The Role of Sse1 in the de Novo Formation and Variant Determination of the [PSI+] Prion

Abstract

Yeast prions are a group of non-Mendelian genetic elements transmitted as altered and self-propagating conformations. Extensive studies in the last decade have provided valuable information on the mechanisms responsible for yeast prion propagation. How yeast prions are formed de novo and what cellular factors are required for determining prion ''strains'' or variants-a single polypeptide capable of existing in multiple conformations to result in distinct heritable phenotypes-continue to defy our understanding. We report here that Sse1, the yeast ortholog of the mammalian heat-shock protein 110 (Hsp110) and a nucleotide exchange factor for Hsp70 proteins, plays an important role in regulating ½PSI

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).…”

“…Other possible targets include Hsp104, Hsp70, and cochaperones that modulate the ATPase activity of Hsp70. Mutations in all of these factors produce a similar reversion of the [PSI ϩ ] phenotype (Chernoff et al, 1995;Jones and Masison, 2003;Fan et al, 2007;Kryndushkin and Wickner, 2007;Sadlish et al, 2008), but several lines of evidence suggest that these proteins are not the crucial target(s) of NatA. 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).…”

“…Much of our mechanistic understanding of the Sup35/ [PSI ϩ ] in vivo prion cycle has its origins in a variety of genetic screens, which identified key modulators of this process. Although a number of mutagenic (Young and Cox, 1971;Jung et al, 2000), overexpression (Chernoff et al, 1993;Chernoff et al, 1995;Kryndushkin et al, 2002), and interaction screens (Bailleul et al, 1999) have been conducted to date, additional factors impacting prion propagation continue to be identified by candidate gene approaches (Chernoff et al, , 2003Newman et al, 1999;Ganusova et al, 2006;Park et al, 2006;Fan et al, 2007;Kryndushkin and Wickner, 2007;Sadlish et al, 2008), suggesting that our current understanding of prion cycle regulation in vivo is incomplete.In an attempt to elucidate additional factors and processes governing efficient prion propagation in vivo, we took advantage of the fact that N-terminal acetylation is critical for the normal function of many proteins (Polevoda and Sherman, 2003). Approximately 50% of the yeast proteome is cotranslationally acetylated on its N-terminus by the collective action of three enzyme complexes: NatA, NatB, and NatC (Polevoda and Sherman, 2003).…”

Cytoplasmic expression of mouse prion protein causes severe toxicity in Caenorhabditis elegans

“…Crude protein extracts prepared from C. elegans expressing MoPrP(23-231)-CFP, MoPrP (Q167R)(23-231)-CFP, and MoPrP(23-231)-CFP and MoPrP(Q167R)(23-231)-YFP were treated with the Sarkosyl sample buffer (50 mM Tris-HCl (pH 6.8), 5% glycerol, 2% Sarkosyl, and 0.05% bromophenol blue) at room temperature for 7 min and separated on 1.5% agarose gels supplemented with 0.1% SDS as described [12]. After transferring to a polyvinylidene difluoride membrane (Millipore), membranes were probed with anti-PrP antibody (3F4) and detected with ECL kit (Amersham).…”

Hierarchical Functional Specificity of Cytosolic Heat Shock Protein 70 (Hsp70) Nucleotide Exchange Factors in Yeast

“…Functionally, Sse1 and Fes1 have both been shown to be involved in prion formation and curing, because Sse1 is required for [PSIϩ] propagation, and deletion of either SSE1 or FES1 blocks [URE3] propagation (35,36). Sse1 has been implicated in Hsp70-mediated protein folding at the ribosome, Hsp90 chaperoning of signal transduction, and post-translational translocation of pre-pro ␣-factor (21,22,37,38).…”