RESUMO
Prions in Saccharomyces cerevisiae are inherited ordered aggregates reliant upon the disaggregase Hsp104 for stable maintenance. The function of other factors in the natural prion cycle is unclear. We constructed yeast-bacterial chimeric chaperones to resolve the roles of Hsp104 domains, and by extension chaperones that interact with these domains, in prion propagation. Our results show that, as with amorphous aggregate dissolution, the Hsp70/40 system recruits prion substrates to Hsp104 via its top ring. By adapting our chimera to couple to an inactive protease "trap," we monitored the reaction products of prion propagation in vivo. We find that prion maintenance is accompanied by translocation of prion proteins through Hsp104 hexamers and that both processes critically rely upon the Hsp40 Sis1. Our data suggest that yeast prion replication is a natural extension of chaperone activity in dissolving amorphous aggregates, distinguished from its ancestral reaction by the ordered, self-propagating structure of the substrate.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Replicação do DNA , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Modelos Biológicos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Príons/química , Príons/genética , Ligação Proteica/genética , Conformação Proteica , Estrutura Terciária de Proteína/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidade por SubstratoRESUMO
Prion proteins can adopt multiple infectious strain conformations. Here we investigate how the sequence of a prion protein affects its capacity to propagate specific conformations by exploiting our ability to create two distinct infectious conformations of the yeast [PSI(+)] prion protein Sup35, termed Sc4 and Sc37. PNM2, a G58D point mutant of Sup35 that was originally identified for its dominant interference with prion propagation, leads to rapid, recessive loss of Sc4 but does not interfere with propagation of Sc37. PNM2 destabilizes the amyloid core of Sc37 and causes compensatory effects that slow prion growth but aid prion division and result in robust propagation of Sc37. By contrast, PNM2 does not affect the structure or chaperone-mediated division of Sc4 but interferes with its delivery to daughter cells. Thus, effective delivery of infectious particles during cell division is a crucial and conformation-dependent step in prion inheritance.