The Pub1 and Upf1 Proteins Act in Concert to Protect Yeast from Toxicity of the [PSI⁺] Prion

Abstract

The [PSI⁺] nonsense-suppressor determinant of Saccharomyces cerevisiae is based on the formation of heritable amyloids of the Sup35 (eRF3) translation termination factor. [PSI⁺] amyloids have variants differing in amyloid structure and in the strength of the suppressor phenotype. The appearance of [PSI⁺], its propagation and manifestation depend primarily on chaperones. Besides chaperones, the Upf1/2/3, Siw14 and Arg82 proteins restrict [PSI⁺] formation, while Sla2 can prevent [PSI⁺] toxicity. Here, we identify two more non-chaperone proteins involved in [PSI⁺] detoxification. We show that simultaneous lack of the Pub1 and Upf1 proteins is lethal to cells harboring [PSI⁺] variants with a strong, but not with a weak, suppressor phenotype. This lethality is caused by excessive depletion of the Sup45 (eRF1) termination factor due to its sequestration into Sup35 polymers. We also show that Pub1 acts to restrict excessive Sup35 prion polymerization, while Upf1 interferes with Sup45 binding to Sup35 polymers. These data allow consideration of the Pub1 and Upf1 proteins as a novel [PSI⁺] detoxification system.

Keywords: Pub1; Saccharomyces cerevisiae; Sup35; Sup45; Upf1; [PSI+] prion toxicity; translation termination factors.

Figure 1

Plasmid-encoded Pub1∆C slightly compensates the effect of chromosomal pub1-∆ on prion polymerization of Sup35 in [PSI⁺]_S7 cells and alleviates the synthetic lethal interaction between pub1-Δ, upf1-Δ and [PSI⁺]_S7. (a) SDD-AGE analysis of polymerized Sup35 in transformants of the 74-D694 [PSI⁺]_S7 strain, with PUB1 deletion carrying multicopy plasmids encoding wild-type Pub1 (PUB1), Pub1∆C (pub1-∆C), or empty vector (pub1-∆). The transformants were grown in liquid Sc-Ura medium selective for the plasmid marker. Blots were probed with the polyclonal antibody against Sup35NM. Equal amounts of total protein from the compared cell lysates were serially diluted in two-fold decrements. Undiluted samples contained ~180 µg of total protein per lane. Four independent transformants of each type were analyzed and representative blot images are presented. Abundances of polymerized Sup35 (percent of the level in the pub1-∆ strain ± SEM) were calculated after densitometry of blots and are shown on the histograms. Statistically significant differences of polymerized Sup35 in compared transformants (marked by trapezoid), determined by Student’s t-test, are indicated by asterisks (* p < 0.05). (b) Growth of the transformants on the solid SC-Ura medium. The transformants of the pub1-Δ upf1-Δ [PSI⁺]_S7 strain with multicopy YEplac195-PUB1 (Multi-PUB1), YEplac195-PUB1ΔC (Multi-pub1-ΔC) and centromeric pRS316-PUB1 (CEN-PUB1), pRS316-PUB1ΔC (CEN-pub1-ΔC) plasmids were grown in liquid SC-Ura medium and after 12 h incubation, cell suspensions were diluted to an OD₆₀₀ of 0.3, spotted onto plates with the same medium and incubated for four days at 30 °C. Four serial three-fold dilutions of cell suspensions are shown.

Figure 2

Deletion of PUB1 slightly increases the levels of Sup35 polymers in cells with weak [PSI⁺]. SDD-AGE analysis of polymerized Sup35: The strains were grown in liquid YPD medium; blots were probed with the polyclonal antibody against Sup35NM; equal amounts of total protein from compared cell lysates were serially diluted in twofold decrements; undiluted samples contained ~180 µg of total protein per lane. Four clones of [PSI⁺]_WS2 and [PSI⁺]_W2 derivatives of the 74-D694 strain with deleted (pub1-Δ) or without PUB1 deletion (PUB1) grown in liquid YPD were studied, and the abundance of Sup35 polymers in pub1-Δ and PUB1 strains was calculated as described in the legend to Figure 1 and shown as the percent of the level in the strain bearing wild-type PUB1± SEM in the histograms. A statistically significant increase in the amount of Sup35 polymers caused by pub1-Δ (determined by Student’s t-test) and indicated by an asterisk (*) was observed for the strain with [PSI⁺]_WS2 (p < 0.05), but not with [PSI⁺]_W2 (p > 0.3). Typical blot images are presented.

Figure 3

Deletion of UPF1 but not of PUB1, UPF2 or UPF3 increases Sup45 aggregation in cells with [PSI⁺]_S7. Cell lysates were fractionated by ultracentrifugation as described in Materials and Methods. Samples were loaded onto the gel in a volume corresponding to equal amounts of starting lysates, which contained ~200 µg of soluble protein. Blots were probed with polyclonal antibody against Sup45 and levels of Sup45 in fractions were determined by densitometric analysis of blots. Four clones of each strain were analyzed. S, soluble fraction; P, pellet. The relative abundances of Sup45 in these fractions estimated by densitometry of blots, were calculated as ratios of its signal intensity in the pellet fraction versus the sum of signal intensities in the pellet and soluble fractions and is shown on the histograms as the percent of the level in the strain wild-type for analyzed gene. Typical blot images are presented. The statistical significance of differences in the amount of aggregated Sup45 in compared strains was estimated by Student’s t-test. (a) Deletion of PUB1 (pub1-Δ) does not cause a statistically significant increase in the amount of aggregated Sup45 (p > 0.08), (b) deletion of UPF1 (upf1-Δ) causes a statistically significant (p < 0.001) increase of the amount of aggregated Sup45 in cells with [PSI⁺]_S7, indicated by two asterisks (**), but not in cells with either [PSI⁺]_WS2 (p > 0.2) or [PSI⁺]_W2 (p > 0.7), (c) simultaneous deletion of UPF2 (upf2-Δ) and UPF3 (upf3-Δ) in [PSI⁺]_S7 cells does not influence Sup45 aggregation (p > 0.6).

Figure 4

Schematic representation of the suggested mechanism mediating the effect of Upf1 on binding of Sup45 to polymeric Sup35. Upf1 and Sup45 interact with monomeric Sup35 independently. Sup45 binds to Sup35 involved in a polymer only if Sup35 is not bound to Upf1.