Binding and Degradation of Heterodimeric Substrates by ClpAP and ClpXP

Suveena Sharma,Joel R Hoskins,Sue Wickner

doi:10.1074/jbc.m412411200

Suveena Sharma, Joel R Hoskins + Show 1 more

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https://doi.org/10.1074/jbc.m412411200

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Abstract

ClpA and ClpX function both as molecular chaperones and as the regulatory components of ClpAP and ClpXP proteases, respectively. ClpA and ClpX bind substrate proteins through specific recognition signals, catalyze ATP-dependent protein unfolding of the substrate, and when in complexes with ClpP translocate the unfolded polypeptide into the cavity of the ClpP peptidase for degradation. To examine the mechanism of interaction of ClpAP with dimeric substrates, single round binding and degradation experiments were performed, revealing that ClpAP degraded both subunits of a RepA homodimer in one cycle of binding. Furthermore, ClpAP was able to degrade both protomers of a RepA heterodimer in which only one subunit contained the ClpA recognition signal. In contrast, ClpXP degraded both subunits of a dimeric substrate only when both protomers contained a recognition signal. These data suggest that ClpAP and ClpXP may recognize and bind substrates in significantly different ways.

Highlights

In the presence of ATP, Clp ATPases self-assemble into oligomeric rings
The results demonstrate that ClpAP degrades both subunits of a RepA heterodimer when only one subunit contains the N-terminal recognition signal
One possible mechanism for the action of ClpAP on heterodimers is that the interaction between ClpA and the tagged subunit brings the untagged subunit into close proximity to ClpA, where it is bound through low affinity secondary recognition signals

Summary

Introduction

In the presence of ATP, Clp ATPases self-assemble into oligomeric rings. The individual subunits are markedly similar in structure to the subunits of classic AAAϩ ATPases (10 –13). ClpA and ClpX translocate substrates in an ATP-dependent reaction from binding sites on the ATPase component to ClpP in a directional manner, providing further experimental evidence for this model (20 –24). The recognition signal that directs RepA to ClpA is located within the first 15 amino acids of RepA This signal is both necessary and sufficient to target a protein fused to the peptide for unfolding by ClpA and degradation by ClpAP [35]. Another well characterized ClpA and ClpX recognition signal is SsrA. In E. coli, both ClpAP and ClpXP degrade SsrA-tagged proteins

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