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

Abstract

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 peptidebinding 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.

The ribosome-associated complex antagonizes prion formation in yeast

“…35,36 Neither the peptide binding domain nor the ATPase activity of Ssz1 is required for RAC function in vivo. 53,54 Thus, the importance of ribosome-association and Ssb stimulation for the anti-prionogenic function of the RAC can be assessed by deleting those 2 functional regions of Zuo1. Previous studies have shown that variants of Zuo1 with deletions of those regions are expressed at similar levels to wildtype Zuo1 but are not functional in vivo; specifically, deletion of the charged region eliminates the association of Zuo1 with ribosomes, whereas deletion of the J domain abolishes function without affecting ribosome association.…”

The chaperones MPP11 and Hsp70L1 form the mammalian ribosome-associated complex

“…The Hsp70 homolog Ssz1p together with zuotin forms an unusual dimeric ribosome-associated complex (RAC) (7). It is now established that the two subunits of RAC and Ssb1͞2p form a chaperone triad, and that each of the chaperones is essential for functionality in vivo and in vitro (6,8,9). The mechanism of the yeast chaperone triad, however, remains poorly defined.…”

Functional Characterization of the Atypical Hsp70 Subunit of Yeast Ribosome-associated Complex

“…At first, Ssz1 contains an unusually short peptide binding domain and lacks the variable C-terminal domain (8). In fact, the function of Ssz1 does not strictly depend on substrate binding as a truncated version lacking nearly the entire peptide binding domain was reported to support growth as efficiently as the wild type protein (7). Moreover, Ssz1 also contains a particular ATPase domain.…”

“…The three chaperones are genetically linked and form a functional triad. Lack of SSZ1, ZUO1, or SSB1/2 results in slow growth, cold sensitivity, and pronounced hypersensitivity against aminoglycosides such as paromomycin (6,7). Ssz1 and Zuo1 assemble into a stable heterodimeric complex termed ribosome-associated complex (RAC), 4 which is anchored to the ribosome via its Zuo1 subunit (8).…”