Functional Proteolytic Complexes of the Human Mitochondrial ATP-dependent Protease, hClpXP

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) (ATPγS), indicating that a symmetry mismatch is a universal feature of Clp proteases. hClpXP displays both ATP-dependent proteolytic activity and ATP- or ATPγS-dependent peptidase activity. hClpXP cannot degrade λO 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. 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) (ATPγS), indicating that a symmetry mismatch is a universal feature of Clp proteases. hClpXP displays both ATP-dependent proteolytic activity and ATP- or ATPγS-dependent peptidase activity. hClpXP cannot degrade λO 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. Protein remodeling and protein degradation carried out by the Clp family of molecular chaperones and their proteolytic complexes with ClpP have important physiological functions in many organisms. Clp proteases, together with other ATP-dependent proteases, such as the Lon protease and the membrane-associated protease FtsH are responsible for most intracellular protein degradation in eubacteria, helping to maintain proper protein homeostasis, contributing to protein quality control, and modulating the intracellular concentration of important global regulatory proteins (1Gottesman S. Maurizi M.R. Microbiol. Rev. 1992; 56: 592-621Crossref PubMed Google Scholar, 2Suzuki C.K. Rep M. van Dijl J.M. Suda K. Grivell L.A. Schatz G. Trends Biochem. Sci. 1997; 22: 118-123Abstract Full Text PDF PubMed Scopus (208) Google Scholar, 3Schirmer E.C. Glover J.R. Singer M.A. Lindquist S. Trends Biochem. Sci. 1996; 21: 289-296Abstract Full Text PDF PubMed Scopus (574) Google Scholar). The ATPase/chaperone components of ATP-dependent proteases target unique substrates for degradation, although some overlap exists in the recognition of misfolded or other abnormal forms of proteins (4Gottesman S. Clark W.P. de Crecy-Lagard V. Maurizi M.R. J. Biol. Chem. 1993; 268: 22618-22626Abstract Full Text PDF PubMed Google Scholar, 5Levchenko I. Luo L. Baker T.A. Genes Dev. 1995; 9: 2399-2408Crossref PubMed Scopus (245) Google Scholar, 6Wojtkowiak D. Georgopoulos C. Zylicz M. J. Biol. Chem. 1993; 268: 22609-22617Abstract Full Text PDF PubMed Google Scholar, 7Gottesman S. Roche E. Zhou Y. Sauer R.T. Genes Dev. 1998; 12: 1338-1347Crossref PubMed Scopus (646) Google Scholar). In the bacterial Clp system, two separate ATPases, ClpX and ClpA, interact with different protein substrates and confer specific protein degrading ability to ClpP (8Gottesman S. Wickner S. Jubete Y. Singh S.K. Kessel M. Maurizi M.R. Cold Spring Harbor Symp. Quant. Biol. 1995; 60: 533-548Crossref PubMed Scopus (33) Google Scholar). Clp proteases, Lon, and FtsH are conserved in most eukaryotes, where they are found in organelles, such as mitochondria, peroxisomes, and chloroplasts (2Suzuki C.K. Rep M. van Dijl J.M. Suda K. Grivell L.A. Schatz G. Trends Biochem. Sci. 1997; 22: 118-123Abstract Full Text PDF PubMed Scopus (208) Google Scholar, 9Langer T. Neupert W. Experientia. 1996; 52: 1069-1076Crossref PubMed Scopus (73) Google Scholar, 10Adam Z. Adamska I. Nakabayashi K. Ostersetzer O. Haussuhl K. Manuell A. Zheng B. Vallon O. Rodermel S.R. Shinozaki K. Clarke A.K. Plant Physiol. 2001; 125: 1912-1918Crossref PubMed Scopus (181) Google Scholar). In yeast, Lon and FtsH homologs have been shown to play important roles in assembly and quality control over membrane protein complexes (11Arlt H. Tauer R. Feldmann H. Neupert W. Langer T. Cell. 1996; 85: 875-885Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar) and in the degradation of misfolded proteins (9Langer T. Neupert W. Experientia. 1996; 52: 1069-1076Crossref PubMed Scopus (73) Google Scholar, 12Stuart R.A. Cyr D.M. Neupert W. Experientia. 1994; 50: 1002-1011Crossref PubMed Scopus (40) Google Scholar). In humans, mutations in paraplegin, an FtsH homolog, have been implicated in the progressive loss of mitochondria function in the hereditary neurodegenerative disease spastic paraplegia (13Pearce D.A. Hum. Genet. 1999; 104: 443-448Crossref PubMed Scopus (13) Google Scholar). The function of mitochondrial Clp proteins has not been identified. The human genome encodes both ClpP, on chromosome 19 (14), and ClpX, on chromosome 15 (15). mRNAs for both human (h) 1The abbreviations used are: hhumaneE. coliATPγSadenosine 5′-O-(thiotriphosphate)mmouseGFPgreen fluorescent protein1The abbreviations used are: hhumaneE. coliATPγSadenosine 5′-O-(thiotriphosphate)mmouseGFPgreen fluorescent protein ClpP and hClpX have been found in greatest abundance in liver, heart, and testes, all of which are mitochondrially rich tissues (14Corydon T.J. Bross P. Holst H.U. Biochem. J. 1998; 331: 309-316Crossref PubMed Scopus (57) Google Scholar, 15Corydon T.J. Wilsbech M. Jespersgaard C. Andresen S. L. Bross P. PubMed Scopus Google Scholar, S. D. A. E. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The human and mitochondria S. D. A. E. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, M.R. I. S. S. J. J. Biol. 1999; PubMed Scopus Google Scholar). The of hClpP by of human mitochondria that the protein has amino from the of the M.R. I. S. S. J. J. Biol. 1999; PubMed Scopus Google of ClpX of amino from the S. D. A. E. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The of the human proteins to their Escherichia coli and in a and in a of all of the of the E. coli hClpX and hClpP are to interact to a proteolytic protease or peptidase activity has been for hClpP or hClpXP complexes. human E. coli adenosine fluorescent protein human E. coli adenosine fluorescent protein A unique feature of bacterial the complexes is the symmetry mismatch between the and the Complexes are by the interaction of rings of ClpX and rings of a symmetry mismatch exists in the between the of the 19 regulatory and the of the such are not the in ATP-dependent for is a between rings of both mismatch is in ATP-dependent dependent proteases such as Lon and which are of with and protease The of symmetry mismatch are not and has not been is a universal feature of or proteases. Clp proteases play important and regulatory roles in many have an of the activity and substrate of the human mitochondrial ClpXP. Our results confirm that symmetry mismatch between ClpX and ClpP is conserved in human complexes. also that between the two components are conserved to between heterologous components from E. coli and that the bacterial and human proteases degrade different proteins and that ClpX is responsible for substrate A with an of by C. Genet. 1993; PubMed Scopus Google Scholar) to have a to E. coli used to by a human The from the were as by in The of the by and an of The on both by the on an universal and as as The confirmed the presence of the for hClpP. hClpP and forms were the in a that hClpP in E. coli of with A hClpP by the and which in and sites the and of the of hClpP. The between the and sites of to the of hClpP by to the of hClpP. The used with a for The with and and hClpP lacking the C-terminal with the and a which 28 of hClpP and the E. coli (e) ClpP C-terminal for and The to the mutant to or were the as in the used as the The for to were and The for to were and The were with in in the presence of The were to and to and for an The were by and the were in of and The were in a and the by for The of hClpP is S. G. M. T. M. R. and T. in of the over a to the used to bound by on a with the a of The protein with a of with in with and over a of in the The a and with a of hClpX and in E. were The for hClpX a human from P. Bross and for a from S. of the were and the were and The and sites the and and were to the ClpX protein from of the hClpX or of the The were between and sites of and with a in and the or were as in the presence of and the the were and in of and The were by and the by The in of and of by The protein by of the in the presence of and the by The a with and and with a of The hClpX or were and a with and The protein with a of and the with were The by The used for most with and and or ATP were The for protein degradation and activity were by R. Kessel M. Maurizi M.R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google degradation of and and the were M.R. Singh S.K. 1994; PubMed Scopus Google Scholar, Maurizi M.R. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). The protein concentration by the P. Andresen I. T.A. 1995; PubMed Scopus Google Scholar). to the of PubMed Scopus Google Scholar). with a The were with with or The were in of of the and the were The protein and the were The for of electron and were J. Singh S.K. Maurizi M.R. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). hClpP of in and were ClpX, were that to the hClpXP complexes were with hClpX and hClpP in the presence of and or for The were for symmetry the E. M. 1995; 60: PubMed Scopus Google Scholar). of the of with E. J. Biol. 1996; PubMed Scopus Google Scholar). were symmetry or symmetry for ClpX or ClpP and two symmetry to and to for complexes. ClpP by ClpP in and different the and the protein from by with of ClpP in and over a in to and of hClpP with the amino of E. that hClpP an extension of amino and this extension displays of mitochondrial (14Corydon T.J. Bross P. Holst H.U. Biochem. J. 1998; 331: 309-316Crossref PubMed Scopus (57) Google Scholar, G. J. J. Biochem. PubMed Scopus Google Scholar, Y. G. Protein PubMed Scopus Google Scholar). on with E. coli ClpP between the and the for the Our hClpP differs from the which were amino (14Corydon T.J. Bross P. Holst H.U. Biochem. J. 1998; 331: 309-316Crossref PubMed Scopus (57) Google Scholar) or M.R. I. S. S. J. J. Biol. 1999; PubMed Scopus Google Scholar). of hClpP in E. coli a with an molecular of on most of the expressed protein found to be hClpP to over confirmed that the amino indicating of the in not also expressed and a mutant of hClpP, which the C-terminal 28 amino of hClpP expressed hClpP and hClpP and hClpX proteins were on in the presence of and with hClpP of the have as the molecular molecular hClpX that hClpP or expressed hClpP not degrade such as (14Corydon T.J. Bross P. Holst H.U. Biochem. 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Chem. 1994; Full Text PDF PubMed Google Scholar). in a activity in of with of human or E. coli or were for ATP and in The coli or human as and substrate proteins were as each of ClpX and ClpP with each of ClpX and ClpP with of ClpX and ClpP with or with ClpP and ClpX. The of activity for protein degradation are ClpP and for peptidase activity are ClpP and have been and T.J. Wilsbech M. Jespersgaard C. Andresen S. L. Bross P. PubMed Scopus Google Scholar, S. D. A. E. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google and the proteins have been shown to be in from shown to to a protein on a S. D. A. E. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google that ClpP and ClpX interact in However, in some eukaryotes, such as the ClpX and not ClpP is L. M. Neupert W. Langer T. 1998; PubMed Scopus Google Scholar). the and enzymatic of the hClpXP expressed and the with eClpX from of hClpX from the or from and enzymatic of both forms were a to but to the human hClpX in a but of of a that as a on a Electron of hClpX in the presence of were for and a symmetry found of the of rings to with also confirmed that hClpP symmetry and rings in the as M.R. I. S. S. J. J. Biol. 1999; PubMed Scopus Google ClpP rings of of hClpX and hClpP over a in the presence of as a molecular Electron of hClpXP complexes in the presence of side with two rings of hClpP in the on both by a ring of hClpX complexes were between hClpP and which the of is identical to that of and of forms and complexes of hClpX and hClpP. of proteins were and as and a of hClpX in the presence of the of the of most of which to with the to the a of hClpXP most of which are from the side as or the that by to the in their hClpX in the presence of hClpXP in the presence of hClpP. heterologous of and hClpP in the presence of heterologous of eClpX and hClpP in the presence of hClpP also stable complexes with and the complexes were identical in eClpA not to hClpP by electron microscopy, or enzymatic also not the heterologous between hClpX and by of The of hybrid eClpX-hClpP complexes to the activity and of homologous and heterologous complexes. hClpXP complexes were for activity λO protein and GFP-SsrA, protein substrates by activity However, hClpXP to degrade a substrate not by hClpXP also and neither of which is by degradation shown in carried out with indicating that the of hClpP is not required for interaction with hClpX or for enzymatic activity. to by hClpXP with an of but with the of as ClpX The degradation were when ClpX activity or ClpP peptidase activity used a that is by when by eClpX R. 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The hybrid eClpX-hClpP on λO protein and in the presence of The of eClpX and hClpP both proteins in the presence of ATP and in the presence of as with degradation when used in of hClpP not proteolytic activity in the presence of eClpA not of to hClpP with eClpX and The of to that with the the heterologous has and activities to of the homologous E. coli the ability of hClpX to interact with and activate and electron microscopy to stable interaction between hClpX not proteolytic activity the λO protein or GFP-SsrA, but also not of the by hClpXP and not activate in the presence of which is the most for interaction between Clp and with hClpP for to a of over hClpP to degradation in which hClpX of degradation indicating that not for to hClpX and of hClpP of ClpX with ClpP but not ATP the ClpX concentration and ClpP, between different of the human and E. coli eClpX and the eClpX and hClpX for hClpP, but both of with eClpXP eClpX and hClpX for for the hClpP The activity of hClpP in is to with ClpX. interaction between hClpX and hClpP between and the by Maurizi M.R. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). with hClpP or when by hClpX or the of hClpP, of with in the and were used are all the by both and hClpP not most were by hClpP and hClpP more to the presence of a in the or the hClpP not with a The does not the of The of by are to with Maurizi M.R. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). by hClpP were the hClpX or eClpX used for not of by peptidase were to the of and of of and of with or for with hClpXP or eClpXP in the presence of and of the were between and The were to the of and of Maurizi M.R. J. Biol. Chem. 1994; Full Text PDF PubMed Google with or in a for with hClpXP or eClpXP in the presence of and of the were between and as a the by hClpXP and eClpXP were but a were hClpP not but hClpP and have in by both and hClpP that of the is not and that the the the in the for of the hClpP and protein that the of many proteases and conserved in hClpP. with the of of hClpP with the of the is to that target of proteases, hClpP is to such or is in loss of activity of hClpP with or in the presence of and hClpP with for not hClpP also to and The which the found to to be of the proteolytic and not to the of hClpP. G. and M. R. in confirm that is the of hClpP, used to this with or of mutant a to the of hClpP, and the expressed proteins were by of and λO by to hClpP and stable interaction of with eClpX by not and were in the presence of hClpX with as substrate and in the presence of eClpX with λO and as substrates. The mutant were on all substrates not as a for GFP-SsrA, which is and the of by eClpX S.K. R. J.R. Wickner S. Maurizi M.R. Sci. S. A. PubMed Scopus Google Scholar). In the presence of also but not degrade indicating that the mutant protein forms a with eClpX by proteolytic activity. data that hClpP is a protease and that is the for the of the in hClpP The and proteolytic functions of Clp proteases are conserved and, in many functions for or for to or in have been for human ClpX on chromosome T.J. Wilsbech M. Jespersgaard C. Andresen S. L. Bross P. PubMed Scopus Google Scholar) and for human ClpP on chromosome 19 proteins to be to Our data establish that ClpX and ClpP to a ATP-dependent protease that has and enzymatic to the bacterial ClpX to be the Clp in with to of ClpA are but the human genome does not have a all of the of in enzymatic out between hClpXP and the peptidase and protease activities of hClpXP are of of not have a for the activity. The activity is also in the with such as and an for that in a with the of hClpP are not all in an is that hClpX of to the of hClpP, but the of this also has of with Singh S.K. Maurizi M.R. J. Biol. Chem. 1994; Full Text PDF PubMed Google the from the that all of the sites of are in the J. J.M. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). activity of all sites of ClpP on the of the with ClpX or on the presence of substrate or other be in the bacterial where the are of but may play a in the of the by the ClpXP. that hClpP and hClpX are both are also to hClpXP expressed in to and different the enzymatic The in enzymatic is that hClpX and eClpX different substrates. by Clp proteases is dependent on the Clp ATPase. In E. ClpA and ClpX target different proteins for degradation both in and in although ClpA also has a ability to degrade most ClpX substrates. In is that of target proteins by mutations in ClpX on the of heterologous complexes of ClpX and ClpP from different have been that eClpX can specific substrates in the of a heterologous with hClpP and can target substrates to hClpP. Human ClpX, does not the substrates recognized by although can target other specific proteins to hClpP for recognition not to be dependent on a homologous interaction and not to be specific for the or substrate of a ClpP does the or of degradation, the be conserved between E. coli and human The of hClpX in the activity in the substrates used in are not for In the heterologous λO protein and as as eClpXP and of indicating that when substrates are recognized by the and to hClpP, they can be physiological substrates and or recognized by hClpX in of the enzymatic of hClpP and also in for the substrate for the is overlap in the in substrates by hClpP and the of the and to Human ClpP may an side the and a the of of a and a of The of that the an that interacts with to the in to be are the of to the with that found in also be important to the in by hClpP has a physiological The the of the most substrates in mitochondria in to in E. the to for their the of hClpP be for of mitochondrial The or of they are the mitochondria or out of the by the found in the membrane L. K. T. J. Langer T. 2001; PubMed Scopus Google Scholar). eClpX and eClpA interact with but eClpX can to hClpP. eClpA not activate or for hClpP. The heterologous cannot be data indicate that the of between Clp and ClpP may more of Clp ATPase. of the Clp that interact with ClpP have a conserved found in a of and in the the with the S.K. J. J. J. Maurizi M.R. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, I. K. Sauer R.T. Baker T.A. Biol. 2001; PubMed Scopus Google Scholar). In ClpA, this in a on the that interacts with M. R. L. and D. The in hClpX is in a in to more and for In the homologous interaction of hClpX with hClpP is also that between The of eClpA to to hClpP that is in or that some between eClpA and hClpP that is in the eClpX-hClpP between the two proteins indicate that are in the in hClpP and as as a C-terminal extension of 28 amino in hClpP. the of both ClpP and ClpA have is that they also to interaction are the of to and activity of complexes between Clp and the ClpP The of and the of and proteolytic activity of human to a for proteins in protein quality control and in the of regulatory protein physiological substrates or in functions of hClpXP have been other ATP-dependent proteases play a in mitochondrial In yeast, the Lon protease C.K. Suda K. Schatz G. 1994; PubMed Scopus Google I. H. van L. Langer T. Neupert W. J. 1994; PubMed Scopus Google Scholar) and the membrane-associated proteases, and L. Langer T. Cell. Sci. 1999; 56: PubMed Scopus Google are required for assembly of membrane complexes in mitochondria, as as to misfolded Lon protease also has a regulatory L. Neupert W. Langer T. Genes Dev. 1998; 12: PubMed Scopus Google Scholar). may human such as spastic in which the is in the for a of the mitochondrial membrane protease M. S. M. M. G. A. S. R. A. B. A. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In ClpP has been shown to be important for degradation of the W. Vallon O. Plant Cell. 12: Google and of ClpP activity in a ability to to of that is to have important functions in human mitochondria as has also been to the of mitochondria not as a but as a for that to and and control of regulatory proteins and mitochondrial are important to the that the ClpX and ClpP proteins be for proteins in mitochondrial to physiological roles for human ClpXP.