The Cotranslational Function of Ribosome-Associated Hsp70 in Eukaryotic Protein Homeostasis

Abstract

In eukaryotic cells a molecular chaperone network associates with translating ribosomes, assisting the maturation of emerging nascent polypeptides. Hsp70 is perhaps the major eukaryotic ribosome-associated chaperone and the first reported to bind cotranslationally to nascent chains. However, little is known about the underlying principles, and function of this interaction. Here, we use a sensitive and global approach to define the cotranslational substrate specificity of the yeast Hsp70 SSB. We find that SSB binds to a subset of nascent polypeptides whose intrinsic properties and slow translation rates hinder efficient cotranslational folding. The SSB-ribosome cycle and substrate recognition is modulated by its ribosome-bound co-chaperone RAC. Deletion of SSB leads to widespread aggregation of newly synthesized polypeptides. Thus, cotranslationally acting Hsp70 meets the challenge of folding the eukaryotic proteome by stabilizing its longer, more slowly translated, and aggregation prone nascent polypeptides.

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

“…In addition Fes1 has been directly implicated in recognition of misfolded proteins during stress conditions. A possible explanation for this distribution of NEF dependence could be differential interaction with the two classes of cytosolic Hsp70 in yeast: Ssa is involved in all the processes we investigated, whereas Ssb likely only plays a significant role during protein translation, interacting with nascent chains by virtue of its association with the ribosome (68,69). To test this theory, we took advantage of previously developed co-immunoprecipitation assays using fully functional FLAG-tagged NEF alleles expressed in yeast.…”

The ribosome-associated complex antagonizes prion formation in yeast

“…These observations are consistent with previous studies that demonstrated suppression of Sup35 polymerization into amyloid by purified Zuo1, Ssz1 and Ssb in vitro 64 and identified nascent Sup35 as a substrate of the RAC/Ssb chaperones in vivo. 30 In addition, cells depleted of Ssb also exhibit elevated rates of prion formation. 38 Indeed, here we have shown that the RAC must be capable of ribosome association and Ssb activation to suppress prion formation (Fig.…”

“…Localized crowding of nascent polypeptides along polysomes further enhances the probability of misfolding and aggregation, necessitating co-translational stabilization by chaperones for a substantial fraction of nascent chains in both prokaryotes and eukaryotes. [28][29][30] In eukaryotes, 2 ribosomeassociated complexes interact with nascent chains: the Nascent Chain Associated Complex (NAC), and the Ribosome-Associated Complex (RAC). The NAC is a heterodimeric complex that associates with ribosomes near the polypeptide exit tunnel.…”

Oligosaccharyltransferase Subunits Bind Polypeptide Substrate to Locally Enhance N-glycosylation

“…Transient binding of short, hydrophobic stretches of nascent polypeptide by Ost3p and Ost6p is reminiscent of the activity of co-translational Hsp70 chaperones such as cytoplasmic SSA/SSB (29) and ER lumenal Kar2p (BiP) (30), which assist co-translational folding (31,32). As binding to Ost3p or Ost6p is independent of the presence of a nearby glycosylation site, all stretches of nascent polypeptides with sufficient affinity to either Ost3p or Ost6p are likely transiently sequestered.…”