Aneuploidy causes proteotoxic stress in yeast

Abstract

Gains or losses of entire chromosomes lead to aneuploidy, a condition tolerated poorly in all eukaryotes analyzed to date. How aneuploidy affects organismal and cellular physiology is poorly understood. We found that aneuploid budding yeast cells are under proteotoxic stress. Aneuploid strains are prone to aggregation of endogenous proteins as well as of ectopically expressed hard-to-fold proteins such as those containing polyglutamine (polyQ) stretches. Protein aggregate formation in aneuploid yeast strains is likely due to limiting protein quality-control systems, since the proteasome and at least one chaperone family, Hsp90, are compromised in many aneuploid strains. The link between aneuploidy and the formation and persistence of protein aggregates could have important implications for diseases such as cancer and neurodegeneration.

Figure 1.

Disomic yeast strains harbor an increased protein aggregate load. (A) Wild-type (WT) and disomic yeast strains containing an HSP104-eGFP fusion were grown to exponential phase in YEPD, and the percentage of cells harboring Hsp104-eGFP foci was determined. n = 3; SEM, n = 100 cells per time point; (**) P < 0.005; (***) P < 0.0005; Student's t-test. Strains, in order, are A31392, A31393, A31394, A31395, A31396, A31397, A31398, A31399, A31400, A31401, A31402, A31403, A31404, and A31405. (B) Images of Hsp104-eGFP aggregates. Aggregates are in green, and DNA is in blue. (C,D) Wild-type (A25654), cdc23-1 (A29766), and cdc28-4 (A29765) strains (C) and strains harboring YACs containing 580 kb (A28922) or 670 kb (A28925) of human DNA (D) were grown as in A to determine the percentage of cells with Hsp104-eGFP aggregates. (E) Quantification of Hsp104-eGFP foci in trisomic yeast strains grown as in A. Strains, in order, are A31406, A31407, A31408, A31409, and A31410. Note that the number of cells with Hsp104-eGFP aggregates in diploid cultures is lower than in haploid cultures. The basis for this is unclear at present. (F) Strains grown at 25°C were shifted to 37°C. The percentage of cells with Hsp104-eGFP aggregates was determined at the indicated times after temperature shift. n = 100 cells per time point. Two replicas of this experiment are shown in Supplemental Figure S1. The strains are the same as in A.

Figure 2.

The heat-shock response is intact in disomic yeast strains. Wild-type (WT) and disomic yeast strains were grown at 25°C and shifted to 37°C. RNA samples were taken 0, 5, 15, and 30 min after shift. RNA extracted from wild-type cells grown continuously at 25°C was used as reference for all samples. Data were mined for genes involved in the heat-shock response (Gasch et al. 2000), and those present in the extra chromosome were removed from the analysis (gray boxes). The data set was split into those genes that are up-regulated and those that are down-regulated in the heat-shock response. (A) The expression of genes involved in the heat-shock response in disomic yeast after shifting to 37°C. Yellow shows up-regulated genes, and blue shows down-regulated genes. Shown are the unclustered data after being zero-transformed. (B) The average expression changes of up-regulated (above the X-axis) and down-regulated (below the X-axis) genes displayed in A are shown.

Figure 3.

Meiotic and mitotic chromosome nondisjunction causes increased Hsp104-eGFP focus formation. (A) The percentage of cells with Hsp104-eGFP aggregates was analyzed in the progeny of diploid (A28220) or triploid (A28219) strains 3 d after germination. n = 100 cells per strain. Note that although many of the progeny from the triploid meioses will harbor multiple aneuploidies, some will also be euploid or will have become euploid as they proliferate. (B,C) Wild-type (WT) (A5244), ndc10-1 (A28204), and ipl1-321 (A16154) mutants harboring a GFP-marked chromosome IV, and wild-type (A25654), ndc10-1 (A27681), and ipl1-321 (A27682) mutants harboring Hsp104-eGFP were arrested in G1 with pheromone at 25°C, followed by release at 30°C in either the presence (10 μg/mL; Arrested) or absence (Dividing) of nocodazole. Samples were taken after 3 h to determine the percentage of cells that correctly segregated chromosome IV (B) and that harbored Hsp104-eGFP foci (C). We note that the temperature shift during this experiment may inflate the percentage of cells harboring aggregates in all strains, as they will be adapting to the temperature shift.

Figure 4.

Aneuploid yeast display hallmarks of impaired protein quality control. (A) Wild-type (WT) and disomic yeast strains deleted for the multidrug transporter PDR5 and expressing a VHL-GFP fusion were grown in YEP 2% raffinose and 2% galactose at 25°C, and the percentage of cells with VHL-GFP foci was determined. n = 3; SEM; n = 300 cells per time point; (*) P < 0.05; (**) P < 0.005; (***) P < 0.0005; Student's t-test. Strains, in order, are A32076, A32077, A32078, A32079, A32080, A32081, A32082, A32083, A32084, A32085, A32086, A32087, A32088, and A32089. (B) Strains described in A were analyzed after a 2-h incubation at 37°C. n = 100. (C,D) cdc23-1 (A30461), cdc28-4 (A30462), and wild-type strains harboring YACs carrying human DNA (A29969 and A29971) were grown at 25°C (C) or shifted for 2 h to 37°C (D) to analyze VHL-GFP focus formation. (E) The percentage of cells with VHL-GFP foci was determined in the progeny of diploid (A28388) or triploid (A28389) strains 4 d after germination. n = 100 per colony. (F) Images of VHL-GFP aggregates. Aggregates are in green, and DNA is in blue.

Figure 5.

Increased proteasome activity decreases aggregate burden in disomic strains. Wild-type (WT) (A3369), disome II (A33370), disome V (A33371), and disome IX (A33372) cells harboring a deletion of UBP6 and the HSP104-eGFP fusion were grown to exponential phase in YEPD, and the percentage of cells harboring Hsp104-eGFP foci was determined. n = 3; SEM, n = 100 cells per time point.

Figure 6.

Hsp90 folding capacity is limiting in many disomic strains. (A) Disomic yeast harboring a deletion of the multidrug transporter PDR5 were grown in YEPD or YEPD containing 70 μM radicicol at 30°C to determine their doubling time. The mean and SEM of three replicates are shown. Strains, in order, are A15549, A15551, A15553, A15555, A15557, A15559, A15561, A15563, A15566, A15567, A15569, A15571, and A15573. (B) Schematic of v-src/c-src Hsp90 assay. Hsp90 is required to fold c-src and toxic v-src. A reduction in Hsp90 activity results in misfolded v-src and restores cell viability. (C) Wild-type (WT) and disomic yeast strains carrying c-src or v-src under the galactose-inducible GAL1-10 promoter were grown under conditions in which expression is repressed (−URA 2% glucose) or induced (−URA 2% raffinose and galactose). Tenfold dilutions were plated. C-src strains, in order, are A32090, A32091, A32092, A32093, A32094, A32095, A32096, A32097, A32098, A32099, A32100, and A32101. V-src strains, in order, are A32102, A32103, A32104, A32105, A32106, A32107, A32108, A32109, A32110, A32111, A32112, and A32113. (D,E) Wild-type and disomic yeast strains harboring the GAL-v-src fusion were grown in YEP + 2% raffinose. Galactose was added, and the relative amount of v-src RNA (E) and total tyrosine phosphorylation (D) was determined before and after 2 h of v-src induction. V-src strains are in the same order as in C.

Figure 7.

Disomic yeast strains show increased expression of the Sup35 prion reporter. (A) Single colonies of wild-type (WT) and disomic strains carrying the SUP35-R2E2 allele and a GFP construct preceded by three stop codons were inoculated into SC medium, and the percentage of fluorescent cells was determined after 0, 8, and 24 h by flow cytometry. Shown is the ratio of GFP⁺ cells after 8 and 24 h of growth to GFP⁺ cells immediately after inoculation (0 h). The mean and SEM of at least 12 single colonies are depicted. Strains, in order, are A31114, A29843, A29845, A29846, A29847, A29848, A31110, A31111, A31112, A29450, and A31113. (B) Strains in A and A22361 (no GFP control) were grown for 24 h, and total protein was extracted. GFP protein levels were analyzed by Western blot analysis. Pgk1 was used as a loading control.

Figure 8.

Disomic yeast strains exhibit increased sensitivity to Huntingtin polyQ aggregates. (A) Wild-type (WT) and disomic strains harboring a GAL-Flag-HTT1(17AA)25QΔpro-CFP, GAL-Flag-HTT1(17AA)46QΔpro-CFP, or GAL-Flag-HTT1(17AA)72QΔpro-CFP construct were grown under conditions in which expression is repressed (YEPD) or induced (YPRG). Tenfold dilutions were plated. 25Q strains, in order, are A32114, A32115, A32116, A32117, A32118, A32119, and A32120. 46Q strains, in order, are A32121, A32122, A32123, A32124, A32125, A32126, A32127, and A32128. 72Q strains in order are A32129, A32130, A32131, A32132, A32133, A32134, and A32135. (B) Wild-type and disomic strains harboring the GAL-Flag-HTT1(17AA)46QΔpro-CFP construct were grown for 8 h in the presence of galactose to determine the percentage of cells with Htt1-46Q-CFP foci. n = 100. Shown are the mean and SEM of three independent experiments. (C) Expression of the GAL-HTT1(17aa)-Flag- nQ-CFP constructs. Strains were grown in YEP 2% raffinose to OD₆₀₀ = 0.2 when 2% galactose was added. RNA was extracted from samples taken after 2 h, and the amount of HTT1-nQ-CFP RNA was determined via Northern blot analysis.