The ribosome-associated complex antagonizes prion formation in yeast

Abstract

ABSTRACT. The number of known fungal proteins capable of switching between alternative stable conformations is steadily increasing, suggesting that a prion-like mechanism may be broadly utilized as a means to propagate altered cellular states. To gain insight into the mechanisms by which cells regulate prion formation and toxicity we examined the role of the yeast ribosome-associated complex (RAC) in modulating both the formation of the [PSI C ] prion -an alternative conformer of Sup35 protein -and the toxicity of aggregation-prone polypeptides. The Hsp40 RAC chaperone Zuo1 anchors the RAC to ribosomes and stimulates the ATPase activity of the Hsp70 chaperone Ssb. We found that cells lacking Zuo1 are sensitive to over-expression of some aggregation-prone proteins, including the Sup35 prion domain, suggesting that co-translational protein misfolding increases in Dzuo1 strains. Consistent with this finding, Dzuo1 cells exhibit higher frequencies of spontaneous and induced prion formation. Cells expressing mutant forms of Zuo1 lacking either a C-terminal charged region required for ribosome association, or the J-domain responsible for Ssb ATPase stimulation, exhibit similarly high frequencies of prion formation. Our findings are consistent with a role for the RAC in chaperoning nascent Sup35 to regulate folding of the N-terminal prion domain as it emerges from the ribosome.

Prion-Associated Toxicity is Rescued by Elimination of Cotranslational Chaperones

“…However, we have demonstrated that Ssb deletion is toxic in the presence of Sup35 overexpression, indicating that chaperone function is necessary. Further, perturbations that prevent Ssb association with the ribosome have been shown to enhance yeast sensitivity to Sup35 overexpression [36]. We suggest that the toxicity rescue observed in the NAC deletion strains is due to a shift in Ssb activity to nascent proteins.…”

Prion-dependent proteome remodeling in response to environmental stress is modulated by prion variant and genetic background

“…Plates were incubated at 30°C for 3 days (without ZnCl 2 ) or 4 days (with ZnCl 2 ) and then photographed. Colony area (pixels^2) for 161-326 isolated, single colonies was measured as previously described [48] [49].…”

“…Cells to be transformed were first grown sequentially (three times) on agar medium supplemented with 5 mM guanidine hydrochloride (ACROS Organics) to eliminate [PSI + ] and [RNQ + ] prions (elimination of [RNQ + ] by this treatment in these strains has been previously confirmed by lack of visible GFP foci in cells over-expressing Rnq1-GFP [48]; and our unpublished data). Cells were then treated with 100U of lyticase (Sigma) in 1 M sorbitol + 10 mM Tris pH 7.5 at 30°C for 1 hour to generate spheroplasts.…”

“…Relative protein abundance was determined by flow cytometry of strains expressing proteins with C-terminal GFP fusions as described previously [48], with the exception that cells were harvested from SD agar plates (with or without 5 mM ZnCl 2 ) grown for 3 days at 30°C. For each strain, GFP fluorescence intensity was measured for 50,000 cells using a BD FACS Canto II flow cytometer and analyzed using the FITC 'area' parameter.…”

Combined tRNA modification defects impair protein homeostasis and synthesis of the yeast prion protein Rnq1

“…Interestingly, however, different defects of the ribosome associated chaperone complex (RAC) induced in zuo1, ssz1 and ssb1 ssb2 mutants all increased de novo generation of the [PSI C ] prion. [24][25][26] This may suggest that interference with translation at the level of tRNA functionality might impact on the tendency to form spontaneous amyloid aggregates as well, since similarities in protein homeostasis defects with the RAC mutants exist. However, dual tRNA modification loss affecting tRNA …”