Proteomic Analysis of Rat Liver Peroxisome

Subcellular proteomics, which includes isolation of subcellular components prior to a proteomic analysis, is advantageous not only in characterizing large macro-molecular complexes such as organelles but also in elucidating mechanisms of protein transport and organelle biosynthesis. Because of the high sensitivity achieved by the present proteomics technology, the purity of samples to be analyzed is important for the interpretation of the results obtained. In the present study, peroxisomes isolated from rat liver by usual cell fractionation were further purified by immunoisolation using a specific antibody raised against a peroxisomal membrane protein, PMP70. The isolated peroxisomes were analyzed by SDS-PAGE combined with liquid chromatography/mass spectrometry. Altogether 34 known peroxisomal proteins were identified in addition to several mitochondrial and microsomal proteins. Some of the latter may reside in the peroxisomes as well. Analysis of membrane fractions identified all known peroxins except for Pex7. Two new peroxisomal proteins of unknown function were of high abundance. One is a bi-functional protein consisting of an aminoglycoside phosphotransferase-domain and an acyl-CoA dehydrogenase domain. The other is a newly identified peroxisome-specific isoform of Lon protease, an ATP-dependent protease with chaperone-like activity. The peroxisomal localization of the protein was confirmed by immunological techniques. The peroxisome-type Lon protease, which is distinct from the mitochondrial isoform, may play an important role in the peroxisomal biogenesis. Subcellular proteomics, which includes isolation of subcellular components prior to a proteomic analysis, is advantageous not only in characterizing large macro-molecular complexes such as organelles but also in elucidating mechanisms of protein transport and organelle biosynthesis. Because of the high sensitivity achieved by the present proteomics technology, the purity of samples to be analyzed is important for the interpretation of the results obtained. In the present study, peroxisomes isolated from rat liver by usual cell fractionation were further purified by immunoisolation using a specific antibody raised against a peroxisomal membrane protein, PMP70. The isolated peroxisomes were analyzed by SDS-PAGE combined with liquid chromatography/mass spectrometry. Altogether 34 known peroxisomal proteins were identified in addition to several mitochondrial and microsomal proteins. Some of the latter may reside in the peroxisomes as well. Analysis of membrane fractions identified all known peroxins except for Pex7. Two new peroxisomal proteins of unknown function were of high abundance. One is a bi-functional protein consisting of an aminoglycoside phosphotransferase-domain and an acyl-CoA dehydrogenase domain. The other is a newly identified peroxisome-specific isoform of Lon protease, an ATP-dependent protease with chaperone-like activity. The peroxisomal localization of the protein was confirmed by immunological techniques. The peroxisome-type Lon protease, which is distinct from the mitochondrial isoform, may play an important role in the peroxisomal biogenesis. Peroxisome is a ubiquitous organelle present in nearly all eukaryotic cells and involved in various oxidative enzymatic reactions such as the α- and β-oxidation of fatty acids. 1The peroxisome website, http://www.peroxisome.org. 1The peroxisome website, http://www.peroxisome.org. Enzymes catalyzing these reactions are well characterized and found in the matrix of the organelle. These matrix proteins are synthesized in the cytoplasm and imported across its membrane. Proteins localized in the peroxisomal membranes are also known, and some of them are involved in the transport of small metabolites and peroxisomal proteins. Biogenesis of the organelle proceeds via a cellular machinery consisting of more than 20 proteins (peroxins) that effect the recognition, targeting and import of peroxisomal proteins (1.Deleted in proofGoogle Scholar). In human, dysfunction of the machinery results in inherited metabolic disorders and peroxisome biogenesis disorders such as Zellweger syndrome. Some of the genes responsible for the disease have yet to be identified. To characterize the protein composition of the organelle, therefore, is important not only in studying the proteins involved in the functions of the organelle, but also in elucidating the molecules responsible for the organelle's biogenesis. Because of the rapid expansion of protein sequence databases, mass spectrometry (MS) 2The abbreviations used are: MS, mass spectrometry; ER, endoplasmic reticulum; LC, liquid chromatography; PMP70, 70-kDa peroxisomal membrane protein; PMP22, 22-kDa peroxisomal membrane protein; CBB, Coomassie Brilliant Blue; AAA, ATPases associated with diverse cellular activities; PTS-1, peroxisomal targeting signal-1; ACDH, acyl-CoA dehydrogenase; ACBP, acyl-CoA-binding protein. 2The abbreviations used are: MS, mass spectrometry; ER, endoplasmic reticulum; LC, liquid chromatography; PMP70, 70-kDa peroxisomal membrane protein; PMP22, 22-kDa peroxisomal membrane protein; CBB, Coomassie Brilliant Blue; AAA, ATPases associated with diverse cellular activities; PTS-1, peroxisomal targeting signal-1; ACDH, acyl-CoA dehydrogenase; ACBP, acyl-CoA-binding protein.-based identification of proteins, which relies on the sequence data base, is becoming more realistic and more reliable (2.O'Donovan C. Bairoch A. Apweiler R. Trends Biotechnol. 2001; 19: 178-181Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 3.Shevchenko A. Jensen O.N. Podtelejnikov A.V. Sagliocco F. Wilm M. Vorm O. Mortensen P. Shevchenko A. Boucherie H. Mann M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14440-14445Crossref PubMed Scopus (1283) Google Scholar, 4.Pandey A. Mann M. Nature. 2000; 405: 837-846Crossref PubMed Scopus (1899) Google Scholar). One important field of application of this emerging technique is to analyze proteins in whole cells and tissues, and thousands of proteins expressed can be detected and identified (5.Washburn M.P. Wolters D. Yates J.R. Nat. Biotechnol. 2001; 19: 242-247Crossref PubMed Scopus (4029) Google Scholar, 6.Wu C.C. Yates J.R. Nat. Biotechnol. 2003; 21: 262-267Crossref PubMed Scopus (499) Google Scholar). The same technique can be used to analyze subcellular fractions ranging from organelles to large macromolecular complexes such as ribosomes. Analyzing subcellular fractions reduces the complexity of the samples to be analyzed and should yield more information on the minor protein components that would otherwise escape detection (7.Jung E. Heller M. Sanchez J.C. Hochstrasser D.F. Electrophoresis. 2000; 21: 3369-3377Crossref PubMed Scopus (161) Google Scholar, 8.Bell A.W. Ward M.A. Blackstock W.P. Freeman H.N.M. Choudhary J.S. Lewis A.P. Chotai D. Fazel A. Gushue J.N. Paiement J. Palcy S. Chevet E. Lafrenière-Roula M. Solari R. Thomas D.Y. Rowley A. Bergeron J.J.M. J. Biol. Chem. 2001; 276: 5152-5165Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 9.Andersen J.S. Lyon C.E. Fox A.H. Leung A.K.L. Lam Y.W. Steen H. Mann M. Lamond A.I. Cur. Biol. 2002; 12: 1-11Abstract Full Text Full Text PDF PubMed Scopus (805) Google Scholar, 10.Hanson B.J. Schulenberg B. Patton W.F. Capaldi R.A. Electrophoresis. 2001; 22: 950-959Crossref PubMed Scopus (87) Google Scholar). A classical way of cell fractionation, i.e. differential centrifugation after homogenization, is clearly not adequate for such analyses, since the analytical methods employed may detect minor contaminants. Methods are necessary that allow purification of such macromolecular complexes without losing the structural or functional integrity of the complex. Peroxisomes are usually purified by combining the classical differential centrifugation and density gradient centrifugation. These fractions still contain other contaminating organelles such as mitochondria. In the present study, we combined the Nycodenz density gradient centrifugation with immunoisolation using anti-PMP70 (70-kDa peroxisomal membrane protein) antibody. The fractions obtained were practically free from contaminating mitochondria. MS-based proteomic analyses (11.Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Nat. Biotechnol. 1999; 17: 994-999Crossref PubMed Scopus (4293) Google Scholar) gave an image of the “proteome” of the liver peroxisomes with the composition and the relative abundance of the protein components. In addition to the known peroxisomal proteins, several endoplasmic reticulum (ER) and mitochondrial proteins, which seem to exist in the organelle, and a few new proteins of unknown function were identified. One of them is a peroxisome-specific isozyme of Lon protease, which acts as molecular chaperone. The protein may play an important role in the biogenesis of the peroxisome. Materials—Polyclonal antibody against rat PMP70 was raised using a synthetic peptide corresponding to the C-terminal 15 amino acids (12.Imanaka T. Shiina Y. Takano T. Hashimoto T. Osumi T. J. Biol. Chem. 1996; 271: 3706-3713Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Specific antibody was purified from rabbit antiserum by affinity chromatography using an antigen-bound column (SulfoLink kit, Pierce). Goat anti-rabbit IgG (Fc fragment-specific) was obtained from the same source. Nycodenz was obtained from Daiichi Pure Chemicals Co., Ltd. (Tokyo, Japan), while Dynabeads M-500 Subcellular were from DYNAL (Oslo, Norway). Protease inhibitor mixture tablets were purchased from Roche Diagnostics (Mannheim, Germany). Other chemicals and biochemicals were from Wako Pure Chemical Industries (Osaka, Japan). Preparation of Rat Liver Peroxisomes—The peroxisomal fraction from rat liver was prepared by conventional differential centrifugation in sucrose followed by isopycnic centrifugation in Nycodenz (13.Imanaka T. Lazarow P.B. Takano T. Biochim. Biophys. Acta. 1991; 1062: 264-270Crossref PubMed Scopus (18) Google Scholar) with some modifications. About 0.6 ml of a light mitochondrial fraction-containing peroxisomes was layered onto 10 ml of linear Nycodenz gradient (density span from 1.15 to in a Japan). The gradient on a of Nycodenz in was for of ml were and the fraction was by the of (13.Imanaka T. Lazarow P.B. Takano T. Biochim. Biophys. Acta. 1991; 1062: 264-270Crossref PubMed Scopus (18) Google Scholar). of specific antibody was to using an anti-rabbit antibody as a antibody to the The fractions from the Nycodenz density gradient of protein) were with the in for complexes were by the in a The was with Peroxisomes were by in of for SDS-PAGE and 20 The by in a was to membranes were obtained by the peroxisomes with for complexes were with and and with the as with purified peroxisomal fraction was to SDS-PAGE and with of the A. Wilm M. Vorm O. Mann M. Chem. 1996; PubMed Scopus Google Scholar) or with Coomassie Brilliant of the the proteomic a from was of was small and the of a Japan). The were by in of were by with 10 in for and with in for in the were with and in a were with 15 of in was were with were in and in The peptide obtained were to and mass was in a mass with a H. M. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, H. E. H. T. H. Y. U. Y. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). A linear gradient of was a and the was a column The column was a with a The was in a peptide a were to the obtained from the were used to proteins using against the C-terminal amino acids of was raised in Japan). The of the protein subcellular fractions was by of protein of differential centrifugation and light mitochondrial were to SDS-PAGE and onto a membrane. The membrane was with the antibody and by of rat liver cells with Lon protease antibody was as T. T. R. R. M. S. J. 2001; PubMed Scopus Google Scholar). of was T. Y. T. Y. T. Y. PubMed Scopus Google Scholar). used in this were from a peroxisomal protein Y. M. S. S. M. T. Hashimoto T. J. Full Text Full Text PDF PubMed Scopus Google Scholar) and a Zellweger T. Y. T. Y. J. 2003; PubMed Scopus (18) Google Scholar). of Peroxisomes by fractions obtained from the conventional differential centrifugation were further by Nycodenz gradient centrifugation. peroxisomes were on the of a Peroxisome was further purified by immunoisolation with with against PMP70 as samples obtained and after the were a few were the while some were clearly Some of the protein that were and to the One protein was found of mitochondrial A few proteins of small molecular were from the cytoplasm not should be that mitochondrial and proteins such as and were not contaminating mitochondrial and proteins seem to be by the the other of the peroxisomal membrane proteins, PMP22, was clearly the peroxisomal matrix proteins were not The of the matrix protein, to while the corresponding to matrix protein, clearly in its These results that to be some of matrix proteins the purification of the composition was Proteins in the in the SDS-PAGE obtained from the purified peroxisomal fractions was of the of and to the The peptide were analyzed by as was used to proteins in the to 10 proteins were identified in a Proteins identified are in of identified for mitochondrial were Proteins known to be localized in peroxisomes are than to the from the for the protein in from the were to for was for molecular by with the molecular and with a molecular of involved in peroxisome to peroxisome biogenesis biogenesis membrane protein membrane protein protein membrane membrane protein acyl-CoA acyl-CoA peroxisomal acyl-CoA acyl-CoA of fatty peroxisomal dehydrogenase A amino mitochondrial dehydrogenase microsomal membrane protein, associated protein A membrane protein, associated protein and to expressed sequence to protein Proteins for mitochondrial were Proteins known to be localized in peroxisomes are than to the from the for the protein in from the were to for The was for molecular by with the molecular and with a molecular of proteins in a new 34 known peroxisomal proteins, and proteins were of unknown mitochondrial proteins and proteins were identified in addition to the proteins from other subcellular the known peroxisomal proteins, proteins were peroxisomal membrane proteins. In addition to the matrix proteins, and as proteins of the peroxisomal matrix were found in the of them are involved in proteins were peroxins involved in the various of the peroxisome biogenesis. mitochondrial and proteins identified in the present can be to be peroxisomal proteins than contaminating proteins from other for this be in we only of and peroxisomal membrane proteins, we have isolated peroxisomal membranes by the peroxisome with The membranes were from the and to the same The peroxins and peroxisomal membrane proteins identified are in and only and are in the is not since these proteins are is to the relative of these proteins found in the whole and membrane fractions of the organelle. is the peroxins in the whole but the of the protein relative to other peroxins to be by results that of the protein is associated with the peroxisome and can be with the other which have with a small associated with the were found in the membrane The of the protein, therefore, is to that of proteins. the peroxins that are and were in the membrane the of the isolation other membrane proteins of the organelle, which were not detected in the whole peroxisomal were found in the from the the relative abundance of and with that of the peroxisomal membrane proteins such as PMP70 and of proteins identified in peroxisomal involved in peroxisome membrane protein to biogenesis to peroxisomal biogenesis membrane to Peroxisome protein 10 biogenesis biogenesis to membrane protein to peroxisome biogenesis membrane membrane protein A membrane protein peroxisomal membrane protein to membrane protein, 34 membrane protein in a new of Rat Liver of protein in SDS-PAGE are with the image of a the of the proteins can be are proteins are or are on the of the In some more than protein of were identified in a the the and proteins of the proteins were found not only in a but also in the proteins were detected in the molecular mass from the mass from the known proteins in detected well or the mass from complexes that in the of or from In these only the in which of the proteins were detected were the of of is a from a cell or that of an organelle, is important to the abundance of protein in that specific this is not an we that the by the is with the of protein in that specific The for protein identified is a of peptide detected in the should be that the more a protein the more that from the protein are to be In other a large of protein for the analysis, the sequence and the of detected is the other only a small of protein, such as the the detection is to analysis, few are the for peptide is a function of its more of high are from from proteins. To the the and the relative we the with the of the by image The was for molecular by by the molecular and with a molecular of proteins. of proteins that are found in were proteins are identified were in the is from the is a the Other proteins, which are not in the and and should be localized the should be that the is a of the protein the is and the on the protein. and can be as of proteins. from the a protein is or from the can be have these in the of the peroxisomal proteins The proteins are in of the the and the a image of the of liver peroxisomes can be obtained. In addition to the matrix proteins, and several in the are of high abundance. Some of the membrane proteins such as and PMP70 are proteins, to the matrix proteins. the other the peroxins are minor components in the organelle. In this proteins known to be localized in other subcellular fractions should be Some of them have high with the peroxisomal proteins, that these proteins are peroxisomal proteins than contaminating dehydrogenase and to the proteins in the organelle. may not be to such in Proteins involved in the such as proteins were also identified in the of a of Lon of the proteins of unknown function identified to Lon protease and an associated with diverse cellular A of of Lon of various that exist in One isoform characterized as mitochondrial Lon protease, which is to R. A. C.E. S.A. Google Scholar, O. A. B. O. 2001; PubMed Scopus Google Scholar). The present isoform is an of the structural with an and the C-terminal a protease The are more diverse with the C-terminal should be that the peroxisomal O. A. B. O. 2001; PubMed Scopus Google Scholar, S. F. Biol. PubMed Scopus Google Scholar) with a peroxisomal targeting the protease structural that are the and that the protein is to the To the peroxisomal localization of the Lon we have the cellular localization of the protein in that we have raised a specific antibody against a synthetic peptide on the C-terminal from the sequence only and with the protein. The localization of the protease was by subcellular fractionation, and In the corresponding to was detected only in the fraction were with the the was found in a for proteins A was cells were with antibody not These results that the proteins identified in the and or are the isozyme of Lon protease, which is distinct from the known mitochondrial The same antibody was used for in were associated with the peroxisome an of the peroxisomal a more analysis, of several subcellular components were The density in the was while that in the peroxisomes was the peroxisome the density in the peroxisomal was than that in the matrix that the protein specific localization in the peroxisomal from the and the the present peroxisomal isozyme is an peroxisomal proteins. of a protein of unknown function identified in the present a acyl-CoA dehydrogenase Two are for the protein; and a sequence as to The of the to the aminoglycoside In the present MS-based analysis, from and C-terminal of the sequence were identified. the sequence to be a These proteins with amino or a to as J. 2002; PubMed Scopus Google Scholar). These results the of a bi-functional with an in the is the that an is present not only in the but also in the acyl-CoA to be associated with the organelle P.B. C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Other Proteins of other proteins are in to protein in a high to protein. an acyl-CoA protein in the that characterized as a protein involved in the acyl-CoA In the present analysis, this protein was detected in of molecular 20 and only from the C-terminal were only the C-terminal which the to be localized in the peroxisome. is to proteins are proteins of unknown but are and to which is for mitochondrial J. Biol. 2000; PubMed Scopus Google Scholar). protein is to which some to a J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (18) Google Scholar). In in which the was is an and the amino sequence of of an mitochondrial the C-terminal sequence of the protein, found in this study, is and may function as a Because of the the peroxisomal localization of these proteins should be by other functional proteomics, which is becoming a way to analyze cellular is advantageous in is the complexity of the samples to be and the other is the information on the subcellular localization of protein components such analyses have without of the methods and purity of samples to (7.Jung E. Heller M. Sanchez J.C. Hochstrasser D.F. Electrophoresis. 2000; 21: 3369-3377Crossref PubMed Scopus (161) Google Scholar, 8.Bell A.W. Ward M.A. Blackstock W.P. Freeman H.N.M. Choudhary J.S. Lewis A.P. Chotai D. Fazel A. Gushue J.N. Paiement J. Palcy S. Chevet E. Lafrenière-Roula M. Solari R. Thomas D.Y. Rowley A. Bergeron J.J.M. J. Biol. Chem. 2001; 276: 5152-5165Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 9.Andersen J.S. Lyon C.E. Fox A.H. Leung A.K.L. Lam Y.W. Steen H. Mann M. Lamond A.I. Cur. Biol. 2002; 12: 1-11Abstract Full Text Full Text PDF PubMed Scopus (805) Google Scholar). the other the analytical methods employed are becoming more and more detection of components. In the present study, peroxisomal fractions obtained by differential centrifugation were further purified by density gradient and by from the of several mitochondrial and proteins, the to be practically free from the of the subcellular several proteins, present in the of ER, have proteins such as were not is that some of these proteins are also localized in the One is which is an endoplasmic reticulum protein. from the abundance of the protein, this protein may well be an peroxisomal protein involved in the peroxisomal oxidative protein in in the peroxisomal membranes R. S. A. M. Y. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). and which of the microsomal have to be localized to the endoplasmic reticulum and the membrane of mitochondria. The present that these proteins may also be localized to the of the peroxisomal membranes S. J. A. H. J. M. P. J. Biol. PubMed Scopus Google Scholar). peroxisome proteins were identified that are involved in important functions of peroxisome. the of identified peroxins and that of peroxisomal membrane proteins was proteins that are known to be localized to the peroxisomal matrix were not identified that in the analyzed be The of the present analytical methods from the in the relative abundance of the proteins in a is in of the peroxisomal the peroxisomal membranes with we of the matrix proteins and the proteins. In addition to all the we detect and that are associated with the membrane. to be more to the membrane than These results the of the purification in organelle proteins were identified in the present The was identified as the peroxisomal isozyme of Lon Lon protease is an ATP-dependent protease with an domain. is various and J. PubMed Scopus Google Scholar). with other such as Lon protease is to function as molecular in Methods PubMed Scopus Google Scholar). In these functions are by more but a Lon protease found in of and F. J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Lon in are and R. A. C.E. S.A. Google Scholar). the various Lon The is well in the C-terminal of the that the domain. The are found in the the Lon protease The high with the peroxisomal isoform, are to the mitochondrial Lon The contain the C-terminal for the peroxisomal the isoform as mitochondrial Lon protease R. A. C.E. S.A. Google we can that the isoform is the peroxisome-specific isoform in the in is to be a isoform R. A. C.E. S.A. Google Scholar). this is the have distinct Lon in and peroxisome. In other proteins the are known and are involved in peroxisomal biogenesis and the import of peroxisomal matrix proteins. Lon protease in is in the mitochondrial matrix and is involved in of and PubMed Scopus Google Scholar). functions of Lon protease in peroxisome yet to be is that the protease is involved in the of the matrix proteins imported across the peroxisomal membranes as molecular or in the of the proteins. unknown protein of is a bi-functional an protein as to The C-terminal sequence of the is which can function as a J. 2002; PubMed Scopus Google Scholar). therefore, that the protein an is an peroxisomal protein. in are involved in the of mitochondrial β-oxidation of fatty acids. to the mitochondrial the peroxisomal a in its is of this bi-functional is involved in the peroxisomal fatty The protein of functional is the protein that is to a protein with a molecular mass of an acyl-CoA-binding protein in the ACBP, a protein that may the of acyl-CoA is from the protein and by peroxisome and the peroxisome T. Jensen P. T. J. J. S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). is to a role of this C-terminal from a in the of In we isolated a peroxisome fraction that is practically free from of other cellular and we identified known peroxisomal proteins. a image of relative of protein; a of rat liver peroxisome. A few proteins were to the of which is the newly identified peroxisome-specific isozyme of Lon from the relative of the protein, the Lon protease is an peroxisomal matrix protein. In rat this protein is associated with the in the peroxisomal the Lon function as the peroxisome-specific Lon isozyme is involved in peroxisome biogenesis. The peroxisomal isozyme distinct from the mitochondrial is found not only in but also in is that is a distinct isoform for of the and peroxisome. yet to be the by Lon in organelle are clearly