, 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...

Tyrosinase is a melanocyte-specific enzyme critical for the synthesis of melanin, a process normally restricted to a post-Golgi compartment termed the melanosome. Loss-of-function mutations in tyrosinase are the cause of oculocutaneous albinism, demonstrating the importance of the enzyme in pigmentation. In the present study, we explored the possibility that trafficking of albino tyrosinase from the endoplasmic reticulum (ER) to the Golgi apparatus and beyond is disrupted. Toward this end, we analyzed the common albino mouse mutation Tyr(C85S), the frequent human albino substitution TYR(T373K), and the temperature-sensitive tyrosinase TYR(R402Q)͞Tyr(H402A) found in humans and mice, respectively. Intracellular localization was monitored in albino melanocytes carrying the native mutation, as well as in melanocytes ectopically expressing green fluorescent protein-tagged tyrosinase. Enzymatic characterization of complex glycans and immunofluorescence colocalization with organelle-specific resident proteins established that all four mutations produced defective proteins that were retained in the ER. TYR(R402Q)͞Tyr(H402A) Golgi processing and transport to melanosomes were promoted at the permissive temperature of 32°C, but not at the nonpermissive 37°C temperature. Furthermore, evidence of protein misfolding was demonstrated by the prolonged association of tyrosinase mutants with calnexin and calreticulin, known ER chaperones that play a key role in the quality-control processes of the secretory pathway. From these results we concluded that albinism, at least in part, is an ER retention disease.calnexin ͉ protein folding ͉ quality control

Sis1 and Ydj1, functionally distinct heat shock protein (Hsp)40 molecular chaperones of the yeast cytosol, are homologs of Hdj1 and Hdj2 of mammalian cells, respectively. Sis1 is necessary for propagation of the Saccharomyces cerevisiae prion [RNQ + ]; Ydj1 is not. The ability to function in [RNQ + ] maintenance has been conserved, because Hdj1 can function to maintain Rnq1 in an aggregated form in place of Sis1, but Hdj2 cannot. An extended glycine-rich region of Sis1, composed of a region rich in phenylalanine residues (G/F) and another rich in methionine residues (G/M), is critical for prion maintenance. Single amino acid alterations in a short stretch of amino acids of the G/F region of Sis1 that are absent in the otherwise highly conserved G/F region of Ydj1 cause defects in prion maintenance. However, there is some functional redundancy within the glycine-rich regions of Sis1, because a deletion of the adjacent glycine/methionine (G/M) region was somewhat defective in propagation of [RNQ + ] as well. These results are consistent with a model in which the glycine-rich regions of Hsp40s contain specific determinants of function manifested through interaction with Hsp70s.

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.