Huntington toxicity in yeast model depends on polyglutamine aggregation mediated by a prion-like protein Rnq1

Abstract

The cause of Huntington's disease is expansion of polyglutamine (polyQ) domain in huntingtin, which makes this protein both neurotoxic and aggregation prone. Here we developed the first yeast model, which establishes a direct link between aggregation of expanded polyQ domain and its cytotoxicity. Our data indicated that deficiencies in molecular chaperones Sis1 and Hsp104 inhibited seeding of polyQ aggregates, whereas ssa1, ssa2, and ydj1-151 mutations inhibited expansion of aggregates. The latter three mutants strongly suppressed the polyQ toxicity. Spontaneous mutants with suppressed aggregation appeared with high frequency, and in all of them the toxicity was relieved. Aggregation defects in these mutants and in sis1-85 were not complemented in the cross to the hsp104 mutant, demonstrating an unusual type of inheritance. Since Hsp104 is required for prion maintenance in yeast, this suggested a role for prions in polyQ aggregation and toxicity. We screened a set of deletions of nonessential genes coding for known prions and related proteins and found that deletion of the RNQ1 gene specifically suppressed aggregation and toxicity of polyQ. Curing of the prion form of Rnq1 from wild-type cells dramatically suppressed both aggregation and toxicity of polyQ. We concluded that aggregation of polyQ is critical for its toxicity and that Rnq1 in its prion conformation plays an essential role in polyQ aggregation leading to the toxicity.

Figure 1.

Accumulation of 103Q in yeast cells inhibits yeast growth. (A) Cells transformed with 25Q or 103Q expression vector grew on selective glucose plates for 3 d or on galactose plates for 5 d. (B) Flow cytometry analysis of fluorescence of the cells induced for 20 h. The peaks at the left represent cells, which do not express polyQ. Note, the scale of the fluorescence of accumulated GFP fusion is logarithmic. (C) Accumulation of 103Q does not affect cell cycle. FACS^® analysis of yeast cultures expressing 25Q or 103Q after 20 h induction in selective galactose medium.

Figure 2.

Molecular chaperones are involved in aggregation of 103Q in yeast cells. (A) Fluorescent confocal micrographs of yeast cells expressing polyQ polypeptides after induction for 7 h. (B and C) Levels of polyQ polypeptides (20 h induction) in soluble and insoluble fractions of yeast cells lysates. Sup, soluble fraction; Pel, pellets. (D) Confocal micrographs of yeast strains expressing 103Q. ssa1, 2 and sis1–85 mutants (top); hsp104 deletion mutant (bottom). (The picture with combined fluorescent and phase–contrast microscopy on the right makes cells with one large aggregate and no diffused fluorescence visible.)

Figure 3.

Deletion in hsp104 does not impede growth of preformed IBs. Fluorescent images of wild-type and mutant cells with 103Q (expressed for 3 h before the start of the observation) taken at different time points. The IB formation in the mutant cell was recorded five times; the typical recording is presented.

Figure 4.

Deprivation of yeast cells of certain chaperones relieves 103Q-associated toxicity. Growth (4 d) of hsp104 mutant and the corresponding wild type (top), both expressing 103Q and ssa1,ssa2 mutant and the corresponding wild type (bottom) expressing 103Q or 25Q.

Figure 5.

103Q aggregation and toxicity depend on yeast prions. (A) Fluorescent confocal micrographs of yeast strains expressing 103Q. (B) Growth (4 d) of wild-type, guanidine-pretreated and rnq1mutant cells expressing103Q. (C) Increase in the cellular levels and solubility of 103Q (expressed for 24 h) in the cells presented in Fig. 5 B; Gua, GuHCl pretreated; rnq, rnq1 mutant. Note that (a) 103Q is represented by the lowest band and (b) most of insoluble 103Q in GuHCl-treated and rnq1 cells represents the polypeptide from the fraction of cells with IBs. (D) Distribution of Rnq1 between supernatant (S) and pellet (P) after a centrifugation at 100,000 g of homogenates of the original W303 and JN54 strains and their derivatives.