Unfolding and internalization of proteins by the ATP-dependent proteases ClpXP and ClpAP

ClpX and ClpA are molecular chaperones that interact with specific proteins and, together with ClpP, activate their ATP-dependent degradation. The cha perone activity is thought to convert proteins into an extended conformatio n that can access the sequestered active sites of ClpP. We now show that Cl pX can catalyze unfolding of a green fluorescent protein fused to a ClpX re cognition motif (GFP-SsrA), Unfolding of GFP-SsrA depends on ATP hydrolysis , GFP-SsrA unfolded either by ClpX or by treatment with denaturants binds t o ClpX in the presence of adenosine 5'-O-(3-thiotriphosphate) and is releas ed slowly (t(1/2) approximate to 15 min). Unlike ClpA, ClpX cannot trap unf olded proteins in stable complexes unless they also have a high-affinity bi nding motif. Addition of ATP or ADP accelerates release (t(1/2) approximate to 1 min), consistent with a model in which ATP hydrolysis induces a confo rmation of ClpX with low affinity for unfolded substrates. Proteolytically inactive complexes of ClpXP and ClpAP unfold GFP-SsrA and translocate the p rotein to ClpP, where it remains unfolded. Complexes of ClpXP with transloc ated substrate within the ClpP chamber retain the ability to unfold GFP-Ssr A. Our results suggest a bipartite mode of interaction between ClpX and sub strates. ClpX preferentially targets motifs exposed in specific proteins. A s the protein is unfolded by ClpX, additional motifs are exposed that facil itate its retention and favor its translocation to ClpP for degradation.