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 chaperone activity is thought to convert proteins into an extended conformation that can access the sequestered active sites of ClpP. We now show that ClpX can catalyze unfolding of a green fluorescent protein fused to a ClpX recognition motif (GFP-SsrA). Unfolding of GFP-SsrA depends on ATP hydrolysis. GFP-SsrA unfolded either by ClpX or by treatment with denaturants binds to ClpX in the presence of adenosine 5'-O-(3-thiotriphosphate) and is released slowly (t(1/2) approximately 15 min). Unlike ClpA, ClpX cannot trap unfolded proteins in stable complexes unless they also have a high-affinity binding motif. Addition of ATP or ADP accelerates release (t(1/2) approximately 1 min), consistent with a model in which ATP hydrolysis induces a conformation of ClpX with low affinity for unfolded substrates. Proteolytically inactive complexes of ClpXP and ClpAP unfold GFP-SsrA and translocate the protein to ClpP, where it remains unfolded. Complexes of ClpXP with translocated substrate within the ClpP chamber retain the ability to unfold GFP-SsrA. Our results suggest a bipartite mode of interaction between ClpX and substrates. ClpX preferentially targets motifs exposed in specific proteins. As the protein is unfolded by ClpX, additional motifs are exposed that facilitate its retention and favor its translocation to ClpP for degradation.
Laboratory of Cell Biology and Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
S K
SK
Singh
R
R
Grimaud
J R
JR
Hoskins
S
S
Wickner
M R
MR
Maurizi
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D002384:Catalysis; D004705:Endocytosis; D049071:Endopeptidase Clp; D049452:Green Fluorescent Proteins; D006868:Hydrolysis; D008164:Luminescent Proteins / Q000378:metabolism; D017510:Protein Folding; D012329:RNA, Bacterial / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity
D008164:Luminescent Proteins; D012329:RNA, Bacterial; C096222:tmRNA; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D012697:Serine Endopeptidases; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases