Guanidine hydrochloride blocks a critical step in the propagation of the prion-like determinant [ PSI ⁺ ] of Saccharomyces cerevisiae

Abstract

The cytoplasmic heritable determinant [PSI ؉ ] of the yeast Saccharomyces cerevisiae reflects the prion-like properties of the chromosome-encoded protein Sup35p. This protein is known to be an essential eukaryote polypeptide release factor, namely eRF3. In a [PSI ؉ ] background, the prion conformer of Sup35p forms large oligomers, which results in the intracellular depletion of functional release factor and hence inefficient translation termination. We have investigated the process by which the [ curing ͉ Sup35p ͉ cytoplasmic determinant R ecent biochemical evidence (1-5) has supported the hypothesis that the [PSI ϩ ] phenotype of Saccharomyces cerevisiae reflects the prion-like properties of the SUP35 gene product (6, 7). The essential chromosome-encoded protein Sup35p is known to be one of two eukaryote polypeptide release factors, namely eRF3 (8, 9). Sup35p associates with Sup45p (eRF1) in vivo to mediate translation termination (8). In vitro, Sup35p forms highly ordered fibers, whose appearance resembles that of fibrils formed by other amyloidogenic polypeptides (3,4 , a second prion determinant of S. cerevisiae (7). Other reagents including methanol, ethylene glycol, and hypertonic conditions, have been reported to exhibit curing properties; however, none of these cure with the neartotal efficiency of GuHCl (13,14).Two hypotheses have been proposed to account for the curing properties of GuHCl. First, the elimination of the prion might arise directly from the ability of GuHCl to denature proteins. However, the concentrations of GuHCl effective in curing [PSI ϩ ] are in the millimolar range, rather than the molar range typically required for the denaturation of proteins in vitro (13). Alternatively, GuHCl may actually promote the expression of an ancillary factor, namely the stress protein Hsp104p, which indirectly results in the reactivation of Sup35p and consequently the loss of the prion (15). Unlike other heat shock proteins, Hsp104p does not act to protect proteins against stress (i.e., heat denaturation), rather Hsp104p actively promotes the recovery of stress-denatured aggregated proteins by facilitating their refolding back into functional, native conformations (16,17). Overexpression of Hsp104p might lead to the total refolding of Sup35p from the aberrant prion conformation to its native structure, thereby mediating prion loss. To test both hypotheses, we have examined the kinetics of prion elimination upon growth in the presence of GuHCl and assessed the influence of stress on the curing process. Materials and MethodsStrain. The genotype of the strain used in this study was BSC783͞4a: , MATa. Growth Media. BSC783͞4a was grown at 30°C on 1 ⁄4YEPD solid medium [4% (wt͞vol) glucose, 1% (wt͞vol) Bacto-peptone, 0.25% (wt͞vol) yeast extract, 2% (wt͞vol) agar]. Most liquid cultures also were grown at 30°C in YEPD complete medium [2% (wt͞vol) glucose, 1% (wt͞vol) Bacto-peptone, 1% (wt͞vol) yeast extract], with or without 3 mM GuHCl. For studies using ethanol-supplemented media, strains were grown in f...

Curing of yeast [PSI + ] prion by guanidine inactivation of Hsp104 does not require cell division

“…Because Gdn inactivates Hsp104 immediately, kinetics of [PSI ϩ ] curing after this inactivation can be monitored straightforwardly. When growing cells are exposed to Gdn there is a lag phase during which no curing occurs followed by a linear increase in the number of cured cells over time (25).These kinetic studies have supported the model that Hsp104 acts by breaking up prion polymers, thereby generating new prion ''seeds.'' During the lag phase that occurs after Hsp104 activity is inhibited, the prion polymers or seeds are thought to grow in size but, failing to replicate, become diluted among the increasing number of dividing cells.…”

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

“…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).…”

“…In the presence of GdnHCl, propagon replication is inhibited, and the templates existing in a given cell are redistributed to daughter cells upon division. Under these conditions, [PSI ϩ ] cultures begin to switch to the [psi Ϫ ] state after a lag phase, the length of which is directly related to the number of propagons (Eaglestone et al, 2000). On treatment with GdnHCl, wild-type and NatA mutant [PSI ϩ ] strains were cured of the prion state after a similar lag time (Supplemental Figure S6), suggesting that the number of prion templates is not affected by NatA function.…”

“…At the indicated generation number (as determined by optical density measurements at 600 nm), aliquots of the cultures were plated on solid YPD media, and the percentage of [PSI ϩ ] colonies was determined by their color phenotype as previously described (Eaglestone et al, 2000). Sectored colonies were scored as [PSI ϩ ].…”

The yeast prion protein Ure2: Structure, function and folding

“…In particular, the three best characterized S. cerevisiae prions, Sup35, Ure2 and Rnq1, all require normal expression levels of the chaperone Hsp104 in order to be propagated efficiently [13,77,78]. In the case of Sup35, inhibition of the ATPase activity of Hsp104 similarly interferes with prion propagation [79][80][81] and overexpression of Hsp104 also results in curing of the prion phenotype [77]. To account for these observations, it is proposed that Hsp104 is required to produce new seeds from existing prion aggregates; while [72]; and the mutants K127E or V271E when combined with point mutations in the N-terminus (see text) [73].…”