J-protein co-chaperone Sis1 required for generation of [RNQ+] seeds necessary for prion propagation

Abstract

Yeast prions are protein-based genetic elements capable of self-perpetuation. One such prion, [RNQ(+)], requires the J-protein Sis1, an Ssa Hsp70 co-chaperone, as well as the AAA+ ATPase, Hsp104, for its propagation. We report that, upon depletion of Sis1, as well as upon inactivation of Hsp104, Rnq1 aggregates increased in size. Subsequently, cells having large aggregates, as well as an apparently soluble pool of Rnq1, became predominant in the cell population. Newly synthesized Rnq1 localized to both aggregates and bulk cytosol, suggesting that nascent Rnq1 partitioned into pools of prion and nonprion conformations, and implying that these large aggregates were still active as seeds. Ultimately, soluble Rnq1 predominated, and the prion was lost from the population. Our data suggest a model in which J-protein:Hsp70 machinery functions in prion propagation, in conjunction with Hsp104. Together, these chaperones facilitate fragmentation of prion polymers, generating a sufficient number of seeds to allow efficient conversion of newly synthesized Rnq1 into the prion conformation.

Chaperone-dependent amyloid assembly protects cells from prion toxicity

“…This library includes, among many other chaperones, Ydj1, which is a member of the large Hsp40 family that is closely related to Sis1. It also includes Hsp70 Ssa1 (Hsp70) and Hsp104, which assist in shearing [RNQ ϩ ] prions to form seeds required for the propagation of the [RNQ ϩ ] state (28). Therefore, the effect of Sis1 on the toxicity of Rnq1 overexpression is unique.…”

The ribosome-associated complex antagonizes prion formation in yeast

“…We thus assessed the fitness consequences of ectopically expressing 3 different aggregation-prone sequences in wildtype and Dzuo1 cells: polyglutamine (62Q), 55 the Sup35 NM domains, or the Rnq1 protein 51 (Fig. 4A) of the aggregation-prone proteins (not shown), and thus the lack of toxicity we observed may simply reflect lower expression levels under the growth conditions we used.…”

“…We induced moderate Rnq1-GFP expression for 90 minutes and then examined the cells by fluorescence confocal microscopy ( to incomplete curing (similar to the "diffuseCdot" cells described previously). 51 To address the latter possibility, we unequivocally eliminated [RNQ C ] by deleting the Rnq1 gene in wildtype and Dzuo1 strains, then treated the Drnq1 cells with GuHCl to eliminate any other prions that may have been present. We then transformed the Drnq1 cells with the plasmid expressing Rnq1-GFP, induced moderate Rnq1-GFP expression for 90 minutes and then examined the cells by fluorescence confocal microscopy (Fig.…”

“…Plasmids pRS426 Cup1pr-Sup35NM-GFP, 55 pRS426 Cup1pr-62Q-M-GFP 55 and pRS316 Cup1pr-RNQ1-GFP 51 were generous gifts from J. Weissman and J. Hines, and have been described previously.…”

“…51,55 Dilutions of overnight cultures were plated onto medium lacking uracil and supplemented with 100 mM CuSO 4 (Sigma-Aldrich). Plates were incubated at 30 C for 2-4 d and then photographed.…”