A – Polyglutamine constructs used in this work. All constructs were under the control of the galactose-inducible promoter (P_GAL), and contained the FLAG epitope, N-terminal 17 amino acid residues and poly-Q stretch of human Htt, and were fused to the gene coding for green fluorescent protein (GFP) at C-terminus. Numbers indicate length of poly-Q stretch. Poly-QP constructs also contained the proline-rich region of Htt (designated as P), immediately following the poly-Q stretch. B – Expanded poly-Q without a P-rich region (103Q), expressed under the P_GAL promoter on -Ura/Gal medium, is toxic in the presence of either [PIN⁺] or [PSI⁺] (or both), with two prions showing an additive effect. In contrast, expanded poly-Q with a P-rich region (103QP) is toxic only in the presence of [PSI⁺]. The 25Q construct, not exhibiting toxicity under these conditions, is shown as a control. The 25QP construct (not shown) behaved in the same way as 25Q. C – 103Q and 103QP form multiple peripheral aggregates and single aggregate (aggresome), respectively, in cells containing either or both prions ([PIN⁺] and/or [PSI⁺]), as visualized by fluorescence microscopy. Perinuclear location of aggresome (not shown) was confirmed by DAPI staining as described previously [42]. D – Overexpressed Sup35NM-DsRed (red) forms large clumps in the [PSI⁺] cells, that overlap with the 103QP-GFP aggresome (green), as pointed by arrows. E - Expression of 103Q or 103QP promotes aggregation of Sup35 in the [psi⁻] strain as seen by an increase of pellet (P) versus supernatant (S) fraction, in comparison to the respective strain expressing 25Q. Centrifugation analysis was followed by Western blotting and immunostaining with the Sup35 antibody. F - Expression of the Sup35 derivative, lacking the prion and middle domains (Sup35C), decreases 103Q and 103QP toxicity in the [PIN⁺ PSI⁺] strain but does not influence 103Q toxicity in the [PIN⁺ psi⁻] strain. SUP35C gene was under control of the endogenous SUP35 promoter. Serial decimal dilutions were spotted onto -Ura/Gal medium.

A and B – Typical aggregation patterns of 103Q (multiple dots, A) and 103QP (single clump, B) are not affected by AQT, as confirmed by fluorescence microscopy. C – AQT mutant ameliorates toxicity of excess Sup35 or Sup35N in the [PSI⁺] strain. Sup35 and Sup35N proteins were expressed from centromeric plasmid under control of the galactose-inducible promoter. Cells were grown on the -Ura/glucose medium selective for the plasmid for 1 day. Serial decimal dilutions were plated onto -Ura/Gal medium.

A - An extra copy of SUP45 gene, located on the centromeric plasmid under endogenous promoter, ameliorates toxicity of 103Q in the ubc4Δ [PSI⁺] strain, as seen from serial decimal dilutions plated onto the galactose medium selective for both poly-Q and SUP45 (or control) plasmids. B – Amelioration of [PSI⁺]-dependent polyglutamine toxicity by a plasmid-borne extra copy of SUP45 gene is detected for both endogenous (CEN-SUP45) galactose-inducible (GAL-SUP45) promoters, for both 103Q and 103QP constructs, and in both ubc4Δ and UBC4⁺ strains. Antitoxic effect of the plasmid-borne SUP45 gene in the ubc4Δ strain is comparable to antitoxic effect of AQT. Toxicity was scored on the galactose medium selective for both poly-Q and SUP45 (or control) plasmids. C and D – Centromeric plasmids with SUP45 gene under endogenous (C) or galactose-inducible P_GAL (D) promoters increase levels of Sup45 protein (Sup45p) both [UBC4⁺] and ubc4Δ strains. Cultures were grown in liquid -Ura -Leu glucose (C) or -Ura -Leu galactose/raffinose (D) medium. Ade2 (Ade2p) protein is shown as a loading control. E and F – Plasmids, expressing the SUP45 alleles with either C-terminal deletion, SUP45ΔC19 (that abolishes Sup45 function and interaction with Sup35) (E) or missense mutation sup45-103, T62C (that impairs Sup45 function) (F) from the endogenous SUP45 promoter, do not ameliorate 103Q and 103QP toxicity, as scored on the galactose medium selective for both plasmids.

A – Ubc4Δ has no significant effect on toxicity of 103Q or 103QP in the [PIN⁺ PSI⁺] background. Serial decimal dilutions were spotted onto -Ura/Gal medium. B – Papillae arise spontaneously in the ubc4Δ [PIN⁺ PSI⁺] strain expressing 103Q, and are able to stably maintain the anti-polyQ-toxic phenotype after colony purification. These papillae were designated as AQT. C – Comparison of the growth curves of [PIN⁺ PSI⁺] ubc4Δ strains that differ by polyglutamine constructs and by the presence or absence of AQT. Growth was measured by optical density at 600 nm in the liquid –Ura medium with galactose and raffinose instead of glucose. At least 3 independent cultures were characterized per each combination. Error bars represent standard deviations. D – AQT ameliorates 103QP toxicity. E – AQT is dominant (all strains are [PIN⁺ PSI⁺] and ubc4Δ homozygotes). F – Reintroduction of the UBC4 gene under galactose-inducible promoter on a multicopy plasmid partly suppresses but does not completely eliminate anti-toxic effect of AQT. -Ura/Gal plates are scored on panels D, E and F.

A – Tetrad analysis of a diploid obtained from mating of the AQT strain bearing the ubc4Δ::HIS3 transplacement, to the strain bearing the ubc4Δ::KanMX transplacement, demonstrates presence of at least 2 copies of the HIS3 gene versus one copy of the KanMX gene. This can be concluded from the fact that majority of tetrads produce more than 2 His⁺ spores, in contrast to the typically 2∶2 segregation by G418 resistance caused by KanMX. All AQT spores in this cross were His⁺ (not shown). B – Hybridization of total DNA to a complete DNA microarray of the S. cerevisiae genome confirms that all the coding material of chromosome II is duplicated in the AQT strain. Comparison is performed according to CLAC (CLuster Along Chromosome) consensus plot. For procedure, see Text S1. C – Sequential deletion mapping of the chromosome II extra copy in the AQT strain. The AQT#7 derivative (see Figure S2B) was used in these experiments. Each numbered region corresponds to a respective deletion. Deletions eliminating the antitoxicity phenotype in the [PSI⁺] background are shown as boxes filled in black. All deletions were verified by PCR. Five ORFs located within region 2.1a were each deleted individually; among those deletions, only deletion of SUP45 eliminated AQT as shown on panels B and C. D – Elimination of the antitoxic effect on 103Q and 103QP by the sup45 deletion in AQT strain. Serial decimal dilutions were spotted onto -Ura/Gal medium. E – Sup45 protein levels are elevated in the AQT strain, more profoundly in ubc4Δ background than in the presence of wild type UBC4 gene (UBC4⁺). Sup45p level is shown relative to the isogenic monosomic (non-AQT) control in each case. Ade2 protein was used as the loading control. At least 3 measurements with independent cultures were performed in each case. Error bars correspond to standard deviations. In each case the difference in Sup45 levels between the AQT and non-AQT strain is statistically significant as confidence limits do not overlap, and differences between the UBC4⁺ and ubc4Δ strains are statistically significant according to t-criterium (P_Ho<0.01).

A and B – Fractionation of the polyQ/QP-GFP (A) and Sup35 (B) polymers by sizes in the ubc4Δ [PSI⁺] strains either with (AQT) or without (WT) AQT. Polymers were separated by semi-denaturing agarose gel electrophoresis (SDD-AGE, see Text S1). Filter obtained from one and the same gel was reacted to either GFP (A) or Sup35C (B) antibodies. Polyglutamines alter distribution of Sup35 polymers, and this effect is counteracted by AQT. Experiment has been repeated with 3 independent cultures per each combination, each time with the same result. C – Expression of 103Q promotes aggregation of Sup45 protein (Sup45p) in the ubc4Δ [PIN⁺ psi⁻] strain, and expression of either 103Q or 103QP increases aggregate-associated fraction of Sup45 in the ubc4Δ [PSI⁺] strain, as detected by an increase in the pellet (P) versus supernatant (S) fraction in comparison to the respective strain expressing 25Q. Centrifugation was followed by Western blotting and reaction to the Sup45 antibody. D – Proportion of soluble (supernatant, S) versus aggregate-associated (pellet, P) Sup45 protein is significantly increased in AQT ubc4Δ [PSI⁺] strain, compared to the identical non-AQT (WT) strain, as determined by centrifugation analysis, followed by Western blotting and reaction to the Sup45 antibody.