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

Abstract

Eukaryotic ribosomes carry a stable chaperone complex termed ribosome-associated complex consisting of the J-domain protein Zuo1 and the Hsp70 Ssz1. Zuo1 and Ssz1 together with the Hsp70 homolog Ssb1/2 form a functional triad involved in translation and early polypeptide folding processes. Strains lacking one of these components display slow growth, cold sensitivity, and defects in translational fidelity. Ssz1 diverges from canonical Hsp70s insofar that neither the ability to hydrolyze ATP nor binding to peptide substrates is essential in vivo. The exact role within the chaperone triad and whether or not Ssz1 can hydrolyze ATP has remained unclear. We now find that Ssz1 is not an ATPase in vitro, and even its ability to bind ATP is dispensable in vivo. Furthermore, Ssz1 function was independent of ribosome-associated complex formation, indicating that Ssz1 is not merely a structural scaffold for Zuo1. Finally, Ssz1 function in vivo was inactivated when both nucleotide binding and Zuo1 interaction via the C-terminal domain were disrupted in the same mutant. The two domains of this protein thus cooperate in a way that allows for severe interference in either but not in both of them.

Structural Analysis of the Ribosome-associated Complex (RAC) Reveals an Unusual Hsp70/Hsp40 Interaction

“…This assumption fits well with a recent deletion analysis of Ssz showing that a fragment encompassing the ATPase domain but lacking the C-terminal substrate binding domain is strongly impaired in Zuo binding (11).…”

“…Other findings also indicate that Ssz cannot be considered a classical Hsp70 chaperone: its C-terminal domain is shorter than that of other family members (5) and it appears to not bind to substrates (7). Moreover, Ssz does not efficiently hydrolyze ATP (11).…”

“…5A). Hygromycin B sensitivity of cells lacking Ssz is less pronounced than that of cells deleted for Zuo, most likely due to residual activity of Zuo in the absence of its complex partner (11). Zuo-⌬N62 thus also displays residual function, but its inability to associate with Ssz results in a phenotype similar to that of complete absence of Ssz.…”

“…We could not specifically test whether ATP binding is required for complex formation or RAC activity because no nucleotide-free Ssz in soluble state could be obtained. However, a recent study showed that a Ssz mutant deficient in ATP binding can complement the phenotype of ssz⌬ cells in vivo, suggesting that the assembly of a functional RAC can occur even in the absence of bound nucleotide (11).…”

“…Unlike other J-proteins, Zuo requires stable association with Ssz to stimulate the ATPase activity of Ssb. Moreover, the Hsp70 Ssz does not require ATP binding and hydrolysis for function in vivo, nor does it seem to associate with nascent polypeptide chains (7,9,11). These atypical traits are not restricted to yeast cells, as it has been recently reported that the J-protein MPP11 associates with Hsp70L1 to form a stable ribosome-associated complex in mammalian cells (12,13).…”