Proteomics Characterization of Mouse Kidney Peroxisomes by Tandem Mass Spectrometry and Protein Correlation Profiling
2007; Elsevier BV; Volume: 6; Issue: 12 Linguagem: Inglês
10.1074/mcp.m700169-mcp200
ISSN1535-9484
AutoresSebastian Wiese, Thomas Gronemeyer, Rob Ofman, Markus Kunze, Cláudia P. Grou, J. A. Afonso de Almeida, Martin Eisenacher, Christoph Stephan, Heiko Hayen, Lukas Schollenberger, Thomas Korosec, Hans R. Waterham, Wolfgang Schliebs, Ralf Erdmann, Johannes Berger, Helmut E. Meyer, Wilhelm W. Just, Jorge E. Azevedo, Ronald J. A. Wanders, Bettina Warscheid, Cláudia P. Grou,
Tópico(s)Cancer, Lipids, and Metabolism
ResumoThe peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by α- and β-oxidation, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals. To fulfil this vast array of metabolic functions, peroxisomes accommodate ∼50 different enzymes at least as identified until now. Interest in peroxisomes has been fueled by the discovery of a group of genetic diseases in humans, which are caused by either a defect in peroxisome biogenesis or the deficient activity of a distinct peroxisomal enzyme or transporter. Although this research has greatly improved our understanding of peroxisomes and their role in mammalian metabolism, deeper insight into biochemistry and functions of peroxisomes is required to expand our knowledge of this low abundance but vital organelle. In this work, we used classical subcellular fractionation in combination with MS-based proteomics methodologies to characterize the proteome of mouse kidney peroxisomes. We could identify virtually all known components involved in peroxisomal metabolism and biogenesis. Moreover through protein localization studies by using a quantitative MS screen combined with statistical analyses, we identified 15 new peroxisomal candidates. Of these, we further investigated five candidates by immunocytochemistry, which confirmed their localization in peroxisomes. As a result of this joint effort, we believe to have compiled the so far most comprehensive protein catalogue of mammalian peroxisomes. The peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by α- and β-oxidation, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals. To fulfil this vast array of metabolic functions, peroxisomes accommodate ∼50 different enzymes at least as identified until now. Interest in peroxisomes has been fueled by the discovery of a group of genetic diseases in humans, which are caused by either a defect in peroxisome biogenesis or the deficient activity of a distinct peroxisomal enzyme or transporter. Although this research has greatly improved our understanding of peroxisomes and their role in mammalian metabolism, deeper insight into biochemistry and functions of peroxisomes is required to expand our knowledge of this low abundance but vital organelle. In this work, we used classical subcellular fractionation in combination with MS-based proteomics methodologies to characterize the proteome of mouse kidney peroxisomes. We could identify virtually all known components involved in peroxisomal metabolism and biogenesis. Moreover through protein localization studies by using a quantitative MS screen combined with statistical analyses, we identified 15 new peroxisomal candidates. Of these, we further investigated five candidates by immunocytochemistry, which confirmed their localization in peroxisomes. As a result of this joint effort, we believe to have compiled the so far most comprehensive protein catalogue of mammalian peroxisomes. Peroxisomes are small organelles present in virtually all eukaryotic cells. They are surrounded by a single membrane and harbor a large set of enzymes that enables them to execute an array of metabolic functions, such as α- and β-oxidation of fatty acids, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals (1Wanders R.J. Waterham H.R. Biochemistry of mammalian peroxisomes revisited.Annu. Rev. Biochem. 2006; 75: 295-332Crossref PubMed Scopus (692) Google Scholar). The biogenesis of peroxisomes includes complex processes such as membrane assembly, import of matrix proteins, and division of mature peroxisomes. These processes require the concerted action of a subcellular machinery composed of more than 20 different proteins, the so-called peroxins (2Heiland I. Erdmann R. Biogenesis of peroxisomes. Topogenesis of the peroxisomal membrane and matrix proteins.FEBS J. 2005; 272: 2362-2372Crossref PubMed Scopus (120) Google Scholar, 3Brown L.A. Baker A. Peroxisome biogenesis and the role of protein import.J. Cell. Mol. Med. 2003; 7: 388-400Crossref PubMed Scopus (55) Google Scholar). Failure in the biogenesis of peroxisomes or deficiencies in the function of single peroxisomal proteins leads to serious diseases in humans, such as Zellweger syndrome and X-linked adrenoleukodystrophy (1Wanders R.J. Waterham H.R. Biochemistry of mammalian peroxisomes revisited.Annu. Rev. Biochem. 2006; 75: 295-332Crossref PubMed Scopus (692) Google Scholar). Although much has been learned about peroxisomes in recent years, crucial aspects of their functional activities and biogenesis still remain a conundrum.The combination of subcellular fractionation and mass spectrometric analysis, referred to as organellar proteomics (for reviews, see Refs. 4Yates III, J.R. Gilchrist A. Howell K.E. Bergeron J.J. Proteomics of organelles and large cellular structures.Nat. Rev. Mol. Cell Biol. 2005; 6: 702-714Crossref PubMed Scopus (343) Google Scholar, 5Andersen J.S. Mann M. Organellar proteomics: turning inventories into insights.EMBO Rep. 2006; 7: 874-879Crossref PubMed Scopus (165) Google Scholar, 6Dreger M. Subcellular proteomics.Mass Spectrom. Rev. 2003; 22: 27-56Crossref PubMed Scopus (134) Google Scholar), is a powerful method that facilitates the comprehensive characterization of subcellular structures, such as peroxisomes. However, the low abundance of peroxisomes combined with the limited ability to purify this organelle has complicated the proteomics analysis of peroxisomes until now (7Saleem R.A. Smith J.J. Aitchison J.D. Proteomics of the peroxisome.Biochim. Biophys. Acta. 2006; 1763: 1541-1551Crossref PubMed Scopus (40) Google Scholar). Several proteomics studies have been performed on peroxisomes from Saccharomyces cerevisiae cultured in oleate-containing medium which induces the formation of peroxisomes (8Schafer H. Nau K. Sickmann A. Erdmann R. Meyer H.E. Identification of peroxisomal membrane proteins of Saccharomyces cerevisiae by mass spectrometry.Electrophoresis. 2001; 22: 2955-2968Crossref PubMed Scopus (80) Google Scholar, 9Yi E.C. Marelli M. Lee H. Purvine S.O. Aebersold R. Aitchison J.D. Goodlett D.R. Approaching complete peroxisome characterization by gas-phase fractionation.Electrophoresis. 2002; 23: 3205-3216Crossref PubMed Scopus (177) Google Scholar, 10Marelli M. Smith J.J. Jung S. Yi E. Nesvizhskii A.I. Christmas R.H. Saleem R.A. Tam Y.Y. Fagarasanu A. Goodlett D.R. Aebersold R. Rachubinski R.A. Aitchison J.D. Quantitative mass spectrometry reveals a role for the GTPase Rho1p in actin organization on the peroxisome membrane.J. Cell Biol. 2004; 167: 1099-1112Crossref PubMed Scopus (128) Google Scholar). To isolate peroxisomes with high purity and in adequate yields from mammalian cells, Kikuchi et al. (11Kikuchi M. Hatano N. Yokota S. Shimozawa N. Imanaka T. Taniguchi H. Proteomic analysis of rat liver peroxisome: presence of peroxisome-specific isozyme of Lon protease.J. Biol. Chem. 2004; 279: 421-428Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar) performed density gradient centrifugation of rat liver preparations using Nycodenz followed by immunoaffinity purification using an antibody against the abundant peroxisomal membrane protein (PMP) 1The abbreviations used are: PMP, peroxisomal membrane protein; GPF, gas-phase fractionation; IPI, International Protein Index; PCP, protein correlation profiling; SIM, selected ion monitoring; FA, formic acid; 3D, three-dimensional; PBE, peroxisomal bifunctional enzyme; EGFP, enhanced green fluorescent protein; ALDP, adrenoleukodystrophy protein; PEX, peroxin; ER, endoplasmic reticulum; ABC, ATP-binding cassette; ALDR, adrenoleukodystrophy-related; BACAT, bile acid-CoA:amino acid N-acyltransferase; PTS1, peroxisomal targeting signal type 1; FALDH, fatty aldehyde dehydrogenase; HCT, high capacity trap; LTQ, linear trap quadrupole; MOCO, molybdenium cofactor; MOSC, molybdenium cofactor sulfurase; AAA, ATPases associated with various cellular activities. 1The abbreviations used are: PMP, peroxisomal membrane protein; GPF, gas-phase fractionation; IPI, International Protein Index; PCP, protein correlation profiling; SIM, selected ion monitoring; FA, formic acid; 3D, three-dimensional; PBE, peroxisomal bifunctional enzyme; EGFP, enhanced green fluorescent protein; ALDP, adrenoleukodystrophy protein; PEX, peroxin; ER, endoplasmic reticulum; ABC, ATP-binding cassette; ALDR, adrenoleukodystrophy-related; BACAT, bile acid-CoA:amino acid N-acyltransferase; PTS1, peroxisomal targeting signal type 1; FALDH, fatty aldehyde dehydrogenase; HCT, high capacity trap; LTQ, linear trap quadrupole; MOCO, molybdenium cofactor; MOSC, molybdenium cofactor sulfurase; AAA, ATPases associated with various cellular activities. 70. Proteomics analysis of these peroxisomal preparations by SDS-PAGE followed by LC/tandem MS resulted in the identification of more than 50 bona fide constituents as well as a new isoform of Lon protease. Further studies of rat liver peroxisomes using two-dimensional gel electrophoretic techniques and MS led to the identification of microsomal glutathione S-transferase (12Islinger M. Luers G.H. Zischka H. Ueffing M. Volkl A. Insights into the membrane proteome of rat liver peroxisomes: microsomal glutathione-S-transferase is shared by both subcellular compartments.Proteomics. 2006; 6: 804-816Crossref PubMed Scopus (52) Google Scholar) as well as nudix hydrolase 19, referred to as RP2 (13Ofman R. Speijer D. Leen R. Wanders R.J. Proteomic analysis of mouse kidney peroxisomes: identification of RP2p as a peroxisomal nudix hydrolase with acyl-CoA diphosphatase activity.Biochem. J. 2006; 393: 537-543Crossref PubMed Scopus (69) Google Scholar). In addition, the known microsomal proteins aldehyde dehydrogenase, cytochrome b5, and its corresponding reductase were detected in peroxisomal preparations from rat liver (11Kikuchi M. Hatano N. Yokota S. Shimozawa N. Imanaka T. Taniguchi H. Proteomic analysis of rat liver peroxisome: presence of peroxisome-specific isozyme of Lon protease.J. Biol. Chem. 2004; 279: 421-428Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 12Islinger M. Luers G.H. Zischka H. Ueffing M. Volkl A. Insights into the membrane proteome of rat liver peroxisomes: microsomal glutathione-S-transferase is shared by both subcellular compartments.Proteomics. 2006; 6: 804-816Crossref PubMed Scopus (52) Google Scholar). However, no information on the origin of these proteins (i.e. whether they are derived from peroxisomes or potentially co-purified microsomes) was provided by the descriptive proteomics strategies applied in these studies. To address this important issue, Aitchison and co-workers (10Marelli M. Smith J.J. Jung S. Yi E. Nesvizhskii A.I. Christmas R.H. Saleem R.A. Tam Y.Y. Fagarasanu A. Goodlett D.R. Aebersold R. Rachubinski R.A. Aitchison J.D. Quantitative mass spectrometry reveals a role for the GTPase Rho1p in actin organization on the peroxisome membrane.J. Cell Biol. 2004; 167: 1099-1112Crossref PubMed Scopus (128) Google Scholar) introduced a relative quantitative MS-based proteomics approach to determine the enrichment or depletion of proteins detected in two peroxisomal membrane preparations from yeast that differed in their degree of purity. By determining the abundance ratios of the proteins identified in these two fractions, they were able to identify new peroxisome-associated proteins. At about the same time, strategies were developed to enable the profiling of hundreds of proteins through various fractions of a density gradient using quantitative MS in combination with (14Dunkley T.P. Dupree P. Watson R.B. Lilley K.S. The use of isotope-coded affinity tags (ICAT) to study organelle proteomes in Arabidopsis thaliana.Biochem. Soc. Trans. 2004; 32: 520-523Crossref PubMed Scopus (57) Google Scholar) or without (15Andersen J.S. Wilkinson C.J. Mayor T. Mortensen P. Nigg E.A. Mann M. Proteomic characterization of the human centrosome by protein correlation profiling.Nature. 2003; 426: 570-574Crossref PubMed Scopus (1039) Google Scholar) stable isotope labels. These quantitative profiling approaches combined with statistical analyses were shown to allow for the reliable cellular location of proteins in a global manner, thereby providing an excellent means by which new insights into the proteomes and functions of subcellular structures can be obtained (15Andersen J.S. Wilkinson C.J. Mayor T. Mortensen P. Nigg E.A. Mann M. 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A. 2006; 103: 6518-6523Crossref PubMed Scopus (435) Google Scholar, 19Lilley K.S. Dupree P. Methods of quantitative proteomics and their application to plant organelle characterization.J. Exp. Bot. 2006; 57: 1493-1499Crossref PubMed Scopus (65) Google Scholar).In the present work, we report the proteomics characterization of mammalian peroxisomes. We used differential and Nycodenz density gradient centrifugation to isolate peroxisomes from mouse kidney with high purity. Application of MS-based proteomics methodologies enabled the identification of virtually all known resident proteins of the matrix as well as the membrane compartment of mammalian peroxisomes. Moreover through localization studies by protein correlation profiling combined with statistical analyses, we identified 15 new candidate peroxisomal proteins in mouse kidney. The presence of five of these candidate proteins (zinc-binding alcohol dehydrogenase domain-containing protein 2, acyl-coenzyme A dehydrogenase family member 11, acyl-CoA-binding protein 5, the RIKEN cDNA clone 2810439K08 designated here as PMP52, and MOCO sulfurase C-terminal domain-containing 2 protein) in peroxisomes was confirmed by in vivo studies. Although the first three proteins appear to reside in the matrix and PMP52 is in all likelihood a new integral membrane component of mammalian peroxisomes of unknown function, the latter protein was shown to be localized in both peroxisomes and mitochondria. As a result, we believe to have compiled the so far most comprehensive catalogue of mammalian peroxisomes.DISCUSSIONSubcellular fractionation combined with MS-based proteomics analysis provides a most powerful means to establish comprehensive protein catalogues of organelles. Because organelles are not static entities but rather dynamic cellular structures, the respective protein components can only be defined for a specific tissue, cell type, and/or metabolic state at a given time. A major benefit of organellar proteomics is that the complexity of an organelle-enriched sample is, in theory, compatible with the sensitivity and dynamic range of current MS-based methods, even allowing for the identification of proteins of low abundance. Yet thorough purification of subcellular structures remains a key factor in obtaining meaningful data in organellar proteomics. This is particularly true for mammalian peroxisomes, which contribute to only 1–5% of the cell volume depending on tissue type and metabolic state. Furthermore mitochondrial and ER membranes, which generally account for the largest fraction of total cellular membranes, may impede the detection of low abundance PMPs (36Gouveia A.M. Reguenga C. Oliveira M.E. Eckerskorn C. Sa-Miranda C. Azevedo J.E. Alkaline density gradient floatation of membranes: polypeptide composition of the mammalian peroxisomal membrane.Anal. Biochem. 1999; 274: 270-277Crossref PubMed Scopus (19) Google Scholar). This problem is further emphasized by the fact that PMPs only contribute to ∼10% of the entire proteome of peroxisomes. Moreover although PMP70 and PMP22 are highly abundant in peroxisomal membranes (as a rough estimate 50% of total PMPs), peroxins such as PEX13 and PEX12 each account for less than 0.1% of total peroxisomal protein (35Reguenga C. Oliveira M.E. Gouveia A.M. Sa-Miranda C. Azevedo J.E. Characterization of the mammalian peroxisomal import machinery: Pex2p, Pex5p, Pex12p, and Pex14p are subunits of the same protein assembly.J. Biol. Chem. 2001; 276: 29935-29942Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar).The current literature lists ∼75 proteins as components of mouse kidney peroxisomes of which 48 reside in the matrix and 27 reside in the membrane. Of these, we identified 42 peroxisomal matrix and 22 peroxisomal membrane proteins (64 proteins in total, 85% coverage) by proteomics investigations of purified mouse kidney peroxisomes. We only failed to detect two ATP-binding cassette (ABC) transporters, two peroxins, and seven matrix proteins that were shown previously to localize to mammalian peroxisomes (Supplemental Tables 1 and 2).Four ABC transporters, namely ALDP, adrenoleukodystrophy-related (ALDR) protein, PMP70, and PMP69, are reported to reside in the membrane of mammalian peroxisomes (1Wanders R.J. Waterham H.R. Biochemistry of mammalian peroxisomes revisited.Annu. Rev. Biochem. 2006; 75: 295-332Crossref PubMed Scopus (692) Google Scholar, 39Theodoulou F.L. Holdsworth M. Baker A. Peroxisomal ABC transporters.FEBS Lett. 2006; 580: 1139-1155Crossref PubMed Scopus (97) Google Scholar, 40Wanders R.J. Visser W.F. van Roermund C.W. Kemp S. Waterham H.R. The peroxisomal ABC transporter family.Pfluegers Arch. Eur. J. Physiol. 2007; 453: 719-734Crossref PubMed Scopus (78) Google Scholar). The functional importance of these transporters is demonstrated by mutations in the ALD gene that encodes for ALDP causing X-linked adrenoleukodystrophy, an inherited neurodegenerative disorder in which saturated, very long-chain fatty acids accumulate because of impaired β-oxidation in peroxisomes (1Wanders R.J. Waterham H.R. Biochemistry of mammalian peroxisomes revisited.Annu. Rev. Biochem. 2006; 75: 295-332Crossref PubMed Scopus (692) Google Scholar, 39Theodoulou F.L. Holdsworth M. Baker A. Peroxisomal ABC transporters.FEBS Lett. 2006; 580: 1139-1155Crossref PubMed Scopus (97) Google Scholar). Interestingly there is evidence that these ABC transporters show some functional redundancy, providing new possibilities for the treatment of X-linked adrenoleukodystrophy patients (41Kemp S. Wanders R.J. X-linked adrenoleukodystrophy: very long-chain fatty acid metabolism, ABC half-transporters and the complicated route to treatment.Mol. Genet. Metab. 2007; 90: 268-276Crossref PubMed Scopus (63) Google Scholar, 42Kemp S. Wei H.M. Lu J.F. Braiterman L.T. McGuinness M.C. Moser A.B. Watkins P.A. Smith K.D. Gene redundancy and pharmacological gene therapy: implications for X-linked adrenoleukodystrophy.Nat. Med. 1998; 4: 1261-1268Crossref PubMed Scopus (206) Google Scholar, 43Netik A. Forss-Petter S. Holzinger A. Molzer B. Unterrainer G. Berger J. Adrenoleukodystrophy-related protein can compensate functionally for adrenoleukodystrophy protein deficiency (X-ALD): implications for therapy.Hum. Mol. Genet. 1999; 8: 907-913Crossref PubMed Scopus (119) Google Scholar). The expression of these membrane proteins was found to vary in different tissues (44Smith K.D. Kemp S. Braiterman L.T. Lu J.F. Wei H.M. Geraghty M. Stetten G. Bergin J.S. Pevsner J. Watkins P.A. X-linked adrenoleukodystrophy: genes, mutations, and phenotypes.Neurochem. Res. 1999; 24: 521-535Crossref PubMed Scopus (152) Google Scholar). For example, a high level of PMP70 but very low ALDR expression was observed in mouse kidney as assessed by mRNA analysis (45Berger J. Albet S. Bentejac M. Netik A. Holzinger A. Roscher A.A. Bugaut M. Forss-Petter S. The four murine peroxisomal ABC-transporter genes differ in constitutive, inducible and developmental expression.Eur. J. Biochem. 1999; 265: 719-727Crossref PubMed Scopus (85) Google Scholar). Using current proteomics methodologies, we could readily identify both PMP70 and ALDP in mouse kidney peroxisomes, whereas ALDR and PMP69 remained elusive. Because we successfully identified PMPs of very low abundance such as PEX13, we hypothesize that both ALDR and PMP69 show very low expression in mouse kidney peroxisomes. The inability to detect the peroxins PEX7 and PEX19 can be rationalized by their mainly cytosolic localization due to their function as shuttling receptor proteins (46Sacksteder K.A. Jones J.M. South S.T. Li X. Liu Y. Gould S.J. PEX19 binds multiple peroxisomal membrane proteins, is predominantly cytoplasmic, and is required for peroxisome membrane synthesis.J. Cell Biol. 2000; 148: 931-944Crossref PubMed Scopus (237) Google Scholar, 47Rehling P. Marzioch M. Niesen F. Wittke E. Veenhuis M. Kunau W.H. The import receptor for the peroxisomal targeting signal 2 (PTS2) in Saccharomyces cerevisiae is encoded by the PAS7 gene.EMBO J. 1996; 15: 2901-2913Crossref PubMed Scopus (141) Google Scholar). If peripherally attached to the membrane, they are in all likelihood removed by carbonate treatment of peroxisome samples as performed in this work. Furthermore we did not detect the peroxisomal matrix proteins PTE1C, bile acid-CoA:amino acid N-acyltransferase (BACAT), polyamine oxidase, malonyl-CoA decarboxylase, MPV17, XDH, and NUDT7. The latter, peroxisomal nudix hydrolase 7, exhibits highest expression in liver but only intermediate expression in kidney (48Gasmi L. McLennan A.G. The mouse Nudt7 gene encodes a peroxisomal nudix hydrolase specific for coenzyme A and its derivatives.Biochem. J. 2001; 357: 33-38Crossref PubMed Scopus (78) Google Scholar). A similar pattern of expression was found for the human orthologue, NUDT7. However, NUDT7 as well as xanthine oxidoreductase, XDH (49Terao M. Cazzaniga G. Ghezzi P. Bianchi M. Falciani F. Perani P. Garattini E. Molecular cloning of a cDNA coding for mouse liver xanthine dehydrogenase. Regulation of its transcript by interferons in vivo.Biochem. J. 1992; 283: 863-870Crossref PubMed Scopus (107) Google Scholar), were not detected in peroxisomal preparations from rat liver by proteomics studies either (11Kikuchi M. Hatano N. Yokota S. Shimozawa N. Imanaka T. Taniguchi H. Proteomic analysis of rat liver peroxisome: presence of peroxisome-specific isozyme of Lon protease.J. Biol. Chem. 2004; 279: 421-428Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 12Islinger M. Luers G.H. Zischka H. Ueffing M. Volkl A. Insights into the membrane proteome of rat liver peroxisomes: microsomal glutathione-S-transferase is shared by both subcellular compartments.Proteomics. 2006; 6: 804-816Crossref PubMed Scopus (52) Google Scholar). Yet Kikuchi et al. (11Kikuchi M. Hatano N. Yokota S. Shimozawa N. Imanaka T. Taniguchi H. Proteomic analysis of rat liver peroxisome: presence of peroxisome-specific isozyme of Lon protease.J. Biol. Chem. 2004; 279: 421-428Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar) were able to detect BACAT, a member of the type I acyl-CoA thioesterases, in rat liver peroxisomes. BACAT was recently shown to be strongly expressed in liver, and moreover the human orthologue was mainly found in the cytosol (50Solaas K. Kase B.F. Pham V. Bamberg K. Hunt M.C. Alexson S.E. Differential regulation of cytosolic and peroxisomal bile acid amidation by PPAR α activation favors the formation of unconjugated bile acids.J. Lipid Res. 2004; 45: 1051-1060Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 51O'Byrne J. Hunt M.C. Rai D.K. Saeki M. Alexson S.E. The human bile acid-CoA:amino acid N-acyltransferase functions in the conjugation of fatty acids to glycine.J. Biol. Chem. 2003; 278: 34237-34244Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Accordingly if expressed at all, BACAT may only be present in vanishing low amounts in mouse kidney peroxisomes. Furthermore low expression of the peroxisomal acyl-CoA thioesterase Ic (PTE1C) in kidney was reported (52Westin M.A. Alexson S.E. Hunt M.C. Molecular cloning and characterization of two mouse peroxisome proliferator-activated receptor α (PPARα)-regulated peroxisomal acyl-CoA thioesterases.J. Biol. Chem. 2004; 279: 21841-21848Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), providing us with a reasonable explanation why this protein was not detected in this work.Recent data initiated a new debate on the cellular localization of MPV17. Although Zwacka et al. (53Zwacka R.M. Reuter A. Pfaff E. Moll J. Gorgas K. Karasawa M. Weiher H. The glomerulosclerosis gene Mpv17 encodes a peroxisomal protein producing reactive oxygen species.EMBO J. 1994; 13: 5129-5134Crossref PubMed Scopus (90) Google Scholar) reported a role for MPV17 in the peroxisomal reactive oxygen metabolism, Spinazzola et al. (54Spinazzola A. Viscomi C. Fernandez-Vizarra E. Carrara F. D'Adamo P. Calvo S. Marsano R.M. Donnini C. Weiher H. Strisciuglio P. Parini R. Sarzi E. Chan A. DiMauro S. Rotig A. Gasparini P. Ferrero I. Mootha V.K. Tiranti V. Zeviani M. MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion.Nat. Genet. 2006; 38: 570-575Crossref PubMed Scopus (340) Google Scholar) just recently demonstrated that this protein is an integral constituent of the mitochondrial inner membrane and that its absence or malfunction causes failure of oxidative phosphorylation. The latter investigation (54Spinazzola A. Viscomi C. Fernandez-Vizarra E. Carrara F. D'Adamo P. Calvo S. Marsano R.M. Donnini C. Weiher H. Strisciuglio P. Parini R. Sarzi E. Chan A. DiMauro S. Rotig A. Gasparini P. Ferrero I. Mootha V.K. Tiranti V. Zeviani M. MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion.Nat. Genet. 2006; 38: 570-575Crossref PubMed Scopus (340) Google Scholar) supports proteomics data, i.e. the failure to detect MPV17 in preparations of mammalian peroxisomes as reported here and in previous studies (11Kikuchi M. Hatano N. Yokota S. Shimozawa N. Imanaka T. Taniguchi H. Proteomic analysis of rat liver peroxisome: presence of peroxisome-specific isozyme of Lon protease.J. Biol. Chem. 2004; 279: 421-428Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 12Islinger M. Luers G.H. Zischka H. Ueffing M. Volkl A. Insights into the membrane proteome of rat liver peroxisomes: microsomal glutathione-S-transferase is shared by both subcellular compartments.Proteomics. 2006; 6: 804-816Crossref PubMed Scopus (52) Google Scholar). Two further proteins were not detected in our study, N1-acetylated polyamine oxidase exhibiting only low expression in kidney peroxisomes (55Wu T. Yankovskaya V. McIntire W.S. Cloning, sequencing, and heterologous expression of the murine peroxisomal flavoprotein, N1-acetylated polyamine oxidase.J. Biol. Chem. 2003; 278: 20514-20525Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) as well as a putative peroxisomal form of malonyl-CoA decarboxylase (56Sacksteder K.A. Morrell J.C. Wanders R.J. Matalon R. Gould S.J. MCD encodes peroxisomal and cytoplasmic forms of malonyl-CoA decarboxylase and is mutated in malonyl-CoA decarboxylase deficiency.J. Biol. Chem. 1999; 274: 24461-24468Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). In view of the above discussion, we argue that we were able to detect virtually all known resident proteins of mouse kidney peroxisomes. This inventory also includes enzymes just recently designated as peroxisomal, such as the acyltransferase ACNAT1 (57Reilly S.J. O'shea E.M. Andersson U. O'Byrne J. Alexson S.E. Hunt M.C. A peroxisomal acyltransferase in mouse identifies a novel pathway for taurine conjugation of fatty acids.FASEB J. 2007; 21: 99-107Crossref PubMed Scopus (29) Google Scholar), RP2 (13Ofman R. Speijer D. Leen R. Wanders R.J. Proteomic analysis of mouse kidney peroxisomes: identification of RP2p as a peroxisomal nudix hydrolase with acyl-CoA diphosphatase activity.Biochem. J. 2006; 393: 537-543Crossref PubMed Scopus (69) Google Scholar), and the Lon protease (11Kikuchi M. Hatano N. Yokota S. Shimozawa N. Imanaka T. Taniguchi H. Proteomic analysis of rat liver peroxisome: presence of peroxisome-specific isozyme of Lon protease.J. Biol. Chem. 2004; 279: 421-428Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar).Through elaborate PCP combined with statistical analyses, we provided a set of 15 new peroxisomal candidates of which four proteins, namely ZADH2, ACAD11, ACBD5, and the RIKEN cDNA clone 2810439K08 (designated as PMP52), were validated by immunocytochemistry (Table II and Fig. 2). Six of these candidates (ACAD11, ACBD5, MDH1, CYB5A, DIA1, and ALDH3A2) were also detected in peroxisome preparations from rat liver (11Kikuchi M. Hatano N. Yokota S. Shimozawa N. Imanaka T. Taniguchi H. Proteomic analysis of rat liver peroxisome: presence of peroxisome-specific isozyme of Lon protease.J. Biol. Chem. 2004; 279: 421-428Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 12Islinger M. Luers G.H. Zischka H. Ueffing M. Volkl A. Insights into the mem
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