The role of the ClpA chaperone in proteolysis by ClpAP.
ClpA, a member of the Clp/Hsp100 family of ATPases, is a molecular chaperone and, in combination with a proteolytic component ClpP, participates in ATP-dependent proteolysis. We investigated the role of ClpA in protein degradation by ClpAP by dissociating the reaction into several discrete steps. In the assembly step, ClpA-ClpP-substrate complexes assemble either by ClpA-substrate complexes interacting with ClpP or by ClpA-ClpP complexes interacting with substrate; ClpP in the absence of ClpA is unable to bind substrates. Assembly requires ATP binding but not hydrolysis. We discovered that ClpA translocates substrates from their binding sites on ClpA to ClpP. The translocation step specifically requires ATP; nonhydrolyzable ATP analogs are ineffective. Only proteins that are degraded by ClpAP are translocated. Characterization of the degradation step showed that substrates can be degraded in a single round of ClpA-ClpP-substrate binding followed by ATP hydrolysis. The products generated are indistinguishable from steady-state products. Taken together, our results suggest that ClpA, through its interaction with both the substrate and ClpP, acts as a gatekeeper, actively translocating specific substrates into the proteolytic chamber of ClpP where degradation occurs. As multicomponent ATP-dependent proteases are widespread in nature and share structural similarities, these findings may provide a general mechanism for regulation of substrate import into the proteolytic chamber.
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
J R
JR
Hoskins
M
M
Pak
M R
MR
Maurizi
S
S
Wickner
D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D004268:DNA-Binding Proteins; D049071:Endopeptidase Clp; D006868:Hydrolysis; D018832:Molecular Chaperones / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity; D015534:Trans-Activators
D004268:DNA-Binding Proteins; D018832:Molecular Chaperones; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D012697:Serine Endopeptidases; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; D004265:DNA Helicases