A functional chaperone triad on the yeast ribosome

Abstract

The chaperones RAC (ribosome-associated complex), consisting of Ssz1p and zuotin, and Ssb1͞2p are associated with ribosomes of yeast. Ssb1͞2p was previously shown to form a crosslink product to polypeptides trapped in ribosome-nascent chain complexes (RNCs) in vitro. Here we show that an efficient crosslink of the nascent chain to Ssb1͞2p depends on the presence of functional RAC. The crosslink to Ssb1͞2p was significantly diminished if (i) RAC was removed from RNCs: a process reversed by addition of purified RAC; (ii) RAC carried a mutation in the J-domain of zuotin, leading to its inactivation in vivo; (iii) RAC's Ssz1p subunit was absent because RNCs were generated in a ⌬ssz1-derived translation extract. In vivo the same specific set of growth defects caused by the absence of any of the three chaperones was also displayed by a ⌬ssb1͞2⌬ssz1⌬zuo1 strain. The combination of in vitro and in vivo data supports a model in which Ssb1͞2p, Ssz1p, and zuotin act in concert on nascent chains while they are being synthesized.

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

“…The ATPase domain is required for this regulation, but the mechanism by which it occurs is not known. This model is consistent with the data of Rospert and colleagues (42). In their system, the Ssz1͞Zuo1 heterodimer is required for Ssb to be crosslinked to nascent chains, suggesting that Zuo1 is the ''J partner'' for Ssb, even though it is stably associated with another Hsp70.…”

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.…”

“…Deletion strains lacking SSZ1 (⌬ssz1: ssz1::LEU2), ZUO1 (⌬zuo1: zuo1::TRP1), or both genes (⌬zuo1⌬ssz1: zuo1::TRP1 ssz1::LEU2) were as described (7). Construction of the J-domain mutant of zuotin was as described (8). SSA1 was expressed from the 2-plasmid pRS423.…”

“…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.…”