[Keywords: Biochemical genomics; protein microarray; proteome; high-throughput expression; galactose lethality; glycosylation] Supplemental material is available at http://www.genesdev.org.

The 70 kDa heat shock proteins (Hsp70s) are a ubiquitous class of molecular chaperones. The Ssbs of Saccharomyces cerevisiae are an abundant type of Hsp70 found associated with translating ribosomes. To understand better the function of Ssb in association with ribosomes, the Ssb-ribosome interaction was characterized. Incorporation of the aminoacyl-tRNA analog puromycin by translating ribosomes caused the release of Ssb concomitant with the release of nascent chains. In addition, Ssb could be cross-linked to nascent chains containing a modified lysine residue with a photoactivatable cross-linker. Together, these results suggest an interaction of Ssb with the nascent chain. The interaction of Ssb with the ribosome-nascent chain complex was stable, as demonstrated by resistance to treatment with high salt; however, Ssb interaction with the ribosome in the absence of nascent chain was salt sensitive. We propose that Ssb is a core component of the translating ribosome which interacts with both the nascent polypeptide chain and the ribosome. These interactions allow Ssb to function as a chaperone on the ribosome, preventing the misfolding of newly synthesized proteins.

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.