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

Two proteins of the Hsp70 class (Ssb and Ssz1) and one of the J-type class (Zuo1) of molecular chaperones reside on the yeast ribosome, with Ssz1 forming a stable heterodimer with Zuo1. We designed experiments to address the roles of these two distantly related ribosome-associated Hsp70s and their functional relationship to Zuo1. Strains lacking all three proteins have the same phenotype as those lacking only one, suggesting that these chaperones all function in the same pathway. The Hsp70 Ssb, whose peptide-binding domain is essential for its in vivo function, can be crosslinked to nascent chains on ribosomes that are as short as 54 amino acids, suggesting that Ssb interacts with nascent chains that extend only a short distance beyond the tunnel of the ribosome. A ssz1 mutant protein lacking its putative peptide-binding domain allows normal growth. Thus, binding of unfolded protein substrates in a manner similar to that of typical Hsp70s is not critical for Ssz1's in vivo function. The three chaperones are present in cells in approximately equimolar amounts compared with ribosomes. The level of Ssb can be reduced only a few-fold before growth is affected. However, a 50- to 100-fold reduction of Ssz1 and Zuo1 levels does not have a substantial effect on cell growth. On the basis of these results, we propose that Ssbs function as the major Hsp70 chaperone for nascent chains on the ribosome, and that Ssz1 has evolved to perform a nonclassical function, perhaps modulating Zuo1's ability to function as a J-type chaperone partner of Ssb.