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

Charlotte Conz,Hendrik Otto,Kristin Peisker,Matthias Gautschi,Tina Wölfle,Matthias P Mayer,Sabine Rospert

doi:10.1074/jbc.m706737200

Charlotte Conz, Hendrik Otto + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m706737200

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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.

Highlights

Chaperones of the Hsp70 family usually consist of an ϳ45kDa N-terminal ATPase, an ϳ15-kDa peptide binding, and an ϳ10-kDa variable C-terminal domain [1]
In the case of classical Hsp70s, ATPase activity can be regulated by a diverse set of co-chaperones, which are essential for Hsp70 functions [1]
Untagged ribosome-associated complex (RAC) Purified from E. coli Does Not Hydrolyze ATP— In most cases, objective evidence for ATP hydrolysis by an Hsp70 is based on the ability of specific co-chaperones to stimulate this reaction that occurs at an intrinsically low rate

Summary

Introduction

Chaperones of the Hsp70 family usually consist of an ϳ45kDa N-terminal ATPase, an ϳ15-kDa peptide binding, and an ϳ10-kDa variable C-terminal domain [1]. Ssz1 function in vivo was inactivated when both nucleotide binding and Zuo1 interaction via the C-terminal domain were disrupted in the same mutant. Mutations in the ATPase domain of Ssz1 were found to fully complement the phenotype of a ⌬ssz1 strain, indicating that ATP hydrolysis was not an essential function of Ssz1 in vivo [10].

Results

Conclusion