 104.703 563.658 Td /F1 9.8 Tf [( and is anchored to the plasma membrane and predicted to be palmitoylated, similar to the human prion )] TJ ET BT 26.250 551.753 Td /F1 9.8 Tf [(protein PrP)] TJ ET BT 75.019 555.641 Td /F1 8.7 Tf [(C )] TJ ET 0.267 0.267 0.267 rg BT 83.686 553.260 Td /F4 8.7 Tf [(31)] TJ ET 0.271 0.267 0.267 rg BT 93.324 551.753 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 532.348 Td /F1 9.8 Tf [(Many highly conserved yeast proteins have prion-like features)] TJ ET 0.267 0.267 0.267 rg BT 293.400 533.855 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 303.037 532.348 Td /F1 9.8 Tf [( , suggesting that human proteins with prion-like domains may )] TJ ET BT 26.250 520.443 Td /F1 9.8 Tf [(behave in a similar manner. Indeed, a recent study has implicated a large number of glutamine and asparagine rich human )] TJ ET BT 26.250 508.539 Td /F1 9.8 Tf [(RNA-binding proteins in the pathology of a range of neurodegenerative diseases)] TJ ET 0.267 0.267 0.267 rg BT 373.096 510.046 Td /F4 8.7 Tf [(32)] TJ ET 0.271 0.267 0.267 rg BT 382.734 508.539 Td /F1 9.8 Tf [(. Thus, it is possible that a suite of )] TJ ET BT 26.250 496.634 Td /F1 9.8 Tf [(aggregation prone proteins that modify the aggregation of mutant HTT exists not only in yeast but in humans as well, which may )] TJ ET BT 26.250 484.729 Td /F1 9.8 Tf [(have implications for HD pathogenesis. Indeed, such proteins could modify the aggregation and misfolding of not only mutant )] TJ ET BT 26.250 472.824 Td /F1 9.8 Tf [(HTT, but other amyloidogenic proteins as well, thus informing the development of therapies for HD as well as for other )] TJ ET BT 26.250 460.920 Td /F1 9.8 Tf [(neurodegenerative diseases caused by protein misfolding.)] TJ ET BT 26.250 424.317 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 404.363 Td /F1 9.8 Tf [(We thank Susan Lindquist \(Whitehead Institute, USA\) for providing the pRS303Htt integrative plasmids. We are grateful to Mick )] TJ ET BT 26.250 392.458 Td /F1 9.8 Tf [(Tuite \(University of Kent, UK\) for the Sup35 antibody. We acknowledge Gary Jones \(NUI Maynooth, Ireland\) for his helpful )] TJ ET BT 26.250 380.553 Td /F1 9.8 Tf [(comments and advice.)] TJ ET BT 26.250 351.451 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 323.997 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 323.997 Td /F1 9.8 Tf [(Bates G. Huntingtin aggregation and toxicity in Huntington's disease. Lancet. 2003 May 10;361\(9369\):1642-4. PubMed )] TJ ET BT 26.250 312.092 Td /F1 9.8 Tf [(PMID:12747895.)] TJ ET BT 26.250 292.687 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 292.687 Td /F1 9.8 Tf [(Ross CA, Tabrizi SJ. Huntington's disease: from molecular pathogenesis to clinical treatment. Lancet Neurol. 2011 )] TJ ET BT 26.250 280.782 Td /F1 9.8 Tf [(Jan;10\(1\):83-98. PubMed PMID:21163446.)] TJ ET BT 26.250 261.378 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 261.378 Td /F1 9.8 Tf [(A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. The )] TJ ET BT 26.250 249.473 Td /F1 9.8 Tf [(Huntington's Disease Collaborative Research Group. Cell. 1993 Mar 26;72\(6\):971-83. PubMed PMID:8458085.)] TJ ET BT 26.250 230.068 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 230.068 Td /F1 9.8 Tf [(DiFiglia M, Sapp E, Chase KO, Davies SW, Bates GP, Vonsattel JP, Aronin N. Aggregation of huntingtin in neuronal )] TJ ET BT 26.250 218.163 Td /F1 9.8 Tf [(intranuclear inclusions and dystrophic neurites in brain. Science. 1997 Sep 26;277\(5334\):1990-3. PubMed PMID:9302293.)] TJ ET BT 26.250 198.759 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 198.759 Td /F1 9.8 Tf [(Hackam AS, Singaraja R, Wellington CL, Metzler M, McCutcheon K, Zhang T, Kalchman M, Hayden MR. The influence of )] TJ ET BT 26.250 186.854 Td /F1 9.8 Tf [(huntingtin protein size on nuclear localization and cellular toxicity. J Cell Biol. 1998 Jun 1;141\(5\):1097-105. PubMed )] TJ ET BT 26.250 174.949 Td /F1 9.8 Tf [(PMID:9606203.)] TJ ET BT 26.250 155.544 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 155.544 Td /F1 9.8 Tf [(Arrasate M, Mitra S, Schweitzer ES, Segal MR, Finkbeiner S. Inclusion body formation reduces levels of mutant huntingtin )] TJ ET BT 26.250 143.640 Td /F1 9.8 Tf [(and the risk of neuronal death. Nature. 2004 Oct 14;431\(7010\):805-10. PubMed PMID:15483602.)] TJ ET BT 26.250 124.235 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 124.235 Td /F1 9.8 Tf [(Duennwald ML, Jagadish S, Muchowski PJ, Lindquist S. Flanking sequences profoundly alter polyglutamine toxicity in yeast. )] TJ ET BT 26.250 112.330 Td /F1 9.8 Tf [(Proc Natl Acad Sci U S A. 2006 Jul 18;103\(29\):11045-50. PubMed PMID:16832050.)] TJ ET BT 26.250 92.925 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 92.925 Td /F1 9.8 Tf [(Bennett EJ, Shaler TA, Woodman B, Ryu KY, Zaitseva TS, Becker CH, Bates GP, Schulman H, Kopito RR. Global changes )] TJ ET BT 26.250 81.021 Td /F1 9.8 Tf [(to the ubiquitin system in Huntington's disease. Nature. 2007 Aug 9;448\(7154\):704-8. PubMed PMID:17687326.)] TJ ET BT 26.250 61.616 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 61.616 Td /F1 9.8 Tf [(Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S, Oroz LG, Scaravilli F, Easton DF, Duden R, O'Kane CJ, Rubinsztein )] TJ ET BT 26.250 49.711 Td /F1 9.8 Tf [(DC. Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of )] TJ ET Q q 15.000 35.425 577.500 741.575 re W n 0.271 0.267 0.267 rg BT 26.250 750.278 Td /F4 12.0 Tf [(Discussion)] TJ ET BT 26.250 730.324 Td /F1 9.8 Tf [(Our studies indicate that overexpression of Rnq1p, Sup35p and Ybr016wp can modulate the formation of mutant HTT )] TJ ET BT 26.250 718.419 Td /F1 9.8 Tf [(aggregates in yeast. Furthermore, the induction of mutant HTT inclusion body formation by both Sup35p and Ybr016wp is in )] TJ ET BT 26.250 706.515 Td /F1 9.8 Tf [(part dependent upon the presence of Rnq1p in the cell. The mechanism\(s\) by which these two aggregation-prone proteins )] TJ ET BT 26.250 694.610 Td /F1 9.8 Tf [(modulate mutant HTT aggregation is not clear, nor is it known if they are acting in a similar manner. In the case of Sup35p it has )] TJ ET BT 26.250 682.705 Td /F1 9.8 Tf [(been proposed that there are two groups of factors that govern both the formation of mutant HTT aggregates and the Sup35p to )] TJ ET BT 26.250 670.800 Td /F1 9.8 Tf [([)] TJ ET BT 28.960 670.800 Td /F5 9.8 Tf [(PSI)] TJ ET BT 44.678 674.689 Td /F5 8.7 Tf [(+)] TJ ET BT 49.739 670.800 Td /F1 9.8 Tf [(] switch in yeast)] TJ ET 0.267 0.267 0.267 rg BT 118.554 672.308 Td /F4 8.7 Tf [(26)] TJ ET 0.271 0.267 0.267 rg BT 128.192 670.800 Td /F1 9.8 Tf [( . The first group is involved in the formation of large mutant HTT aggregates and the switch between )] TJ ET BT 26.250 658.896 Td /F1 9.8 Tf [(Sup35p and [)] TJ ET BT 84.253 658.896 Td /F5 9.8 Tf [(PSI)] TJ ET BT 99.970 662.784 Td /F5 8.7 Tf [(+)] TJ ET BT 105.031 658.896 Td /F1 9.8 Tf [(], while the factors in the second group are crucial for the formation of soluble oligomeric species of mutant )] TJ ET BT 26.250 646.991 Td /F1 9.8 Tf [(HTT, as well as the formation of diffuse [)] TJ ET BT 200.736 646.991 Td /F5 9.8 Tf [(PSI)] TJ ET BT 216.453 650.879 Td /F5 8.7 Tf [(+)] TJ ET BT 221.514 646.991 Td /F1 9.8 Tf [(] aggregates. These findings further support the hypothesis that yeast prions and )] TJ ET BT 26.250 635.086 Td /F1 9.8 Tf [(glutamine-rich amyloidogenic proteins interact with similar molecular partners upon forming larger amyloid structures in yeast)] TJ ET 0.267 0.267 0.267 rg BT 564.284 636.593 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 26.250 623.181 Td /F1 9.8 Tf [(. Based upon this observation it is possible that mutant HTT interacts directly with Sup35p. Indeed, a recent investigation )] TJ ET BT 26.250 611.277 Td /F1 9.8 Tf [(indicates that mutant HTT can sequester Sup35p in [)] TJ ET BT 253.864 611.277 Td /F5 9.8 Tf [(PSI)] TJ ET BT 269.581 615.165 Td /F5 8.7 Tf [(+)] TJ ET BT 274.642 611.277 Td /F1 9.8 Tf [(] cells)] TJ ET 0.267 0.267 0.267 rg BT 299.563 612.784 Td /F4 8.7 Tf [(27)] TJ ET 0.271 0.267 0.267 rg BT 309.200 611.277 Td /F1 9.8 Tf [( . As most established yeast prions have a Q/N-rich sequence )] TJ ET BT 26.250 599.372 Td /F1 9.8 Tf [(and mutant HTT can sequester Q-rich sequences in yeast)] TJ ET 0.267 0.267 0.267 rg BT 276.065 600.879 Td /F4 8.7 Tf [(28)] TJ ET 0.271 0.267 0.267 rg BT 285.702 599.372 Td /F1 9.8 Tf [( , it is possible that mutant HTT can sequester other yeast prions. )] TJ ET BT 26.250 587.467 Td /F1 9.8 Tf [(Ybr016wp is poorly characterised, and the relationship between this protein and mutant HTT described here is novel. Although )] TJ ET BT 26.250 575.562 Td /F1 9.8 Tf [(Ybr016wp has an unknown cellular function it contains a CYSTM domain that is characteristic of eukaryotic proteins involved in )] TJ ET BT 26.250 563.658 Td /F1 9.8 Tf [(stress tolerance)] TJ ET 0.267 0.267 0.267 rg BT 95.066 565.165 Td /F4 8.7 Tf [(30)] TJ ET 0.271 0.267 0.267 rg BT 104.703 563.658 Td /F1 9.8 Tf [( and is anchored to the plasma membrane and predicted to be palmitoylated, similar to the human prion )] TJ ET BT 26.250 551.753 Td /F1 9.8 Tf [(protein PrP)] TJ ET BT 75.019 555.641 Td /F1 8.7 Tf [(C )] TJ ET 0.267 0.267 0.267 rg BT 83.686 553.260 Td /F4 8.7 Tf [(31)] TJ ET 0.271 0.267 0.267 rg BT 93.324 551.753 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 532.348 Td /F1 9.8 Tf [(Many highly conserved yeast proteins have prion-like features)] TJ ET 0.267 0.267 0.267 rg BT 293.400 533.855 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 303.037 532.348 Td /F1 9.8 Tf [( , suggesting that human proteins with prion-like domains may )] TJ ET BT 26.250 520.443 Td /F1 9.8 Tf [(behave in a similar manner. Indeed, a recent study has implicated a large number of glutamine and asparagine rich human )] TJ ET BT 26.250 508.539 Td /F1 9.8 Tf [(RNA-binding proteins in the pathology of a range of neurodegenerative diseases)] TJ ET 0.267 0.267 0.267 rg BT 373.096 510.046 Td /F4 8.7 Tf [(32)] TJ ET 0.271 0.267 0.267 rg BT 382.734 508.539 Td /F1 9.8 Tf [(. Thus, it is possible that a suite of )] TJ ET BT 26.250 496.634 Td /F1 9.8 Tf [(aggregation prone proteins that modify the aggregation of mutant HTT exists not only in yeast but in humans as well, which may )] TJ ET BT 26.250 484.729 Td /F1 9.8 Tf [(have implications for HD pathogenesis. Indeed, such proteins could modify the aggregation and misfolding of not only mutant )] TJ ET BT 26.250 472.824 Td /F1 9.8 Tf [(HTT, but other amyloidogenic proteins as well, thus informing the development of therapies for HD as well as for other )] TJ ET BT 26.250 460.920 Td /F1 9.8 Tf [(neurodegenerative diseases caused by protein misfolding.)] TJ ET BT 26.250 424.317 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 404.363 Td /F1 9.8 Tf [(We thank Susan Lindquist \(Whitehead Institute, USA\) for providing the pRS303Htt integrative plasmids. We are grateful to Mick )] TJ ET BT 26.250 392.458 Td /F1 9.8 Tf [(Tuite \(University of Kent, UK\) for the Sup35 antibody. We acknowledge Gary Jones \(NUI Maynooth, Ireland\) for his helpful )] TJ ET BT 26.250 380.553 Td /F1 9.8 Tf [(comments and advice.)] TJ ET BT 26.250 351.451 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 323.997 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 323.997 Td /F1 9.8 Tf [(Bates G. Huntingtin aggregation and toxicity in Huntington's disease. Lancet. 2003 May 10;361\(9369\):1642-4. PubMed )] TJ ET BT 26.250 312.092 Td /F1 9.8 Tf [(PMID:12747895.)] TJ ET BT 26.250 292.687 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 292.687 Td /F1 9.8 Tf [(Ross CA, Tabrizi SJ. Huntington's disease: from molecular pathogenesis to clinical treatment. Lancet Neurol. 2011 )] TJ ET BT 26.250 280.782 Td /F1 9.8 Tf [(Jan;10\(1\):83-98. PubMed PMID:21163446.)] TJ ET BT 26.250 261.378 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 261.378 Td /F1 9.8 Tf [(A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. The )] TJ ET BT 26.250 249.473 Td /F1 9.8 Tf [(Huntington's Disease Collaborative Research Group. Cell. 1993 Mar 26;72\(6\):971-83. PubMed PMID:8458085.)] TJ ET BT 26.250 230.068 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 230.068 Td /F1 9.8 Tf [(DiFiglia M, Sapp E, Chase KO, Davies SW, Bates GP, Vonsattel JP, Aronin N. Aggregation of huntingtin in neuronal )] TJ ET BT 26.250 218.163 Td /F1 9.8 Tf [(intranuclear inclusions and dystrophic neurites in brain. Science. 1997 Sep 26;277\(5334\):1990-3. PubMed PMID:9302293.)] TJ ET BT 26.250 198.759 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 198.759 Td /F1 9.8 Tf [(Hackam AS, Singaraja R, Wellington CL, Metzler M, McCutcheon K, Zhang T, Kalchman M, Hayden MR. The influence of )] TJ ET BT 26.250 186.854 Td /F1 9.8 Tf [(huntingtin protein size on nuclear localization and cellular toxicity. J Cell Biol. 1998 Jun 1;141\(5\):1097-105. PubMed )] TJ ET BT 26.250 174.949 Td /F1 9.8 Tf [(PMID:9606203.)] TJ ET BT 26.250 155.544 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 155.544 Td /F1 9.8 Tf [(Arrasate M, Mitra S, Schweitzer ES, Segal MR, Finkbeiner S. Inclusion body formation reduces levels of mutant huntingtin )] TJ ET BT 26.250 143.640 Td /F1 9.8 Tf [(and the risk of neuronal death. Nature. 2004 Oct 14;431\(7010\):805-10. PubMed PMID:15483602.)] TJ ET BT 26.250 124.235 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 124.235 Td /F1 9.8 Tf [(Duennwald ML, Jagadish S, Muchowski PJ, Lindquist S. Flanking sequences profoundly alter polyglutamine toxicity in yeast. )] TJ ET BT 26.250 112.330 Td /F1 9.8 Tf [(Proc Natl Acad Sci U S A. 2006 Jul 18;103\(29\):11045-50. PubMed PMID:16832050.)] TJ ET BT 26.250 92.925 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 92.925 Td /F1 9.8 Tf [(Bennett EJ, Shaler TA, Woodman B, Ryu KY, Zaitseva TS, Becker CH, Bates GP, Schulman H, Kopito RR. Global changes )] TJ ET BT 26.250 81.021 Td /F1 9.8 Tf [(to the ubiquitin system in Huntington's disease. Nature. 2007 Aug 9;448\(7154\):704-8. PubMed PMID:17687326.)] TJ ET BT 26.250 61.616 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 61.616 Td /F1 9.8 Tf [(Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S, Oroz LG, Scaravilli F, Easton DF, Duden R, O'Kane CJ, Rubinsztein )] TJ ET BT 26.250 49.711 Td /F1 9.8 Tf [(DC. Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of )] TJ ET Q q 15.000 35.425 577.500 741.575 re W n 0.271 0.267 0.267 rg BT 26.250 750.278 Td /F4 12.0 Tf [(Discussion)] TJ ET BT 26.250 730.324 Td /F1 9.8 Tf [(Our studies indicate that overexpression of Rnq1p, Sup35p and Ybr016wp can modulate the formation of mutant HTT )] TJ ET BT 26.250 718.419 Td /F1 9.8 Tf [(aggregates in yeast. Furthermore, the induction of mutant HTT inclusion body formation by both Sup35p and Ybr016wp is in )] TJ ET BT 26.250 706.515 Td /F1 9.8 Tf [(part dependent upon the presence of Rnq1p in the cell. The mechanism\(s\) by which these two aggregation-prone proteins )] TJ ET BT 26.250 694.610 Td /F1 9.8 Tf [(modulate mutant HTT aggregation is not clear, nor is it known if they are acting in a similar manner. In the case of Sup35p it has )] TJ ET BT 26.250 682.705 Td /F1 9.8 Tf [(been proposed that there are two groups of factors that govern both the formation of mutant HTT aggregates and the Sup35p to )] TJ ET BT 26.250 670.800 Td /F1 9.8 Tf [([)] TJ ET BT 28.960 670.800 Td /F5 9.8 Tf [(PSI)] TJ ET BT 44.678 674.689 Td /F5 8.7 Tf [(+)] TJ ET BT 49.739 670.800 Td /F1 9.8 Tf [(] switch in yeast)] TJ ET 0.267 0.267 0.267 rg BT 118.554 672.308 Td /F4 8.7 Tf [(26)] TJ ET 0.271 0.267 0.267 rg BT 128.192 670.800 Td /F1 9.8 Tf [( . The first group is involved in the formation of large mutant HTT aggregates and the switch between )] TJ ET BT 26.250 658.896 Td /F1 9.8 Tf [(Sup35p and [)] TJ ET BT 84.253 658.896 Td /F5 9.8 Tf [(PSI)] TJ ET BT 99.970 662.784 Td /F5 8.7 Tf [(+)] TJ ET BT 105.031 658.896 Td /F1 9.8 Tf [(], while the factors in the second group are crucial for the formation of soluble oligomeric species of mutant )] TJ ET BT 26.250 646.991 Td /F1 9.8 Tf [(HTT, as well as the formation of diffuse [)] TJ ET BT 200.736 646.991 Td /F5 9.8 Tf [(PSI)] TJ ET BT 216.453 650.879 Td /F5 8.7 Tf [(+)] TJ ET BT 221.514 646.991 Td /F1 9.8 Tf [(] aggregates. These findings further support the hypothesis that yeast prions and )] TJ ET BT 26.250 635.086 Td /F1 9.8 Tf [(glutamine-rich amyloidogenic proteins interact with similar molecular partners upon forming larger amyloid structures in yeast)] TJ ET 0.267 0.267 0.267 rg BT 564.284 636.593 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 26.250 623.181 Td /F1 9.8 Tf [(. Based upon this observation it is possible that mutant HTT interacts directly with Sup35p. Indeed, a recent investigation )] TJ ET BT 26.250 611.277 Td /F1 9.8 Tf [(indicates that mutant HTT can sequester Sup35p in [)] TJ ET BT 253.864 611.277 Td /F5 9.8 Tf [(PSI)] TJ ET BT 269.581 615.165 Td /F5 8.7 Tf [(+)] TJ ET BT 274.642 611.277 Td /F1 9.8 Tf [(] cells)] TJ ET 0.267 0.267 0.267 rg BT 299.563 612.784 Td /F4 8.7 Tf [(27)] TJ ET 0.271 0.267 0.267 rg BT 309.200 611.277 Td /F1 9.8 Tf [( . As most established yeast prions have a Q/N-rich sequence )] TJ ET BT 26.250 599.372 Td /F1 9.8 Tf [(and mutant HTT can sequester Q-rich sequences in yeast)] TJ ET 0.267 0.267 0.267 rg BT 276.065 600.879 Td /F4 8.7 Tf [(28)] TJ ET 0.271 0.267 0.267 rg BT 285.702 599.372 Td /F1 9.8 Tf [( , it is possible that mutant HTT can sequester other yeast prions. )] TJ ET BT 26.250 587.467 Td /F1 9.8 Tf [(Ybr016wp is poorly characterised, and the relationship between this protein and mutant HTT described here is novel. Although )] TJ ET BT 26.250 575.562 Td /F1 9.8 Tf [(Ybr016wp has an unknown cellular function it contains a CYSTM domain that is characteristic of eukaryotic proteins involved in )] TJ ET BT 26.250 563.658 Td /F1 9.8 Tf [(stress tolerance)] TJ ET 0.267 0.267 0.267 rg BT 95.066 565.165 Td /F4 8.7 Tf [(30)] TJ ET 0.271 0.267 0.267 rg BT 104.703 563.658 Td /F1 9.8 Tf [( and is anchored to the plasma membrane and predicted to be palmitoylated, similar to the human prion )] TJ ET BT 26.250 551.753 Td /F1 9.8 Tf [(protein PrP)] TJ ET BT 75.019 555.641 Td /F1 8.7 Tf [(C )] TJ ET 0.267 0.267 0.267 rg BT 83.686 553.260 Td /F4 8.7 Tf [(31)] TJ ET 0.271 0.267 0.267 rg BT 93.324 551.753 Td /F1 9.8 Tf [(.)] TJ ET BT 26.250 532.348 Td /F1 9.8 Tf [(Many highly conserved yeast proteins have prion-like features)] TJ ET 0.267 0.267 0.267 rg BT 293.400 533.855 Td /F4 8.7 Tf [(22)] TJ ET 0.271 0.267 0.267 rg BT 303.037 532.348 Td /F1 9.8 Tf [( , suggesting that human proteins with prion-like domains may )] TJ ET BT 26.250 520.443 Td /F1 9.8 Tf [(behave in a similar manner. Indeed, a recent study has implicated a large number of glutamine and asparagine rich human )] TJ ET BT 26.250 508.539 Td /F1 9.8 Tf [(RNA-binding proteins in the pathology of a range of neurodegenerative diseases)] TJ ET 0.267 0.267 0.267 rg BT 373.096 510.046 Td /F4 8.7 Tf [(32)] TJ ET 0.271 0.267 0.267 rg BT 382.734 508.539 Td /F1 9.8 Tf [(. Thus, it is possible that a suite of )] TJ ET BT 26.250 496.634 Td /F1 9.8 Tf [(aggregation prone proteins that modify the aggregation of mutant HTT exists not only in yeast but in humans as well, which may )] TJ ET BT 26.250 484.729 Td /F1 9.8 Tf [(have implications for HD pathogenesis. Indeed, such proteins could modify the aggregation and misfolding of not only mutant )] TJ ET BT 26.250 472.824 Td /F1 9.8 Tf [(HTT, but other amyloidogenic proteins as well, thus informing the development of therapies for HD as well as for other )] TJ ET BT 26.250 460.920 Td /F1 9.8 Tf [(neurodegenerative diseases caused by protein misfolding.)] TJ ET BT 26.250 424.317 Td /F4 12.0 Tf [(Acknowledgements)] TJ ET BT 26.250 404.363 Td /F1 9.8 Tf [(We thank Susan Lindquist \(Whitehead Institute, USA\) for providing the pRS303Htt integrative plasmids. We are grateful to Mick )] TJ ET BT 26.250 392.458 Td /F1 9.8 Tf [(Tuite \(University of Kent, UK\) for the Sup35 antibody. We acknowledge Gary Jones \(NUI Maynooth, Ireland\) for his helpful )] TJ ET BT 26.250 380.553 Td /F1 9.8 Tf [(comments and advice.)] TJ ET BT 26.250 351.451 Td /F4 12.0 Tf [(References)] TJ ET BT 26.250 323.997 Td /F1 9.8 Tf [(1.)] TJ ET BT 38.132 323.997 Td /F1 9.8 Tf [(Bates G. Huntingtin aggregation and toxicity in Huntington's disease. Lancet. 2003 May 10;361\(9369\):1642-4. PubMed )] TJ ET BT 26.250 312.092 Td /F1 9.8 Tf [(PMID:12747895.)] TJ ET BT 26.250 292.687 Td /F1 9.8 Tf [(2.)] TJ ET BT 38.132 292.687 Td /F1 9.8 Tf [(Ross CA, Tabrizi SJ. Huntington's disease: from molecular pathogenesis to clinical treatment. Lancet Neurol. 2011 )] TJ ET BT 26.250 280.782 Td /F1 9.8 Tf [(Jan;10\(1\):83-98. PubMed PMID:21163446.)] TJ ET BT 26.250 261.378 Td /F1 9.8 Tf [(3.)] TJ ET BT 38.132 261.378 Td /F1 9.8 Tf [(A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. The )] TJ ET BT 26.250 249.473 Td /F1 9.8 Tf [(Huntington's Disease Collaborative Research Group. Cell. 1993 Mar 26;72\(6\):971-83. PubMed PMID:8458085.)] TJ ET BT 26.250 230.068 Td /F1 9.8 Tf [(4.)] TJ ET BT 38.132 230.068 Td /F1 9.8 Tf [(DiFiglia M, Sapp E, Chase KO, Davies SW, Bates GP, Vonsattel JP, Aronin N. Aggregation of huntingtin in neuronal )] TJ ET BT 26.250 218.163 Td /F1 9.8 Tf [(intranuclear inclusions and dystrophic neurites in brain. Science. 1997 Sep 26;277\(5334\):1990-3. PubMed PMID:9302293.)] TJ ET BT 26.250 198.759 Td /F1 9.8 Tf [(5.)] TJ ET BT 38.132 198.759 Td /F1 9.8 Tf [(Hackam AS, Singaraja R, Wellington CL, Metzler M, McCutcheon K, Zhang T, Kalchman M, Hayden MR. The influence of )] TJ ET BT 26.250 186.854 Td /F1 9.8 Tf [(huntingtin protein size on nuclear localization and cellular toxicity. J Cell Biol. 1998 Jun 1;141\(5\):1097-105. PubMed )] TJ ET BT 26.250 174.949 Td /F1 9.8 Tf [(PMID:9606203.)] TJ ET BT 26.250 155.544 Td /F1 9.8 Tf [(6.)] TJ ET BT 38.132 155.544 Td /F1 9.8 Tf [(Arrasate M, Mitra S, Schweitzer ES, Segal MR, Finkbeiner S. Inclusion body formation reduces levels of mutant huntingtin )] TJ ET BT 26.250 143.640 Td /F1 9.8 Tf [(and the risk of neuronal death. Nature. 2004 Oct 14;431\(7010\):805-10. PubMed PMID:15483602.)] TJ ET BT 26.250 124.235 Td /F1 9.8 Tf [(7.)] TJ ET BT 38.132 124.235 Td /F1 9.8 Tf [(Duennwald ML, Jagadish S, Muchowski PJ, Lindquist S. Flanking sequences profoundly alter polyglutamine toxicity in yeast. )] TJ ET BT 26.250 112.330 Td /F1 9.8 Tf [(Proc Natl Acad Sci U S A. 2006 Jul 18;103\(29\):11045-50. PubMed PMID:16832050.)] TJ ET BT 26.250 92.925 Td /F1 9.8 Tf [(8.)] TJ ET BT 38.132 92.925 Td /F1 9.8 Tf [(Bennett EJ, Shaler TA, Woodman B, Ryu KY, Zaitseva TS, Becker CH, Bates GP, Schulman H, Kopito RR. Global changes )] TJ ET BT 26.250 81.021 Td /F1 9.8 Tf [(to the ubiquitin system in Huntington's disease. Nature. 2007 Aug 9;448\(7154\):704-8. PubMed PMID:17687326.)] TJ ET BT 26.250 61.616 Td /F1 9.8 Tf [(9.)] TJ ET BT 38.132 61.616 Td /F1 9.8 Tf [(Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S, Oroz LG, Scaravilli F, Easton DF, Duden R, O'Kane CJ, Rubinsztein )] TJ ET BT 26.250 49.711 Td /F1 9.8 Tf [(DC. Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of )] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(6)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 300 0 obj << /Type /Annot /Subtype /Link /A 301 0 R /Border [0 0 0] /H /I /Rect [ 118.5543 671.5059 128.1917 680.3243 ] >> endobj 301 0 obj << /Type /Action >> endobj 302 0 obj << /Type /Annot /Subtype /Link /A 303 0 R /Border [0 0 0] /H /I /Rect [ 564.2843 635.7917 573.9216 644.6100 ] >> endobj 303 0 obj << /Type /Action >> endobj 304 0 obj << /Type /Annot /Subtype /Link /A 305 0 R /Border [0 0 0] /H /I /Rect [ 299.5631 611.9822 309.2004 620.8005 ] >> endobj 305 0 obj << /Type /Action >> endobj 306 0 obj << /Type /Annot /Subtype /Link /A 307 0 R /Border [0 0 0] /H /I /Rect [ 276.0645 600.0774 285.7018 608.8957 ] >> endobj 307 0 obj << /Type /Action >> endobj 308 0 obj << /Type /Annot /Subtype /Link /A 309 0 R /Border [0 0 0] /H /I /Rect [ 95.0655 564.3632 104.7028 573.1815 ] >> endobj 309 0 obj << /Type /Action >> endobj 310 0 obj << /Type /Annot /Subtype /Link /A 311 0 R /Border [0 0 0] /H /I /Rect [ 83.6862 552.4584 93.3235 561.2767 ] >> endobj 311 0 obj << /Type /Action >> endobj 312 0 obj << /Type /Annot /Subtype /Link /A 313 0 R /Border [0 0 0] /H /I /Rect [ 293.4000 533.0537 303.0373 541.8720 ] >> endobj 313 0 obj << /Type /Action >> endobj 314 0 obj << /Type /Annot /Subtype /Link /A 315 0 R /Border [0 0 0] /H /I /Rect [ 373.0965 509.2442 382.7338 518.0625 ] >> endobj 315 0 obj << /Type /Action >> endobj 316 0 obj << /Type /Annot /Subtype /Link /A 317 0 R /Border [0 0 0] /H /I /Rect [ 118.5543 671.5059 128.1917 680.3243 ] >> endobj 317 0 obj << /Type /Action >> endobj 318 0 obj << /Type /Annot /Subtype /Link /A 319 0 R /Border [0 0 0] /H /I /Rect [ 564.2843 635.7917 573.9216 644.6100 ] >> endobj 319 0 obj << /Type /Action >> endobj 320 0 obj << /Type /Annot /Subtype /Link /A 321 0 R /Border [0 0 0] /H /I /Rect [ 299.5631 611.9822 309.2004 620.8005 ] >> endobj 321 0 obj << /Type /Action >> endobj 322 0 obj << /Type /Annot /Subtype /Link /A 323 0 R /Border [0 0 0] /H /I /Rect [ 276.0645 600.0774 285.7018 608.8957 ] >> endobj 323 0 obj << /Type /Action >> endobj 324 0 obj << /Type /Annot /Subtype /Link /A 325 0 R /Border [0 0 0] /H /I /Rect [ 95.0655 564.3632 104.7028 573.1815 ] >> endobj 325 0 obj << /Type /Action >> endobj 326 0 obj << /Type /Annot /Subtype /Link /A 327 0 R /Border [0 0 0] /H /I /Rect [ 83.6862 552.4584 93.3235 561.2767 ] >> endobj 327 0 obj << /Type /Action >> endobj 328 0 obj << /Type /Annot /Subtype /Link /A 329 0 R /Border [0 0 0] /H /I /Rect [ 293.4000 533.0537 303.0373 541.8720 ] >> endobj 329 0 obj << /Type /Action >> endobj 330 0 obj << /Type /Annot /Subtype /Link /A 331 0 R /Border [0 0 0] /H /I /Rect [ 373.0965 509.2442 382.7338 518.0625 ] >> endobj 331 0 obj << /Type /Action >> endobj 332 0 obj << /Type /Annot /Subtype /Link /A 333 0 R /Border [0 0 0] /H /I /Rect [ 118.5543 671.5059 128.1917 680.3243 ] >> endobj 333 0 obj << /Type /Action >> endobj 334 0 obj << /Type /Annot /Subtype /Link /A 335 0 R /Border [0 0 0] /H /I /Rect [ 564.2843 635.7917 573.9216 644.6100 ] >> endobj 335 0 obj << /Type /Action >> endobj 336 0 obj << /Type /Annot /Subtype /Link /A 337 0 R /Border [0 0 0] /H /I /Rect [ 299.5631 611.9822 309.2004 620.8005 ] >> endobj 337 0 obj << /Type /Action >> endobj 338 0 obj << /Type /Annot /Subtype /Link /A 339 0 R /Border [0 0 0] /H /I /Rect [ 276.0645 600.0774 285.7018 608.8957 ] >> endobj 339 0 obj << /Type /Action >> endobj 340 0 obj << /Type /Annot /Subtype /Link /A 341 0 R /Border [0 0 0] /H /I /Rect [ 95.0655 564.3632 104.7028 573.1815 ] >> endobj 341 0 obj << /Type /Action >> endobj 342 0 obj << /Type /Annot /Subtype /Link /A 343 0 R /Border [0 0 0] /H /I /Rect [ 83.6862 552.4584 93.3235 561.2767 ] >> endobj 343 0 obj << /Type /Action >> endobj 344 0 obj << /Type /Annot /Subtype /Link /A 345 0 R /Border [0 0 0] /H /I /Rect [ 293.4000 533.0537 303.0373 541.8720 ] >> endobj 345 0 obj << /Type /Action >> endobj 346 0 obj << /Type /Annot /Subtype /Link /A 347 0 R /Border [0 0 0] /H /I /Rect [ 373.0965 509.2442 382.7338 518.0625 ] >> endobj 347 0 obj << /Type /Action >> endobj 348 0 obj << /Type /Page /Parent 3 0 R /Contents 349 0 R >> endobj 349 0 obj << /Length 23329 >> stream 0.271 0.267 0.267 rg q 15.000 36.167 577.500 740.833 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(Huntington disease. Nat Genet. 2004 Jun;36\(6\):585-95. PubMed PMID:15146184.)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 748.071 Td /F1 9.8 Tf [(Parker JA, Connolly JB, Wellington C, Hayden M, Dausset J, Neri C. Expanded polyglutamines in Caenorhabditis elegans )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(cause axonal abnormalities and severe dysfunction of PLM mechanosensory neurons without cell death. Proc Natl Acad Sci U )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(S A. 2001 Nov 6;98\(23\):13318-23. PubMed PMID:11687635.)] TJ ET BT 26.250 704.857 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 704.857 Td /F1 9.8 Tf [(Miller VM, Nelson RF, Gouvion CM, Williams A, Rodriguez-Lebron E, Harper SQ, Davidson BL, Rebagliati MR, Paulson HL. )] TJ ET BT 26.250 692.952 Td /F1 9.8 Tf [(CHIP suppresses polyglutamine aggregation and toxicity in vitro and in vivo. J Neurosci. 2005 Oct 5;25\(40\):9152-61. PubMed )] TJ ET BT 26.250 681.048 Td /F1 9.8 Tf [(PMID:16207874.)] TJ ET BT 26.250 661.643 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 661.643 Td /F1 9.8 Tf [(Krobitsch S, Lindquist S. Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the )] TJ ET BT 26.250 649.738 Td /F1 9.8 Tf [(expression of chaperone proteins. Proc Natl Acad Sci U S A. 2000 Feb 15;97\(4\):1589-94. PubMed PMID:10677504.)] TJ ET BT 26.250 630.333 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 630.333 Td /F1 9.8 Tf [(Mason RP, Giorgini F. Modeling Huntington disease in yeast: perspectives and future directions. Prion. 2011 Oct-)] TJ ET BT 26.250 618.429 Td /F1 9.8 Tf [(Dec;5\(4\):269-76. PubMed PMID:22052350.)] TJ ET BT 26.250 599.024 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 599.024 Td /F1 9.8 Tf [(Meriin AB, Zhang X, He X, Newnam GP, Chernoff YO, Sherman MY. Huntington toxicity in yeast model depends on )] TJ ET BT 26.250 587.119 Td /F1 9.8 Tf [(polyglutamine aggregation mediated by a prion-like protein Rnq1. J Cell Biol. 2002 Jun 10;157\(6\):997-1004. PubMed )] TJ ET BT 26.250 575.214 Td /F1 9.8 Tf [(PMID:12058016.)] TJ ET BT 26.250 555.810 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 555.810 Td /F1 9.8 Tf [(Duennwald ML, Jagadish S, Giorgini F, Muchowski PJ, Lindquist S. A network of protein interactions determines )] TJ ET BT 26.250 543.905 Td /F1 9.8 Tf [(polyglutamine toxicity. Proc Natl Acad Sci U S A. 2006 Jul 18;103\(29\):11051-6. PubMed PMID:16832049.)] TJ ET BT 26.250 524.500 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 524.500 Td /F1 9.8 Tf [(Derkatch IL, Bradley ME, Zhou P, Chernoff YO, Liebman SW. Genetic and environmental factors affecting the de novo )] TJ ET BT 26.250 512.595 Td /F1 9.8 Tf [(appearance of the [PSI+] prion in Saccharomyces cerevisiae. Genetics. 1997 Oct;147\(2\):507-19. PubMed PMID:9335589.)] TJ ET BT 26.250 493.191 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 493.191 Td /F1 9.8 Tf [(Derkatch IL, Bradley ME, Hong JY, Liebman SW. Prions affect the appearance of other prions: the story of [PIN\(+\)]. Cell. )] TJ ET BT 26.250 481.286 Td /F1 9.8 Tf [(2001 Jul 27;106\(2\):171-82. PubMed PMID:11511345.)] TJ ET BT 26.250 461.881 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 461.881 Td /F1 9.8 Tf [(Shorter J, Lindquist S. Prions as adaptive conduits of memory and inheritance. Nat Rev Genet. 2005 Jun;6\(6\):435-50. )] TJ ET BT 26.250 449.976 Td /F1 9.8 Tf [(PubMed PMID:15931169.)] TJ ET BT 26.250 430.572 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 430.572 Td /F1 9.8 Tf [(Gong H, Romanova NV, Allen KD, Chandramowlishwaran P, Gokhale K, Newnam GP, Mieczkowski P, Sherman MY, )] TJ ET BT 26.250 418.667 Td /F1 9.8 Tf [(Chernoff YO. Polyglutamine toxicity is controlled by prion composition and gene dosage in yeast. PLoS Genet. )] TJ ET BT 26.250 406.762 Td /F1 9.8 Tf [(2012;8\(4\):e1002634. PubMed PMID:22536159.)] TJ ET BT 26.250 387.357 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 387.357 Td /F1 9.8 Tf [(Giorgini F, Guidetti P, Nguyen Q, Bennett SC, Muchowski PJ. A genomic screen in yeast implicates kynurenine 3-)] TJ ET BT 26.250 375.453 Td /F1 9.8 Tf [(monooxygenase as a therapeutic target for Huntington disease. Nat Genet. 2005 May;37\(5\):526-31. PubMed PMID:15806102.)] TJ ET BT 26.250 356.048 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 356.048 Td /F1 9.8 Tf [(Michelitsch MD, Weissman JS. A census of glutamine/asparagine-rich regions: implications for their conserved function and )] TJ ET BT 26.250 344.143 Td /F1 9.8 Tf [(the prediction of novel prions. Proc Natl Acad Sci U S A. 2000 Oct 24;97\(22\):11910-5. PubMed PMID:11050225.)] TJ ET BT 26.250 324.738 Td /F1 9.8 Tf [(22.)] TJ ET BT 43.553 324.738 Td /F1 9.8 Tf [(Alberti S, Halfmann R, King O, Kapila A, Lindquist S. A systematic survey identifies prions and illuminates sequence )] TJ ET BT 26.250 312.834 Td /F1 9.8 Tf [(features of prionogenic proteins. Cell. 2009 Apr 3;137\(1\):146-58. PubMed PMID:19345193.)] TJ ET BT 26.250 293.429 Td /F1 9.8 Tf [(23.)] TJ ET BT 43.553 293.429 Td /F1 9.8 Tf [(Sherman, F., Fink, G. R. & Hicks, J. B. \(1986\). Methods in Yeast Genetics.., New York: Cold Spring Harbor Laboratory )] TJ ET BT 26.250 281.524 Td /F1 9.8 Tf [(Press)] TJ ET BT 26.250 262.119 Td /F1 9.8 Tf [(24.)] TJ ET BT 43.553 262.119 Td /F1 9.8 Tf [(Tauber E, Miller-Fleming L, Mason RP, Kwan W, Clapp J, Butler NJ, Outeiro TF, Muchowski PJ, Giorgini F. Functional gene )] TJ ET BT 26.250 250.215 Td /F1 9.8 Tf [(expression profiling in yeast implicates translational dysfunction in mutant huntingtin toxicity. J Biol Chem. 2011 Jan )] TJ ET BT 26.250 238.310 Td /F1 9.8 Tf [(7;286\(1\):410-9. PubMed PMID:21044956.)] TJ ET BT 26.250 218.905 Td /F1 9.8 Tf [(25.)] TJ ET BT 43.553 218.905 Td /F1 9.8 Tf [(Zhao X, Park YN, Todor H, Moomau C, Masison D, Eisenberg E, Greene LE. Sequestration of Sup35 by aggregates of )] TJ ET BT 26.250 207.000 Td /F1 9.8 Tf [(huntingtin fragments causes toxicity of [PSI+] yeast. J Biol Chem. 2012 Jul 6;287\(28\):23346-55. PubMed PMID:22573320.)] TJ ET BT 26.250 187.596 Td /F1 9.8 Tf [(26.)] TJ ET BT 43.553 187.596 Td /F1 9.8 Tf [(Manogaran AL, Hong JY, Hufana J, Tyedmers J, Lindquist S, Liebman SW. Prion formation and polyglutamine aggregation )] TJ ET BT 26.250 175.691 Td /F1 9.8 Tf [(are controlled by two classes of genes. PLoS Genet. 2011 May;7\(5\):e1001386. PubMed PMID:21625618.)] TJ ET BT 26.250 156.286 Td /F1 9.8 Tf [(27.)] TJ ET BT 43.553 156.286 Td /F1 9.8 Tf [(Toombs JA, McCarty BR, Ross ED. Compositional determinants of prion formation in yeast. Mol Cell Biol. 2010 )] TJ ET BT 26.250 144.381 Td /F1 9.8 Tf [(Jan;30\(1\):319-32. PubMed PMID:19884345.)] TJ ET BT 26.250 124.977 Td /F1 9.8 Tf [(28.)] TJ ET BT 43.553 124.977 Td /F1 9.8 Tf [(Glover JR, Kowal AS, Schirmer EC, Patino MM, Liu JJ, Lindquist S. Self-seeded fibers formed by Sup35, the protein )] TJ ET BT 26.250 113.072 Td /F1 9.8 Tf [(determinant of [PSI+], a heritable prion-like factor of S. cerevisiae. Cell. 1997 May 30;89\(5\):811-9. PubMed PMID:9182769.)] TJ ET BT 26.250 93.667 Td /F1 9.8 Tf [(29.)] TJ ET BT 43.553 93.667 Td /F1 9.8 Tf [(Derkatch IL, Uptain SM, Outeiro TF, Krishnan R, Lindquist SL, Liebman SW. Effects of Q/N-rich, polyQ, and non-polyQ )] TJ ET BT 26.250 81.762 Td /F1 9.8 Tf [(amyloids on the de novo formation of the [PSI+] prion in yeast and aggregation of Sup35 in vitro. Proc Natl Acad Sci U S A. )] TJ ET BT 26.250 69.858 Td /F1 9.8 Tf [(2004 Aug 31;101\(35\):12934-9. PubMed PMID:15326312.)] TJ ET BT 26.250 50.453 Td /F1 9.8 Tf [(30.)] TJ ET BT 43.553 50.453 Td /F1 9.8 Tf [(Venancio TM, Aravind L. CYSTM, a novel cysteine-rich transmembrane module with a role in stress tolerance across )] TJ ET Q q 15.000 36.167 577.500 740.833 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(Huntington disease. Nat Genet. 2004 Jun;36\(6\):585-95. PubMed PMID:15146184.)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 748.071 Td /F1 9.8 Tf [(Parker JA, Connolly JB, Wellington C, Hayden M, Dausset J, Neri C. Expanded polyglutamines in Caenorhabditis elegans )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(cause axonal abnormalities and severe dysfunction of PLM mechanosensory neurons without cell death. Proc Natl Acad Sci U )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(S A. 2001 Nov 6;98\(23\):13318-23. PubMed PMID:11687635.)] TJ ET BT 26.250 704.857 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 704.857 Td /F1 9.8 Tf [(Miller VM, Nelson RF, Gouvion CM, Williams A, Rodriguez-Lebron E, Harper SQ, Davidson BL, Rebagliati MR, Paulson HL. )] TJ ET BT 26.250 692.952 Td /F1 9.8 Tf [(CHIP suppresses polyglutamine aggregation and toxicity in vitro and in vivo. J Neurosci. 2005 Oct 5;25\(40\):9152-61. PubMed )] TJ ET BT 26.250 681.048 Td /F1 9.8 Tf [(PMID:16207874.)] TJ ET BT 26.250 661.643 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 661.643 Td /F1 9.8 Tf [(Krobitsch S, Lindquist S. Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the )] TJ ET BT 26.250 649.738 Td /F1 9.8 Tf [(expression of chaperone proteins. Proc Natl Acad Sci U S A. 2000 Feb 15;97\(4\):1589-94. PubMed PMID:10677504.)] TJ ET BT 26.250 630.333 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 630.333 Td /F1 9.8 Tf [(Mason RP, Giorgini F. Modeling Huntington disease in yeast: perspectives and future directions. Prion. 2011 Oct-)] TJ ET BT 26.250 618.429 Td /F1 9.8 Tf [(Dec;5\(4\):269-76. PubMed PMID:22052350.)] TJ ET BT 26.250 599.024 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 599.024 Td /F1 9.8 Tf [(Meriin AB, Zhang X, He X, Newnam GP, Chernoff YO, Sherman MY. Huntington toxicity in yeast model depends on )] TJ ET BT 26.250 587.119 Td /F1 9.8 Tf [(polyglutamine aggregation mediated by a prion-like protein Rnq1. J Cell Biol. 2002 Jun 10;157\(6\):997-1004. PubMed )] TJ ET BT 26.250 575.214 Td /F1 9.8 Tf [(PMID:12058016.)] TJ ET BT 26.250 555.810 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 555.810 Td /F1 9.8 Tf [(Duennwald ML, Jagadish S, Giorgini F, Muchowski PJ, Lindquist S. A network of protein interactions determines )] TJ ET BT 26.250 543.905 Td /F1 9.8 Tf [(polyglutamine toxicity. Proc Natl Acad Sci U S A. 2006 Jul 18;103\(29\):11051-6. PubMed PMID:16832049.)] TJ ET BT 26.250 524.500 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 524.500 Td /F1 9.8 Tf [(Derkatch IL, Bradley ME, Zhou P, Chernoff YO, Liebman SW. Genetic and environmental factors affecting the de novo )] TJ ET BT 26.250 512.595 Td /F1 9.8 Tf [(appearance of the [PSI+] prion in Saccharomyces cerevisiae. Genetics. 1997 Oct;147\(2\):507-19. PubMed PMID:9335589.)] TJ ET BT 26.250 493.191 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 493.191 Td /F1 9.8 Tf [(Derkatch IL, Bradley ME, Hong JY, Liebman SW. Prions affect the appearance of other prions: the story of [PIN\(+\)]. Cell. )] TJ ET BT 26.250 481.286 Td /F1 9.8 Tf [(2001 Jul 27;106\(2\):171-82. PubMed PMID:11511345.)] TJ ET BT 26.250 461.881 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 461.881 Td /F1 9.8 Tf [(Shorter J, Lindquist S. Prions as adaptive conduits of memory and inheritance. Nat Rev Genet. 2005 Jun;6\(6\):435-50. )] TJ ET BT 26.250 449.976 Td /F1 9.8 Tf [(PubMed PMID:15931169.)] TJ ET BT 26.250 430.572 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 430.572 Td /F1 9.8 Tf [(Gong H, Romanova NV, Allen KD, Chandramowlishwaran P, Gokhale K, Newnam GP, Mieczkowski P, Sherman MY, )] TJ ET BT 26.250 418.667 Td /F1 9.8 Tf [(Chernoff YO. Polyglutamine toxicity is controlled by prion composition and gene dosage in yeast. PLoS Genet. )] TJ ET BT 26.250 406.762 Td /F1 9.8 Tf [(2012;8\(4\):e1002634. PubMed PMID:22536159.)] TJ ET BT 26.250 387.357 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 387.357 Td /F1 9.8 Tf [(Giorgini F, Guidetti P, Nguyen Q, Bennett SC, Muchowski PJ. A genomic screen in yeast implicates kynurenine 3-)] TJ ET BT 26.250 375.453 Td /F1 9.8 Tf [(monooxygenase as a therapeutic target for Huntington disease. Nat Genet. 2005 May;37\(5\):526-31. PubMed PMID:15806102.)] TJ ET BT 26.250 356.048 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 356.048 Td /F1 9.8 Tf [(Michelitsch MD, Weissman JS. A census of glutamine/asparagine-rich regions: implications for their conserved function and )] TJ ET BT 26.250 344.143 Td /F1 9.8 Tf [(the prediction of novel prions. Proc Natl Acad Sci U S A. 2000 Oct 24;97\(22\):11910-5. PubMed PMID:11050225.)] TJ ET BT 26.250 324.738 Td /F1 9.8 Tf [(22.)] TJ ET BT 43.553 324.738 Td /F1 9.8 Tf [(Alberti S, Halfmann R, King O, Kapila A, Lindquist S. A systematic survey identifies prions and illuminates sequence )] TJ ET BT 26.250 312.834 Td /F1 9.8 Tf [(features of prionogenic proteins. Cell. 2009 Apr 3;137\(1\):146-58. PubMed PMID:19345193.)] TJ ET BT 26.250 293.429 Td /F1 9.8 Tf [(23.)] TJ ET BT 43.553 293.429 Td /F1 9.8 Tf [(Sherman, F., Fink, G. R. & Hicks, J. B. \(1986\). Methods in Yeast Genetics.., New York: Cold Spring Harbor Laboratory )] TJ ET BT 26.250 281.524 Td /F1 9.8 Tf [(Press)] TJ ET BT 26.250 262.119 Td /F1 9.8 Tf [(24.)] TJ ET BT 43.553 262.119 Td /F1 9.8 Tf [(Tauber E, Miller-Fleming L, Mason RP, Kwan W, Clapp J, Butler NJ, Outeiro TF, Muchowski PJ, Giorgini F. Functional gene )] TJ ET BT 26.250 250.215 Td /F1 9.8 Tf [(expression profiling in yeast implicates translational dysfunction in mutant huntingtin toxicity. J Biol Chem. 2011 Jan )] TJ ET BT 26.250 238.310 Td /F1 9.8 Tf [(7;286\(1\):410-9. PubMed PMID:21044956.)] TJ ET BT 26.250 218.905 Td /F1 9.8 Tf [(25.)] TJ ET BT 43.553 218.905 Td /F1 9.8 Tf [(Zhao X, Park YN, Todor H, Moomau C, Masison D, Eisenberg E, Greene LE. Sequestration of Sup35 by aggregates of )] TJ ET BT 26.250 207.000 Td /F1 9.8 Tf [(huntingtin fragments causes toxicity of [PSI+] yeast. J Biol Chem. 2012 Jul 6;287\(28\):23346-55. PubMed PMID:22573320.)] TJ ET BT 26.250 187.596 Td /F1 9.8 Tf [(26.)] TJ ET BT 43.553 187.596 Td /F1 9.8 Tf [(Manogaran AL, Hong JY, Hufana J, Tyedmers J, Lindquist S, Liebman SW. Prion formation and polyglutamine aggregation )] TJ ET BT 26.250 175.691 Td /F1 9.8 Tf [(are controlled by two classes of genes. PLoS Genet. 2011 May;7\(5\):e1001386. PubMed PMID:21625618.)] TJ ET BT 26.250 156.286 Td /F1 9.8 Tf [(27.)] TJ ET BT 43.553 156.286 Td /F1 9.8 Tf [(Toombs JA, McCarty BR, Ross ED. Compositional determinants of prion formation in yeast. Mol Cell Biol. 2010 )] TJ ET BT 26.250 144.381 Td /F1 9.8 Tf [(Jan;30\(1\):319-32. PubMed PMID:19884345.)] TJ ET BT 26.250 124.977 Td /F1 9.8 Tf [(28.)] TJ ET BT 43.553 124.977 Td /F1 9.8 Tf [(Glover JR, Kowal AS, Schirmer EC, Patino MM, Liu JJ, Lindquist S. Self-seeded fibers formed by Sup35, the protein )] TJ ET BT 26.250 113.072 Td /F1 9.8 Tf [(determinant of [PSI+], a heritable prion-like factor of S. cerevisiae. Cell. 1997 May 30;89\(5\):811-9. PubMed PMID:9182769.)] TJ ET BT 26.250 93.667 Td /F1 9.8 Tf [(29.)] TJ ET BT 43.553 93.667 Td /F1 9.8 Tf [(Derkatch IL, Uptain SM, Outeiro TF, Krishnan R, Lindquist SL, Liebman SW. Effects of Q/N-rich, polyQ, and non-polyQ )] TJ ET BT 26.250 81.762 Td /F1 9.8 Tf [(amyloids on the de novo formation of the [PSI+] prion in yeast and aggregation of Sup35 in vitro. Proc Natl Acad Sci U S A. )] TJ ET BT 26.250 69.858 Td /F1 9.8 Tf [(2004 Aug 31;101\(35\):12934-9. PubMed PMID:15326312.)] TJ ET BT 26.250 50.453 Td /F1 9.8 Tf [(30.)] TJ ET BT 43.553 50.453 Td /F1 9.8 Tf [(Venancio TM, Aravind L. CYSTM, a novel cysteine-rich transmembrane module with a role in stress tolerance across )] TJ ET Q q 15.000 36.167 577.500 740.833 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(Huntington disease. Nat Genet. 2004 Jun;36\(6\):585-95. PubMed PMID:15146184.)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(10.)] TJ ET BT 43.553 748.071 Td /F1 9.8 Tf [(Parker JA, Connolly JB, Wellington C, Hayden M, Dausset J, Neri C. Expanded polyglutamines in Caenorhabditis elegans )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(cause axonal abnormalities and severe dysfunction of PLM mechanosensory neurons without cell death. Proc Natl Acad Sci U )] TJ ET BT 26.250 724.262 Td /F1 9.8 Tf [(S A. 2001 Nov 6;98\(23\):13318-23. PubMed PMID:11687635.)] TJ ET BT 26.250 704.857 Td /F1 9.8 Tf [(11.)] TJ ET BT 43.553 704.857 Td /F1 9.8 Tf [(Miller VM, Nelson RF, Gouvion CM, Williams A, Rodriguez-Lebron E, Harper SQ, Davidson BL, Rebagliati MR, Paulson HL. )] TJ ET BT 26.250 692.952 Td /F1 9.8 Tf [(CHIP suppresses polyglutamine aggregation and toxicity in vitro and in vivo. J Neurosci. 2005 Oct 5;25\(40\):9152-61. PubMed )] TJ ET BT 26.250 681.048 Td /F1 9.8 Tf [(PMID:16207874.)] TJ ET BT 26.250 661.643 Td /F1 9.8 Tf [(12.)] TJ ET BT 43.553 661.643 Td /F1 9.8 Tf [(Krobitsch S, Lindquist S. Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the )] TJ ET BT 26.250 649.738 Td /F1 9.8 Tf [(expression of chaperone proteins. Proc Natl Acad Sci U S A. 2000 Feb 15;97\(4\):1589-94. PubMed PMID:10677504.)] TJ ET BT 26.250 630.333 Td /F1 9.8 Tf [(13.)] TJ ET BT 43.553 630.333 Td /F1 9.8 Tf [(Mason RP, Giorgini F. Modeling Huntington disease in yeast: perspectives and future directions. Prion. 2011 Oct-)] TJ ET BT 26.250 618.429 Td /F1 9.8 Tf [(Dec;5\(4\):269-76. PubMed PMID:22052350.)] TJ ET BT 26.250 599.024 Td /F1 9.8 Tf [(14.)] TJ ET BT 43.553 599.024 Td /F1 9.8 Tf [(Meriin AB, Zhang X, He X, Newnam GP, Chernoff YO, Sherman MY. Huntington toxicity in yeast model depends on )] TJ ET BT 26.250 587.119 Td /F1 9.8 Tf [(polyglutamine aggregation mediated by a prion-like protein Rnq1. J Cell Biol. 2002 Jun 10;157\(6\):997-1004. PubMed )] TJ ET BT 26.250 575.214 Td /F1 9.8 Tf [(PMID:12058016.)] TJ ET BT 26.250 555.810 Td /F1 9.8 Tf [(15.)] TJ ET BT 43.553 555.810 Td /F1 9.8 Tf [(Duennwald ML, Jagadish S, Giorgini F, Muchowski PJ, Lindquist S. A network of protein interactions determines )] TJ ET BT 26.250 543.905 Td /F1 9.8 Tf [(polyglutamine toxicity. Proc Natl Acad Sci U S A. 2006 Jul 18;103\(29\):11051-6. PubMed PMID:16832049.)] TJ ET BT 26.250 524.500 Td /F1 9.8 Tf [(16.)] TJ ET BT 43.553 524.500 Td /F1 9.8 Tf [(Derkatch IL, Bradley ME, Zhou P, Chernoff YO, Liebman SW. Genetic and environmental factors affecting the de novo )] TJ ET BT 26.250 512.595 Td /F1 9.8 Tf [(appearance of the [PSI+] prion in Saccharomyces cerevisiae. Genetics. 1997 Oct;147\(2\):507-19. PubMed PMID:9335589.)] TJ ET BT 26.250 493.191 Td /F1 9.8 Tf [(17.)] TJ ET BT 43.553 493.191 Td /F1 9.8 Tf [(Derkatch IL, Bradley ME, Hong JY, Liebman SW. Prions affect the appearance of other prions: the story of [PIN\(+\)]. Cell. )] TJ ET BT 26.250 481.286 Td /F1 9.8 Tf [(2001 Jul 27;106\(2\):171-82. PubMed PMID:11511345.)] TJ ET BT 26.250 461.881 Td /F1 9.8 Tf [(18.)] TJ ET BT 43.553 461.881 Td /F1 9.8 Tf [(Shorter J, Lindquist S. Prions as adaptive conduits of memory and inheritance. Nat Rev Genet. 2005 Jun;6\(6\):435-50. )] TJ ET BT 26.250 449.976 Td /F1 9.8 Tf [(PubMed PMID:15931169.)] TJ ET BT 26.250 430.572 Td /F1 9.8 Tf [(19.)] TJ ET BT 43.553 430.572 Td /F1 9.8 Tf [(Gong H, Romanova NV, Allen KD, Chandramowlishwaran P, Gokhale K, Newnam GP, Mieczkowski P, Sherman MY, )] TJ ET BT 26.250 418.667 Td /F1 9.8 Tf [(Chernoff YO. Polyglutamine toxicity is controlled by prion composition and gene dosage in yeast. PLoS Genet. )] TJ ET BT 26.250 406.762 Td /F1 9.8 Tf [(2012;8\(4\):e1002634. PubMed PMID:22536159.)] TJ ET BT 26.250 387.357 Td /F1 9.8 Tf [(20.)] TJ ET BT 43.553 387.357 Td /F1 9.8 Tf [(Giorgini F, Guidetti P, Nguyen Q, Bennett SC, Muchowski PJ. A genomic screen in yeast implicates kynurenine 3-)] TJ ET BT 26.250 375.453 Td /F1 9.8 Tf [(monooxygenase as a therapeutic target for Huntington disease. Nat Genet. 2005 May;37\(5\):526-31. PubMed PMID:15806102.)] TJ ET BT 26.250 356.048 Td /F1 9.8 Tf [(21.)] TJ ET BT 43.553 356.048 Td /F1 9.8 Tf [(Michelitsch MD, Weissman JS. A census of glutamine/asparagine-rich regions: implications for their conserved function and )] TJ ET BT 26.250 344.143 Td /F1 9.8 Tf [(the prediction of novel prions. Proc Natl Acad Sci U S A. 2000 Oct 24;97\(22\):11910-5. PubMed PMID:11050225.)] TJ ET BT 26.250 324.738 Td /F1 9.8 Tf [(22.)] TJ ET BT 43.553 324.738 Td /F1 9.8 Tf [(Alberti S, Halfmann R, King O, Kapila A, Lindquist S. A systematic survey identifies prions and illuminates sequence )] TJ ET BT 26.250 312.834 Td /F1 9.8 Tf [(features of prionogenic proteins. Cell. 2009 Apr 3;137\(1\):146-58. PubMed PMID:19345193.)] TJ ET BT 26.250 293.429 Td /F1 9.8 Tf [(23.)] TJ ET BT 43.553 293.429 Td /F1 9.8 Tf [(Sherman, F., Fink, G. R. & Hicks, J. B. \(1986\). Methods in Yeast Genetics.., New York: Cold Spring Harbor Laboratory )] TJ ET BT 26.250 281.524 Td /F1 9.8 Tf [(Press)] TJ ET BT 26.250 262.119 Td /F1 9.8 Tf [(24.)] TJ ET BT 43.553 262.119 Td /F1 9.8 Tf [(Tauber E, Miller-Fleming L, Mason RP, Kwan W, Clapp J, Butler NJ, Outeiro TF, Muchowski PJ, Giorgini F. Functional gene )] TJ ET BT 26.250 250.215 Td /F1 9.8 Tf [(expression profiling in yeast implicates translational dysfunction in mutant huntingtin toxicity. J Biol Chem. 2011 Jan )] TJ ET BT 26.250 238.310 Td /F1 9.8 Tf [(7;286\(1\):410-9. PubMed PMID:21044956.)] TJ ET BT 26.250 218.905 Td /F1 9.8 Tf [(25.)] TJ ET BT 43.553 218.905 Td /F1 9.8 Tf [(Zhao X, Park YN, Todor H, Moomau C, Masison D, Eisenberg E, Greene LE. Sequestration of Sup35 by aggregates of )] TJ ET BT 26.250 207.000 Td /F1 9.8 Tf [(huntingtin fragments causes toxicity of [PSI+] yeast. J Biol Chem. 2012 Jul 6;287\(28\):23346-55. PubMed PMID:22573320.)] TJ ET BT 26.250 187.596 Td /F1 9.8 Tf [(26.)] TJ ET BT 43.553 187.596 Td /F1 9.8 Tf [(Manogaran AL, Hong JY, Hufana J, Tyedmers J, Lindquist S, Liebman SW. Prion formation and polyglutamine aggregation )] TJ ET BT 26.250 175.691 Td /F1 9.8 Tf [(are controlled by two classes of genes. PLoS Genet. 2011 May;7\(5\):e1001386. PubMed PMID:21625618.)] TJ ET BT 26.250 156.286 Td /F1 9.8 Tf [(27.)] TJ ET BT 43.553 156.286 Td /F1 9.8 Tf [(Toombs JA, McCarty BR, Ross ED. Compositional determinants of prion formation in yeast. Mol Cell Biol. 2010 )] TJ ET BT 26.250 144.381 Td /F1 9.8 Tf [(Jan;30\(1\):319-32. PubMed PMID:19884345.)] TJ ET BT 26.250 124.977 Td /F1 9.8 Tf [(28.)] TJ ET BT 43.553 124.977 Td /F1 9.8 Tf [(Glover JR, Kowal AS, Schirmer EC, Patino MM, Liu JJ, Lindquist S. Self-seeded fibers formed by Sup35, the protein )] TJ ET BT 26.250 113.072 Td /F1 9.8 Tf [(determinant of [PSI+], a heritable prion-like factor of S. cerevisiae. Cell. 1997 May 30;89\(5\):811-9. PubMed PMID:9182769.)] TJ ET BT 26.250 93.667 Td /F1 9.8 Tf [(29.)] TJ ET BT 43.553 93.667 Td /F1 9.8 Tf [(Derkatch IL, Uptain SM, Outeiro TF, Krishnan R, Lindquist SL, Liebman SW. Effects of Q/N-rich, polyQ, and non-polyQ )] TJ ET BT 26.250 81.762 Td /F1 9.8 Tf [(amyloids on the de novo formation of the [PSI+] prion in yeast and aggregation of Sup35 in vitro. Proc Natl Acad Sci U S A. )] TJ ET BT 26.250 69.858 Td /F1 9.8 Tf [(2004 Aug 31;101\(35\):12934-9. PubMed PMID:15326312.)] TJ ET BT 26.250 50.453 Td /F1 9.8 Tf [(30.)] TJ ET BT 43.553 50.453 Td /F1 9.8 Tf [(Venancio TM, Aravind L. CYSTM, a novel cysteine-rich transmembrane module with a role in stress tolerance across )] TJ ET Q q 0.000 0.000 0.000 rg BT 291.710 19.825 Td /F1 11.0 Tf [(7)] TJ ET BT 25.000 19.825 Td /F1 11.0 Tf [(PLOS Currents Huntington Disease)] TJ ET Q endstream endobj 350 0 obj << /Type /Page /Parent 3 0 R /Contents 351 0 R >> endobj 351 0 obj << /Length 2629 >> stream 0.271 0.267 0.267 rg q 15.000 687.476 577.500 89.524 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(eukaryotes. Bioinformatics. 2010 Jan 15;26\(2\):149-52. PubMed PMID:19933165.)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(31.)] TJ ET BT 43.553 748.071 Td /F1 9.8 Tf [(Ren J, Wen L, Gao X, Jin C, Xue Y, Yao X. CSS-Palm 2.0: an updated software for palmitoylation sites prediction. Protein )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(Eng Des Sel. 2008 Nov;21\(11\):639-44. PubMed PMID:18753194.)] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(32.)] TJ ET BT 43.553 716.762 Td /F1 9.8 Tf [(King OD, Gitler AD, Shorter J. The tip of the iceberg: RNA-binding proteins with prion-like domains in neurodegenerative )] TJ ET BT 26.250 704.857 Td /F1 9.8 Tf [(disease. Brain Res. 2012 Jun 26;1462:61-80. PubMed PMID:22445064.)] TJ ET Q q 15.000 687.476 577.500 89.524 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(eukaryotes. Bioinformatics. 2010 Jan 15;26\(2\):149-52. PubMed PMID:19933165.)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(31.)] TJ ET BT 43.553 748.071 Td /F1 9.8 Tf [(Ren J, Wen L, Gao X, Jin C, Xue Y, Yao X. CSS-Palm 2.0: an updated software for palmitoylation sites prediction. Protein )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(Eng Des Sel. 2008 Nov;21\(11\):639-44. PubMed PMID:18753194.)] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(32.)] TJ ET BT 43.553 716.762 Td /F1 9.8 Tf [(King OD, Gitler AD, Shorter J. The tip of the iceberg: RNA-binding proteins with prion-like domains in neurodegenerative )] TJ ET BT 26.250 704.857 Td /F1 9.8 Tf [(disease. Brain Res. 2012 Jun 26;1462:61-80. PubMed PMID:22445064.)] TJ ET Q q 15.000 687.476 577.500 89.524 re W n 0.271 0.267 0.267 rg BT 26.250 767.476 Td /F1 9.8 Tf [(eukaryotes. Bioinformatics. 2010 Jan 15;26\(2\):149-52. PubMed PMID:19933165.)] TJ ET BT 26.250 748.071 Td /F1 9.8 Tf [(31.)] TJ ET BT 43.553 748.071 Td /F1 9.8 Tf [(Ren J, Wen L, Gao X, Jin C, Xue Y, Yao X. CSS-Palm 2.0: an updated software for palmitoylation sites prediction. Protein )] TJ ET BT 26.250 736.167 Td /F1 9.8 Tf [(Eng Des Sel. 2008 Nov;21\(11\):639-44. PubMed PMID:18753194.)] TJ ET BT 26.250 716.762 Td /F1 9.8 Tf [(32.)] TJ ET BT 43.553 716.762 Td /F1 9.8 Tf [(King OD, Gitler AD, Shorter J. The tip of the iceberg: RNA-binding proteins with prion-like domains in neurodegenerative )] TJ ET BT 26.250 704.857 Td /F1 9.8 Tf [(disease. Brain Res. 2012 Jun 26;1462:61-80. 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