Single methyl group determines prion propagation and protein degradation activities of yeast heat shock protein (Hsp)-70 chaperones Ssa1p and Ssa2p

Abstract

Organisms encode multiple homologous heat shock protein (Hsp)-70s, which are essential protein chaperones that play the major role in cellular protein "quality control." Although Hsp70s are functionally redundant and highly homologous, many possess distinct functions. A regulatory motif underlying such distinctions, however, is unknown. The 98% identical cytoplasmic Hsp70s Ssa1p and Ssa2p function differently with regard to propagation of yeast [URE3] prions and in the vacuolar-mediated degradation of gluconeogenesis enzymes, such as FBPase. Here, we show that the Hsp70 nucleotide binding domain (NBD) regulates these functional specificities. We find little difference in ATPase, protein refolding, and amyloid inhibiting activities of purified Ssa1p and Ssa2p, but show that interchanging NBD residue alanine 83 (Ssa1p) and glycine 83 (Ssa2p) switched functions of Ssa1p and Ssa2p in [URE3] propagation and FBPase degradation. Disrupting the degradation pathway did not affect prion propagation, however, indicating these are two distinct processes where Ssa1/2p chaperones function differently. Our results suggest that the primary evolutionary pressure for Hsp70 functional distinctions is not to specify interactions of Hsp70 with substrate, but to specify the regulation of this activity. Our data suggest a rationale for maintaining multiple Hsp70s and suggest that subtle differences among Hsp70s evolved to provide functional specificity without affecting overall enzymatic activity.

Prions in Yeast

“…Mutation in SSA1 was shown to antagonize [PSI + ] propagation (Jung et al 2000), and deletion of SSA2, responsible for the major fraction of Ssa in exponentially growing cells, destabilizes a weak [PSI + ] variant (Newnam et al 2011 (Schwimmer and Masison 2002), with a single amino acid change at position 83 being responsible for these differences (Sharma and Masison 2011). Mutation in SSA2 also antagonizes [URE3] (Roberts et al 2004).…”

Biology of the Heat Shock Response and Protein Chaperones: Budding Yeast (Saccharomyces cerevisiae) as a Model System

“…Fascinatingly, despite the high degree of sequence conservation between Ssa1 and Ssa2, only Ssa1 is competent to mediate FBPase degradation. A recent study narrowed the cause for this specificity to a single residue in the ATPase domain, alanine 83 (417). Substitution with glycine (the analogous residue in Ssa2) blocked Ssa1 functions in this biological process, and conversely, the replacement of the glycine with alanine in Ssa2 allowed this chaperone to complement the transport defects in the ssa1⌬ null strain, demonstrating that a methyl group determines the functional delineation between Ssa1 and Ssa2 in this pathway.…”

Cadmium and Secondary Structure-dependent Function of a Degron in the Pca1p Cadmium Exporter

“…There is also mounting evidence for the role of Hsp70p chaperones in the degradation of proteins, including several ERAD substrates (18,(27)(28)(29), which can at least partially resolve how the substrates of degradation pathways are initially recognized.…”