The Hsp70 Ssz1 modulates the function of the ribosome-associated J-protein Zuo1

Abstract

J-proteins are obligate partners of Hsp70s, forming a ubiquitous class of molecular chaperone machinery. The ribosome-associated Hsp70 of yeast Ssb binds nascent polypeptides as they exit the ribosome. Here we report that the ribosome-associated J-protein Zuo1 is the partner of Ssb. However, Zuo1 efficiently stimulates the ATPase activity of Ssb only when in complex with another Hsp70, Ssz1. Ssz1 binds ATP, but none of the 11 different amino acid substitutions in the ATP-binding cleft affected Ssz1 function in vivo, suggesting that neither nucleotide binding nor hydrolysis is required. We propose that Ssz1's predominant function in the cell is to facilitate Zuo1's ability to function as a J-protein partner of Ssb on the ribosome, serving as an example of an Hsp70 family member that has evolved to carry out functions distinct from that of a chaperone.

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

“…Hsp70L1 was able to functionally interact with zuotin. In this context it is important to recall that only the complex of zuotin and Ssz1p (RAC) is able to stimulate the ATPase of the yeast ribosome-bound Hsp70 Ssb1͞2p (6,8). As complementation by zuotin͞Hsp70L1 depended on an intact J domain of zuotin, Hsp70L1 most likely can replace Ssz1p and support zuotin's interaction with Ssb1͞2p (see Introduction).…”

“…4, lane 12). Finally, we used a nonfunctional J-domain mutant of zuotin (zuo1-H128Q), which is known to associate with ribosomes and Ssz1p but fails to functionally interact with Ssb1͞2p (6,8). Expression of Hsp70L1 in the presence of zuo1-H128Q failed to complement growth defects of ⌬zuo1⌬ssz1 (Fig.…”

“…Based on localization and concordant phenotypes of ⌬ssb1⌬ssb2 and ⌬zuo1 strains, it was proposed that Ssb1͞2p and zuotin function together in the same cellular process (5). In vitro, however, zuotin does not stimulate Ssb1͞2p's ATPase as would be expected for a classical J partner (6). This puzzle was solved with the identification of Ssz1p, a third chaperone component on yeast ribosomes.…”

“…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.…”

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.…”