, formed by the aggregated states of the cytosolic proteins Sup35, Rnq1, and Ure2, respectively (1). Sup35 is a translation termination factor; Ure2 is a regulator that acts to repress transcription of a set of genes involved in nitrogen catabolism; the function of Rnq1 is unknown. Prion proteins can form different conformational states resulting in prion strains having different heritable traits. For propagation in the cell population, physical transmission of the prion template, often referred to as the propagon or seed, is required to allow conversion of newly synthesized protein to the prion conformation (2).Somewhat paradoxically, the propagation of yeast prions appears to be inexorably reliant on the function of molecular chaperones, proteins that normally function to prevent protein misfolding (2). Two chaperone systems have been linked to prion propagation: the hexameric AAAϩ ATPase Hsp104 and the J-protein (Hsp40):Hsp70 chaperone machinery, with its associated nucleotide exchange factors (3). Hsp104, like its ortholog ClpB, functions in protein remodeling by threading partially folded proteins through its central pore and is stringently required for the propagation of all identified yeast prions (1,4,5).Hsp70s function with their obligate cochaperones, J-proteins, which act to stimulate Hsp70 ATPase activity and stabilize their interaction with client proteins (6). Although J-proteins are very diverse in sequence and structure, they possess a highly conserved J-domain that is responsible for the stimulation of the ATPase activity of Hsp70s. One cytosolic J-protein, Sis1, is required for propagation of [RNQ ϩ ] (7). In addition, multiple individual amino acid substitutions in the cytosolic Hsp70s Ssa1/2 that impair propagation of [PSI ϩ propagation implies an involvement of an unidentified J-protein as well.The currently favored model for prion propagation posits chaperone-mediated fragmentation of prion complexes to produce sufficient prion seeds to assure consistent transmission of seeds to daughter cells, thus maintaining the prion in the cell population (2,(10)(11)(12)(13)(14). Supporting this model, inhibition of Hsp104 activity results in an increase in the size of Sup35 and Rnq1 prion complexes and subsequent prion loss, which has been shown in the case of [PSI ϩ ] to be dependent on cell division (10,11,15,16). Additional support for this idea comes from reports of fragmentation of prion fibers in vitro by Hsp104 (17) and the apparent decrease in the number of [PSI ϩ ] prion seeds in cells expressing a dominant mutation in the Hsp70 SSA1 gene (8). An increase in the size of Rnq1 polymers, followed by [RNQ ϩ ] loss, also occurs upon depletion of Sis1, a partner of Ssa1 (15). Together these data suggest cooperation between the 2 chaperone systems. Such cooperation has precedent, as Hsp104 is known to function in disaggregation of amorphous protein aggregates in conjunction with J-protein:Hsp70 chaperone machinery, with J-protein/Hsp70 and Hsp104 machineries acting sequentially (4).The yeast cytos...

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.

No abstract