Divergent Functional Properties of the Ribosome-Associated Molecular Chaperone Ssb Compared with Other Hsp70s

Abstract

Ssbs of Saccharomyces cerevisiae are ribosome-associated molecular chaperones, which can be cross-linked to nascent polypeptide chains. Because Ssbs are members of a divergent subclass of Hsp70s found thus far only in fungi, we asked if the structural requirements for in vivo function were similar to those of “classic” Hsp70s. An intact peptide-binding domain is essential and an alteration of a conserved residue in the peptide-binding cleft (V442) affects function. However, Ssb tolerates a number of alterations in the peptide-binding cleft, revealing a high degree of flexibility in its functional requirements. Because binding of Ssb to peptide substrates in vitro was undetectable, we assessed the importance of substrate binding using the chimera BAB, in which the peptide binding domain of Ssb is exchanged for the analogous domain of the more “classical” Hsp70, Ssa. BAB, which binds peptide substrates in vitro, can substitute for Ssb in vivo. Alteration of a residue in the peptide-binding cleft of BAB creates a protein with a reduced affinity for peptide and altered ribosome binding that is unable to substitute for Ssb in vivo. These results indicate that Ssb's ability to bind unfolded polypeptides is likely critical for its function. This binding accounts, in part, for its stable interaction with translating ribosomes, even although it has a low affinity for peptides that detectably bind to other Hsp70s in vitro. These unusual properties may allow Ssb to function efficiently as a chaperone for ribosome-bound nascent chains.

The in vivo function of the ribosome-associated Hsp70, Ssz1, does not require its putative peptide-binding domain

“…All yeast strains used are isogenic with DS10 (his3-11, 15 leu2-3,112 lys1 lys2 ⌬trp1 ura3-52) (19), with the exception mutations of the genes encoding SSB1, SSB2, SSZ1, and͞or ZUO1. Deletions of ZUO1, SSB1, and SSB2 have been described (15,19). A deletion of SSZ1 was made in which the sequence between Ϫ102 and ϩ1,617 was replaced with the LYS2 gene.…”

“…A BamHI-SalI fragment containing the entire coding region of SSB1 was obtained from a plasmid containing a SSB1 gene having a BamHI site immediately upstream of the initiating ATG (19) and cloned into the same sites of p416CYC1. The CYC-SSZ1 plasmid was created by introducing BamHI and SalI sites by using PCR 100 nucleotides upstream and at the end of the SSZ1 gene, respectively, and cloning into the same sites of both the p416CYC1 and p414CYC1 plasmids.…”

“…Ssb can be crosslinked to ribosome-associated nascent polypeptides, suggesting that it acts as a chaperone of these newly synthesized polypeptides (18). Consistent with this idea, Ssb lacking the peptide-binding domain is nonfunctional (19). Ssz, which is encoded by a single gene, SSZ1, is less well studied.…”

Quantitative proteomics and network analysis of SSA1 and SSB1 deletion mutants reveals robustness of chaperone HSP70 network in Saccharomyces cerevisiae

“…The HSP70 chaperone family, including SSA1/SSA2 and SSB1/SSB2 proteins, has been extensively studied in terms of evolutionary similarity 14, 15, 16 and functional similarity/redundancy via knock‐out models 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27. For example, Craig et al.…”

Activation of futile cycles as an approach to increase ethanol yield during glucose fermentation inSaccharomyces cerevisiae

“…The isolated 44 kDa ATPase domain was shown to have higher affinity to ATP with increased rate of reaction compared with the full length protein. 9 ATP-diphosphohydrolases (apyrases) ( EC 3.6.1.5), are enzymes that hydrolyze both the g-and b-phosphates of ATP and ADP. They are distinct from other phosphohydrolases with respect to their specific activity, nucleotide substrate specificity, divalent cation requirement, and sensitivity to inhibitors.…”