Abstract
ClpS is an adaptor protein that interacts with ClpA and promotes degradation of proteins with N-end rule degradation motifs (N-degrons) by ClpAP while blocking degradation of substrates with other motifs. Although monomeric ClpS forms a 1:1 complex with an isolated N-domain of ClpA, only one molecule of ClpS binds with high affinity to ClpA hexamers (ClpA(6)). One or two additional molecules per hexamer bind with lower affinity. Tightly bound ClpS dissociates slowly from ClpA(6) with a t((1/2)) of approximately 3 min at 37 degrees C. Maximum activation of degradation of the N-end rule substrate, LR-GFP(Venus), occurs with a single ClpS bound per ClpA(6); one ClpS is also sufficient to inhibit degradation of proteins without N-degrons. ClpS competitively inhibits degradation of unfolded substrates that interact with ClpA N-domains and is a non-competitive inhibitor with substrates that depend on internal binding sites in ClpA. ClpS inhibition of substrate binding is dependent on the order of addition. When added first, ClpS blocks binding of both high and low affinity substrates; however, when substrates first form committed complexes with ClpA(6), ClpS cannot displace them or block their degradation by ClpP. We propose that the first molecule of ClpS binds to the N-domain and to an additional functional binding site, sterically blocking binding of non-N-end rule substrates as well as additional ClpS molecules to ClpA(6). Limiting ClpS-mediated substrate delivery to one per ClpA(6) avoids congestion at the axial channel and allows facile transfer of proteins to the unfolding and translocation apparatus.
Highlights
ClpS is an adaptor protein that interacts with ClpA and promotes degradation of proteins with N-end rule degradation motifs (N-degrons) by ClpAP while blocking degradation of substrates with other motifs
Either directly recognized by sites in ClpA or ClpX or recognized by one of several adaptor proteins that associate with ClpA or ClpX, and, in some instances, different parts of the tag can be recognized by the chaperone and the adaptor [3,4,5]
Arginine and lysine serve as secondary destabilizing residues, because proteins with these residues at the N terminus are modified by Leu/Phe-tRNA protein transferase to generate an N-degron by post-translational attachment of phenylalanine or leucine [8, 9]
Summary
ClpS is an adaptor protein that interacts with ClpA and promotes degradation of proteins with N-end rule degradation motifs (N-degrons) by ClpAP while blocking degradation of substrates with other motifs. Maximum activation of degradation of the N-end rule substrate, LR-GFPVenus, occurs with a single ClpS bound per ClpA6; one ClpS is sufficient to inhibit degradation of proteins without N-degrons. ClpS competitively inhibits degradation of unfolded substrates that interact with ClpA N-domains and is a non-competitive inhibitor with substrates that depend on internal binding sites in ClpA. In at least one case, an aminopeptidase generates a protein with an N-degron that is degraded by ClpAP [9, 11, 12], but the mechanisms by which N-end rule substrates are formed and the frequency with which they occur are largely unknown. Details of how ClpS interacts with the ClpAP holoenzyme complex and performs its functions are not well understood
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