Role for Hsp70 Chaperone in Saccharomyces cerevisiae Prion Seed Replication

Effects of Ubiquitin System Alterations on the Formation and Loss of a Yeast Prion

“…Ssa protein has been implicated in disassembly of the large Sup35 aggregates into the smaller prion-propagating units (56), and our preliminary data suggest that Ssa can bind at least some cytologically detectable Sup35-GFP aggregates in the yeast cells. 5 (32).…”

The NatA Acetyltransferase Couples Sup35 Prion Complexes to the [PSI+] Phenotype

“…First, our expression profiling, genetic, and biochemical analyses indicate that Hsp104 and Hsp70 are neither the direct nor indirect targets of this pathway (Supplemental Figure S4 and Figure 4, B and C). Second, strains with impaired Hsp104 or Hsp70 activity are defective in the generation of new propagons (Ness et al, 2002;Song et al, 2005), whereas NatA mutants sustain wild-type propagon levels (Supplemental Figure S6). Third, the steady-state size of SDS-resistant Sup35 aggregates increases in [PSI ϩ ] strains deficient in Hsp104, Hsp70, or Hsp70 cochaperones (Eaglestone et al, 2000;Wegrzyn et al, 2001;Cox et al, 2003;Kryndushkin et al, 2003;Jones et al, 2004;Song et al, 2005;Fan et al, 2007;Kryndushkin and Wickner, 2007;SatputeKrishnan et al, 2007;Sadlish et al, 2008), an observation that is completely opposed to our findings for NatA mutants ( Figure 5C).…”

“…Second, strains with impaired Hsp104 or Hsp70 activity are defective in the generation of new propagons (Ness et al, 2002;Song et al, 2005), whereas NatA mutants sustain wild-type propagon levels (Supplemental Figure S6). Third, the steady-state size of SDS-resistant Sup35 aggregates increases in [PSI ϩ ] strains deficient in Hsp104, Hsp70, or Hsp70 cochaperones (Eaglestone et al, 2000;Wegrzyn et al, 2001;Cox et al, 2003;Kryndushkin et al, 2003;Jones et al, 2004;Song et al, 2005;Fan et al, 2007;Kryndushkin and Wickner, 2007;SatputeKrishnan et al, 2007;Sadlish et al, 2008), an observation that is completely opposed to our findings for NatA mutants ( Figure 5C). In addition to these factors, the ubiquitin-conjugating enzyme Ubc4 and the Hsp70 family members Ssb1 and Ssb2 are predicted or proven substrates for NatA (Huang et al, 1987;Polevoda et al, 1999), are modifiers of the [PSI ϩ ] prion cycle Allen et al, 2007), and have demonstrated synthetic interactions with NatA (Gautschi et al, 2003;Pan et al, 2006).…”

“…To rule out this possibility, we monitored the mobility of Sup35-GFP in wild-type and NatA mutant strains using FRAP experiments, which are capable of quantitatively detecting soluble Sup35-GFP in individual cells containing a mixture of protein forms (Satpute-Krishnan et al, 2007). When a [psi Ϫ ] cell expressing Sup35-GFP is mated to an unmarked [psi Ϫ ] cell and the resulting zygote is analyzed by FRAP, fluorescence recovery in the bleached area is rapid, with a half-time (t 1/2 ) ϳ0.7 s. However, when the same Sup35-GFP expressing [psi Ϫ ] strain is mated to an unmarked wild-type [PSI ϩ ] strain, fluorescence recovery is slowed (t 1/2 ϳ3.1 s) because of the incorporation of soluble Sup35-GFP into prion complexes (Song et al, 2005;Satpute-Krishnan et al, 2007). When a [psi Ϫ ] strain expressing Sup35-GFP was mated to an unmarked NatA mutant [PSI ϩ ] strain, the rate of fluorescence recovery by FRAP was indistinguishable from the wild-type [psi Ϫ ] ϫ [PSI ϩ ] cross (t 1/2 ϳ2.7 s, Figure 5E), consistent with the rapid incorporation of soluble Sup35 in this cross ( Figure 5A).…”

“…In strains lacking NatA function, heritable Sup35 prion complexes are uncoupled from their phenotypic effects. In contrast to other known modulators of [PSI ϩ ] propagation that appear to act by altering prion complex dynamics (Jung et al, 2000;Wegrzyn et al, 2001;Kryndushkin et al, 2003;Song et al, 2005;Satpute-Krishnan et al, 2007), NatA null strains efficiently incorporate soluble Sup35 into existing prion complexes, but the nature of this interaction is altered, allowing faithful translation termination. …”