title: Functional proteolytic complexes of the human mitochondrial ATP-dependent protease, hClpXP.
abstract: Human mitochondrial ClpP (hClpP) and ClpX (hClpX) were separately cloned, and the expressed proteins were purified. Electron microscopy confirmed that hClpP forms heptameric rings and that hClpX forms a hexameric ring. Complexes of a double heptameric ring of hClpP with hexameric hClpX rings bound on each side are stable in the presence of ATP or adenosine 5'-(3-thiotriphosphate) (ATPgammaS), indicating that a symmetry mismatch is a universal feature of Clp proteases. hClpXP displays both ATP-dependent proteolytic activity and ATP- or ATPgammaS-dependent peptidase activity. hClpXP cannot degrade lambdaO protein or GFP-SsrA, specific protein substrates recognized by Escherichia coli (e) ClpXP. However, eClpX interacts with hClpP, and, when examined by electron microscopy, the resulting heterologous complexes are indistinguishable from homologous eClpXP complexes. The hybrid eClpX-hClpP complexes degrade eClpX-specific protein substrates. In contrast, eClpA can neither associate with nor activate hClpP. hClpP has an extra C-terminal extension of 28 amino acids. A mutant lacking this C-terminal extension interacts more tightly with both hClpX and eClpX and shows enhanced enzymatic activities but still does not interact with eClpA. Our results establish that human ClpX and ClpP constitute a bone fide ATP-dependent protease and confirm that substrate selection, which differs between human and E. coli ClpX, is dependent solely on the Clp ATPase. Our data also indicate that human ClpP has conserved sites required for interaction with eClpX but not eClpA, implying that the modes of interaction with ClpP may not be identical for ClpA and ClpX.
authors.affiliation: Laboratory of Cell Biology, NCI and Laboratory of Structural Biology, NIAMS, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Sung Gyun
authors.initials: SG
authors.lastname: Kang
authors.forename: Joaquin
authors.initials: J
authors.lastname: Ortega
authors.forename: Satyendra K
authors.initials: SK
authors.lastname: Singh
authors.forename: Nan
authors.initials: N
authors.lastname: Wang
authors.forename: Ning-Na
authors.initials: NN
authors.lastname: Huang
authors.forename: Alasdair C
authors.initials: AC
authors.lastname: Steven
authors.forename: Michael R
authors.initials: MR
authors.lastname: Maurizi
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D049071:Endopeptidase Clp; D004789:Enzyme Activation; D029968:Escherichia coli Proteins; D006801:Humans; D006868:Hydrolysis; D008854:Microscopy, Electron; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D012697:Serine Endopeptidases; C483568:ClpA protease, E coli; C483573:ClpXP protease, E coli; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases