Cloning, Expression, Characterization, and Interaction of Two Components of a Human Mitochondrial Fatty Acid Synthase
2003; Elsevier BV; Volume: 278; Issue: 41 Linguagem: Inglês
10.1074/jbc.m306121200
ISSN1083-351X
AutoresLei Zhang, Anil K. Joshi, Sean C. Smith,
Tópico(s)Chemical Synthesis and Analysis
ResumoThe possibility that human cells contain, in addition to the cytosolic type I fatty acid synthase complex, a mitochondrial type II malonyl-CoA-dependent system for the biosynthesis of fatty acids has been examined by cloning, expressing, and characterizing two putative components. Candidate coding sequences for a malonyl-CoA:acyl carrier protein transacylase (malonyltransferase) and its acyl carrier protein substrate, identified by BLAST searches of the human sequence data base, were located on nuclear chromosomes 22 and 16, respectively. The encoded proteins localized exclusively in mitochondria only when the putative N-terminal mitochondrial targeting sequences were present as revealed by confocal microscopy of HeLa cells infected with appropriate green fluorescent protein fusion constructs. The mature, processed forms of the mitochondrial proteins were expressed in Sf9 cells and purified, the acyl carrier protein was converted to the holoform in vitro using purified human phosphopantetheinyltransferase, and the functional interaction of the two proteins was studied. Compared with the dual specificity malonyl/acetyltransferase component of the cytosolic type I fatty acid synthase, the type II mitochondrial counterpart exhibits a relatively narrow substrate specificity for both the acyl donor and acyl carrier protein acceptor. Thus, it forms a covalent acyl-enzyme complex only when incubated with malonyl-CoA and transfers exclusively malonyl moieties to the mitochondrial holoacyl carrier protein. The type II acyl carrier protein from Bacillus subtilis, but not the acyl carrier protein derived from the human cytosolic type I fatty acid synthase, can also function as an acceptor for the mitochondrial transferase. These data provide compelling evidence that human mitochondria contain a malonyl-CoA/acyl carrier protein-dependent fatty acid synthase system, distinct from the type I cytosolic fatty acid synthase, that resembles the type II system present in prokaryotes and plastids. The final products of this system, yet to be identified, may play an important role in mitochondrial function. The possibility that human cells contain, in addition to the cytosolic type I fatty acid synthase complex, a mitochondrial type II malonyl-CoA-dependent system for the biosynthesis of fatty acids has been examined by cloning, expressing, and characterizing two putative components. Candidate coding sequences for a malonyl-CoA:acyl carrier protein transacylase (malonyltransferase) and its acyl carrier protein substrate, identified by BLAST searches of the human sequence data base, were located on nuclear chromosomes 22 and 16, respectively. The encoded proteins localized exclusively in mitochondria only when the putative N-terminal mitochondrial targeting sequences were present as revealed by confocal microscopy of HeLa cells infected with appropriate green fluorescent protein fusion constructs. The mature, processed forms of the mitochondrial proteins were expressed in Sf9 cells and purified, the acyl carrier protein was converted to the holoform in vitro using purified human phosphopantetheinyltransferase, and the functional interaction of the two proteins was studied. Compared with the dual specificity malonyl/acetyltransferase component of the cytosolic type I fatty acid synthase, the type II mitochondrial counterpart exhibits a relatively narrow substrate specificity for both the acyl donor and acyl carrier protein acceptor. Thus, it forms a covalent acyl-enzyme complex only when incubated with malonyl-CoA and transfers exclusively malonyl moieties to the mitochondrial holoacyl carrier protein. The type II acyl carrier protein from Bacillus subtilis, but not the acyl carrier protein derived from the human cytosolic type I fatty acid synthase, can also function as an acceptor for the mitochondrial transferase. These data provide compelling evidence that human mitochondria contain a malonyl-CoA/acyl carrier protein-dependent fatty acid synthase system, distinct from the type I cytosolic fatty acid synthase, that resembles the type II system present in prokaryotes and plastids. The final products of this system, yet to be identified, may play an important role in mitochondrial function. The existence of a mitochondrial system for the biosynthesis of fatty acids in animals was first reported 40 years ago and postulated to function by a reversal of the process of fatty acid β-oxidation (1Seubert W. Greull G. Lynen F. Angew. Chem. 1959; 69: 359Crossref Google Scholar, 2Wakil S.J. J. Lipid Res. 1961; 2: 1-24Abstract Full Text PDF Google Scholar, 3Whereat A.F. Hull F.E. Orishimo M.W. J. Biol. Chem. 1967; 242: 4013-4022Abstract Full Text PDF PubMed Google Scholar). Indeed one laboratory reported that short and medium chain-length fatty acids could be produced using the partially purified mitochondrial β-oxidation enzymes, β-ketoacyl thiolase, β-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and enoyl-CoA reductase (4Seubert W. Lamberts I. Kramer R. Ohly B. Biochim. Biophys. Acta. 1968; 164: 498-517Crossref PubMed Scopus (111) Google Scholar), and another found that inhibitory antibodies to pig heart 3-ketoacyl-CoA thiolase inhibited, in a parallel fashion, the fatty acid elongation system of pig heart mitochondria (5Staack H. Davidson B. Schulz H. Lipids. 1980; 15: 175-178Crossref PubMed Scopus (5) Google Scholar). The picture was complicated by reports that liver and heart mitochondria have two distinct fatty acid-synthesizing systems, one acetyl-CoA-dependent, possibly involving some of the β-oxidation enzymes, the other malonyl-CoA-dependent and apparently resembling the cytosolic FAS 1The abbreviations used are: FAS, fatty acid synthase; ACP, acyl carrier protein; ACPmit, mitochondrial acyl carrier protein; ACPfas, acyl carrier protein domain of the fatty acid synthase; MTmit, mitochondrial malonyltransferase; MATfas, malonyl/acetyltransferase of the cytosolic fatty acid synthase; PCP, peptidyl carrier protein; PBS, phosphate-buffered saline; GFP, green fluorescent protein. system. Most studies were in agreement that the acetyl-CoA-dependent system was an elongation pathway in which one or more C2 units, derived from acetyl-CoA, were added to preexisting saturated and unsaturated fatty acids of a broad range of chain length (C6–C20). In this pathway, at least one of the β-oxidation enzymes appears to be substituted by a specific biosynthetic enzyme: the FAD-dependent acyl-CoA dehydrogenase being replaced by a more thermodynamically favorable enzyme, enoyl-CoA reductase (6Seubert W. Podack E.R. Mol. Cell. Biochem. 1973; 1: 29-40Crossref PubMed Scopus (84) Google Scholar). The possible presence of a malonyl-CoA-dependent system capable of synthesizing saturated fatty acids C14–C18 de novo in mitochondria was hotly debated with some investigators suggesting that contamination with cytosolic FAS and/or components of a microsomal malonyl-CoA-dependent elongation system was very likely responsible for the widely varying activity of this pathway reported by different laboratories (7Christ E.J.V.J. Biochim. Biophys. Acta. 1968; 152: 50-62Crossref PubMed Scopus (25) Google Scholar, 8Quagliariello E. Landriscina C. Coratelli P. Biochim. Biophys. Acta. 1968; 164: 12-24Crossref PubMed Scopus (62) Google Scholar, 9Whereat A.F. Orishimo M.W. Nelson J. Phillips S.J. J. Biol. Chem. 1969; 244: 6498-6506Abstract Full Text PDF PubMed Google Scholar, 10Podack E.R. Seubert W. Biochim. Biophys. Acta. 1972; 280: 235-247Crossref PubMed Scopus (36) Google Scholar). Perhaps because of these early conflicting reports and inherent difficulties in working with this system, the animal mitochondrial FAS has received little attention over the last 25 years, and almost all of the recent advances in our knowledge have come from studies with fungi and plants. The first notable advance was the discovery that mitochondria of both Neurospora crassa and Saccharomyces cerevisiae contain a small molecular mass phosphopantetheinylated protein that resembles closely the acyl carrier proteins (ACPs) characteristically associated with the type II 2The enzymes of the type II FASs exist as separate discrete proteins, whereas those of type I FASs are covalently linked in multifunctional polypeptides. FAS systems of prokaryotes and plants (11Brody S. Mikolajczyk S. Eur. J. Biochem. 1988; 173: 353-359Crossref PubMed Scopus (55) Google Scholar, 12Schneider R. Massow M. Lisowsky T. Weiss H. Curr. Genet. 1995; 29: 10-17Crossref PubMed Scopus (93) Google Scholar). Unexpectedly this mitochondrial ACP was found to be associated with the NADH:ubiquinone reductase (proton-pumping respiratory complex 1) in N. crassa. This is not the case in S. cerevisiae, however, since this organism lacks complex 1 and respires using an alternative, non-proton-pumping, ubiquinone oxidoreductase. Disruption of the nuclear-encoded gene for ACPmit in both N. crassa and S. cerevisiae produced respiratory-deficient phenotypes (12Schneider R. Massow M. Lisowsky T. Weiss H. Curr. Genet. 1995; 29: 10-17Crossref PubMed Scopus (93) Google Scholar, 13Brody S. Oh C. Hoja U. Schweizer E. FEBS Lett. 1997; 408: 217-220Crossref PubMed Scopus (133) Google Scholar): in N. crassa, complex 1 was improperly assembled, and lysophospholipids accumulated in mitochondrial membranes, and in S. cerevisiae cellular lipoic acid was reduced to less than 10% of that of the wild-type strain. Studies on plants have also revealed that mitochondria are capable of synthesizing both long chain acyl-ACPs and octanoyl-ACP, the direct precursor of lipoic acid (14Gueguen V. Macherel D. Jaquinod M. Douce R. Bourguignon J. J. Biol. Chem. 2000; 275: 5016-5025Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 15Wada H. Shintani D. Ohlrogge J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1591-1596Crossref PubMed Scopus (177) Google Scholar). Meanwhile genes encoding several putative mitochondrial proteins, β-ketoacyl synthase (CEM1), β-ketoacyl reductase (OAR1), malonyl-CoA:ACP transferase (MCT1), and enoyl reductase (YBR026c), that resemble type II FAS enzymes were identified in S. cerevisiae (16Schneider R. Brors B. Massow M. Weiss H. FEBS Lett. 1997; 407: 249-252Crossref PubMed Scopus (50) Google Scholar, 17Harington A. Herbert C.J. Tung B. Getz G.S. Slonimski P.P. Mol. Microbiol. 1993; 9: 545-555Crossref PubMed Scopus (57) Google Scholar, 18Torkko J.M. Koivuranta K.T. Miinalainen I.J. Yagi A.I. Schmitz W. Kastaniotis A.J. Airenne T.T. Gurvitz A. Hiltunen K.J. Mol. Cell. Biol. 2001; 21: 6243-6253Crossref PubMed Scopus (66) Google Scholar). Disruption of each of these genes produced a respiratory deficient phenotype. In summary, fungal and plant mitochondria appear to contain an ACP-dependent lipogenic system that likely is responsible for the production of fatty acids that play essential roles in mitochondrial function. An ACP-like protein was found in animal mitochondria several years ago and shown to be a subunit of respiratory complex 1 (19Runswick M.J. Fearnley I.M. Skehel J.M. Walker J.E. FEBS Lett. 1991; 286: 121-124Crossref PubMed Scopus (159) Google Scholar, 20Triepels R. Smeitink J. Loeffen J. Smeets R. Buskens C. Trijbels F. van den Heuvel L. J. Inherit. Metab. Dis. 1999; 22: 163-173Crossref PubMed Scopus (36) Google Scholar). However, the role of this ACP is not known, and no other components of a putative animal mitochondrial FAS system have been identified or characterized. In an attempt to clarify the role of this system, we have initiated a program to identify and characterize the components of a putative type II mitochondrial FAS in humans. In this communication we report the identification, cloning, and expression of a mitochondrial malonyltransferase and the characterization of its interaction with mitochondrial ACP. Prediction of Mitochondrially Imported Proteins—The software programs PSORT (PSORT.nibb.ac.jp), iPSORT (hypothesiscreator.net/iP-SORT/), and MitoProt II (www.mips.biochem.mpg.de/cgi-bin/proj/medgen/mitofilter) were used to predict mitochondrially targeted proteins (21Bannai H. Tamada Y. Maruyama O. Nakai K. Miyano S. Bioinformatics. 2002; 18: 298-305Crossref PubMed Scopus (595) Google Scholar, 22Claros M.G. Vincens P. Eur. J. Biochem. 1996; 241: 779-786Crossref PubMed Scopus (1388) Google Scholar). Cloning of the Human Mitochondrial Malonyltransferase—Based on the available sequence information (GenBank™ accession number GI 8574363), PCR primers were designed to amplify the putative human mitochondrial MT DNA using expressed sequence tag cDNA clone ATCC 4811508 as the template DNA. The PCR procedure used was essentially as described earlier (23Joshi A.K. Smith S. Biochem. J. 1993; 296: 143-149Crossref PubMed Scopus (41) Google Scholar). Three sets of primers were designed to facilitate expression of the full-length mitochondrial MT (MTmit1), the putative N-terminally processed form lacking the first 21 residues (MTmit22), and an N-terminally truncated form lacking the first 59 residues that is of similar length to the MT of Escherichia coli (MTmit60). Thus, the primer sets MT1.bac.T/B, MT22.bac.T/B, and MT60.bac.T/B were used to amplify constructs encoding MTmit1, MTmit22, and MTmit60, respectively (Table I).Table IPCR primers and linkers used in this study Expression of the Human Mitochondrial Malonyltransferase in E. coli—DNA fragments encoding MTmit22 and MTmit60 were cloned into the pET-29c(+) vector, and expression was attempted in BL21-Codon-Plus cells (Stratagene, La Jolla, CA). Cells were grown at 37 °C in LB medium to A 600 of 0.5–0.6 and induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 3 h. However, in both cases, induction of protein expression resulted in cessation of cell growth within 1 h. The level of protein expression was very low, and almost all of the recombinant protein was recovered in inclusion bodies. Lowering the isopropyl-1-thio-β-d-galactopyranoside concentration, lowering the growth temperature, or using alternative host strains did not improve expression of soluble mitochondrial MT. Expression of the Human Mitochondrial Malonyltransacylase in Insect Sf9 Cells—The amplified constructs encoding MTmit1, MTmit22, and MTmit60 were purified using QIAquick PCR purification kit (Qiagen Inc., Valencia, CA), digested at XbaI and NheI sites that had been engineered into the amplification primers T and B, respectively, and cloned into the modified baculoviral transfer vector pFast Bac 1 (Invitrogen). To facilitate purification of the encoded proteins by metal ion affinity chromatography, an in-frame coding sequence for a C-terminal His6 tag was engineered into the cloned cDNAs. Authenticity of the cloned PCR fragments was confirmed by DNA sequencing, recombinant baculoviral stocks were generated, and the encoded protein was expressed in Sf9 insect cells using the Bac-to-Bac baculoviral expression system (Invitrogen) according to the manufacturer's instructions. The amino acid sequences of the encoded proteins are shown in Supplemental Fig. 1. A construct encoding both His6 and FLAG affinity tags at the C terminus was engineered by digesting the pMTmit22.FB plasmid DNA with the restriction enzymes NheI and NotI and ligating the ct.H6.flag.T/B linker (Table I) in-frame with the coding sequence. Subcellular Fractionation of Sf9 Cells Expressing the Full-length and Putative N-terminally Processed Forms of Human Mitochondrial Malonyltransferase—Sf9 cells were infected with recombinant baculovirus encoding either the MTmit1 or MTmit22 proteins carrying only the His6 affinity tag, harvested by centrifugation at 600 × g for 5 min, washed with PBS, and resuspended at 4 °C in 5 volumes of mitochondrial isolation buffer (210 mm mannitol, 70 mm sucrose, 5 mm HEPES-KOH, pH 7.35, 1 mm EDTA) containing protease inhibitors (5 μg/ml leupeptin, 10 μm trans-epoxysuccinyl-l-leucylamido(4-guanidino)-butane, 1 μg/ml pepstatin, 5 μg/ml antitrypsin). The cellular suspension was disrupted using a Teflon/glass homogenizer and centrifuged at 800 × g for 10 min at 4 °C to remove cell debris. The supernatant was then centrifuged at 10,000 × g for 10 min at 4 °C. The supernatant was collected and further centrifuged at 100,000 × g for 30 min at 4 °C. The 100,000 × g supernatant (cytosol) was collected, and the pellet (microsomal fraction) was resuspended in isolation buffer to the same volume as that of the cytosolic fraction. The 10,000 × g pellet (mitochondrial fraction) was washed twice with isolation buffer and resuspended in isolation buffer in the same volume as cytosol. Purification of the Human Mitochondrial Malonyltransferase—Sf9 cells were infected with baculoviruses encoding either MTmit22 or MTmit60. For purification of the cytosolic form, insect cells were homogenized at 4 °C in buffer A (50 mm HEPES, pH 8.0, 300 mm NaCl, 10% glycerol) containing protease inhibitors and centrifuged at 140,000 × g for 60 min at 4 °C. The supernatant was filtered (0.45 μm) and loaded at 20 °C onto a HiTrap Chelating HP column (5-ml bed volume, Amersham Biosciences). The column was washed with 50 mm imidazole in buffer A. The bound proteins were eluted with 250 mm imidazole in buffer A. For further purification of the doubly tagged MTmit22, fractions containing the enzyme were pooled and applied to a column containing 2 ml of anti-FLAG M2 agarose (Sigma), which was equilibrated with buffer A. The column was washed with 20 ml of buffer A, and the bound proteins were eluted with 1.5 mg/ml FLAG peptide in buffer A. Cloning, Expression, Purification, and Phosphopantetheinylation of the Human Mitochondrial ACP—The putative ACPmit sequence (20Triepels R. Smeitink J. Loeffen J. Smeets R. Buskens C. Trijbels F. van den Heuvel L. J. Inherit. Metab. Dis. 1999; 22: 163-173Crossref PubMed Scopus (36) Google Scholar) was identified and cloned from a human expressed sequence tag using the PCR. The coding sequence for the putative processed form lacking the mitochondrial targeting sequence (ACPmit68) was cloned into a baculoviral vector that also encoded a C-terminal His6 tag, and the recombinant protein was expressed in insect Sf9 cells. The soluble ACPmit68 protein was purified by a combination of nickel ion affinity chromatography and anion exchange chromatography and phosphopantetheinylated in vitro with purified recombinant human phosphopantetheinyltransferase. The details of these procedures are described elsewhere (24Joshi A.K. Zhang L. Rangan V.S. Smith S. J. Biol. Chem. 2003; 278: 33142-33149Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Expression and Phosphopantetheinylation of the Peptidyl Carrier Protein (PCP) and ACP-A from Bacillus brevis and Bacillus subtilis, Respectively—E. coli cells harboring plasmids encoding C-terminally His6-tagged versions of ACP-A (M15 pQE60[acpA], Ref. 25Mootz H.D. Finking R. Marahiel M.A. J. Biol. Chem. 2001; 276: 37289-37298Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) and PCP (M15 pQE70[TycC3PCP], Ref. 26Weber T. Baumgartner R. Renner C. Marahiel M.A. Holak T.A. Struct. Fold. Des. 2000; 8: 407-418Abstract Full Text Full Text PDF Scopus (164) Google Scholar) were obtained from Dr. Mohamed A. Marahiel at the Institut fur Biochemie, Fachbereich Chemie, Philipps-Universitat Marburg, Marburg, Germany. Cells were grown at 37 °C to a density equivalent to A 600 of 0.7, and protein expression was induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 3–4 h at either 37 °C (M15) or 30 °C (BL21). Proteins were purified by nickel ion affinity chromatography and phosphopantetheinylated in vitro using human phosphopantetheine transferase (24Joshi A.K. Zhang L. Rangan V.S. Smith S. J. Biol. Chem. 2003; 278: 33142-33149Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). A typical phosphopantetheinylation reaction contained 100 μm apo-ACP-A or apo-PCP, 0.2 mm CoA, 10 mm MgCl2, 1 mm Tris(2-carboxyethyl)phosphine hydrochloride, and 1 μm phosphopantetheinyltransferase. The reaction components were incubated at 37 °C overnight. The holo-ACP-A or holo-PCP were separated from phosphopantetheinyltransferase by nickel ion affinity chromatography. Confirmation that at least 90% of the apoforms had been converted to the corresponding holoforms was obtained by incubation of a portion of the product with [1-14C]acetyl-CoA and human phosphopantetheinyltransferase and assaying the incorporation of radiolabel into the ACP (only residual apoform is radiolabeled by [1-14C]acetyl-phosphopantetheine transfer (24Joshi A.K. Zhang L. Rangan V.S. Smith S. J. Biol. Chem. 2003; 278: 33142-33149Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar)). Expression and Purification of the Malonyl/Acetyltransferase and Acyl Carrier Protein Domains of Mammalian FASs—A cDNA fragment encoding the ACP domain of the human cytosolic FAS (amino acid residues 2117–2205) was cloned into the expression vector pQE80 L (Qiagen) and expressed as a soluble protein in E. coli. The recombinant ACP was purified to homogeneity by a combination of nickel ion affinity chromatography and gel filtration. Details are described elsewhere (24Joshi A.K. Zhang L. Rangan V.S. Smith S. J. Biol. Chem. 2003; 278: 33142-33149Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The malonyl/acetyltransferase domain of the rat cytosolic FAS (amino acid residues 488–809) was expressed in E. coli inclusion bodies, refolded in vitro, and purified as described earlier (27Rangan V.S. Serre L. Witkowska H.E. Bari A. Smith S. Protein Eng. 1997; 10: 561-566Crossref PubMed Scopus (13) Google Scholar). Verification That the Putative N-terminal Mitochondrial Recognition Elements Direct the Mitochondrial FAS Proteins into Mitochondria—A 1170-bp fragment encoding MTmit1 was amplified using PCR primer set MT1.gfp.T/B (Table I) and directionally cloned into the KpnI/BamHI sites of pEGFP-N3 vector (Clontech) to facilitate expression as the N-terminal partner of a MTmit1-green fluorescent protein (GFP) fusion protein. A second fusion protein construct (primer set MT22.gfp.T/B), lacking the coding sequence for the N-terminal 21 amino acids (putative targeting sequence), was similarly engineered as a control. Authenticity of the cloned fragments was confirmed by DNA sequencing. Expression vectors encoding the fusion proteins were transfected into HeLa cells using FuGENE 6 reagent (Roche Applied Science), and the cells were cultured for 60 h. Cells were then fixed with paraformaldehyde in PBS, treated with 50 mm NH4Cl (in PBS), permeabilized with 0.1% Triton X-100 (in PBS), washed with PBS, and treated with bovine serum albumin blocking solution. The cells were then treated with the primary antibody (purified mouse anti-cytochrome c monoclonal antibody), washed with PBS, and treated with the fluorescent secondary antibody (Cy3-conjugated affinity-purified goat anti-mouse IgG, Jackson ImmunoResearch Labs., West Grove, PA). Finally the washed cells were mounted in glycerol/PBS and examined for green and red fluorescence in a Sony confocal fluorescence microscope. The ACPmit-GFP fusion constructs were generated in a similar manner. The two ACP-specific PCR-amplified fragments (ACP1.gfp.T/B and ACP68.gfp.T/B primer set products for constructs with and without the putative mitochondrial targeting sequence, respectively; Table I) were cloned into the EcoRI/KpnI sites of pEGFP-N3 vector, and authenticity of the cloned fragments was confirmed by DNA sequencing. Transfection into HeLa cells was carried out using FuGENE 6 reagent as above except that ACPmit-GFP fusion constructs were cotransfected with plasmid pDsRed2-Mito (Clontech) for fluorescent labeling of mitochondria. Cells were cultured for 60 h, fixed with paraformaldehyde as above, and directly analyzed for green and red fluorescence by confocal microscopy. Formation of a Covalent Malonyl-O-malonyltransferase Intermediate—MTmit22 (250 ng) was incubated with either 36 μm [2-14C]malonyl-CoA (51 Ci/mol) or 66 μm [1-14C]acetyl-CoA (54 Ci/mol) in 50 mm potassium phosphate buffer, pH 6.8 for 5 min at 20 °C and subjected to SDS-PAGE on 12% acrylamide gels. Gels were either stained with Coomassie Brilliant Blue, or the proteins were electroblotted onto a polyvinylidene difluoride membrane and analyzed both by phosphorimaging and by Western blotting using mouse anti-PentaHis monoclonal antibody (Qiagen) as primary antibody and alkaline phosphatase-coupled goat anti-mouse IgG antibodies (Bio-Rad) as secondary antibody. Assay of Human Mitochondrial Malonyltransferase Activity—Typical assay systems contained 83 mm potassium phosphate buffer, pH 6.8, 20 μm [2-14C]malonyl-CoA, 10–20 μm holo-ACP, and 50 ng of MTmit22. Incubations were carried out at 20 °C for 2 min. Proteins were precipitated with acid, washed, and assayed for radioactivity. Blank reactions were performed without enzyme. Kinetic parameters were determined using EnzymeKinetics (Trinity Software); values represent the means ± S.D. for calculations using six different methods. Identification of Sequences of the Putative Mitochondrial ACP and Malonyltransferase Proteins—BLAST searches of the eukaryotic sequence data bases, using as probe sequences malonyltransferases from either prokaryotic, type II, or eukaryotic type I FAS systems, identified likely malonyltransferase sequences with N-terminal extensions in several species (see Supplemental Fig. 1A). Sequences for putative malonyltransferases from human, rat, mouse, Drosophila, and Neurospora were predicted by several computational methods (PSORT, iPSORT, and MitoProt II) to have a high probability of containing N-terminal mitochondrial targeting sequences (see Supplemental Fig. 1A). In addition, all of these sequences contained three positionally conserved features that are characteristic of malonyl and malonyl/acetyltransferases: a Gly-Xaa-Ser-Xaa-Gly serine esterase active site motif, a conserved Arg residue that interacts with the 3-carboxylate of the malonyl substrate, and a conserved His residue that is required for activation of the Ser nucleophile (27Rangan V.S. Serre L. Witkowska H.E. Bari A. Smith S. Protein Eng. 1997; 10: 561-566Crossref PubMed Scopus (13) Google Scholar, 28Serre L. Verbree E.C. Dauter Z. Stuitje A.R. Derewenda Z.S. J. Biol. Chem. 1995; 270: 12961-12964Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 29Rangan V.S. Smith S. J. Biol. Chem. 1996; 271: 31749-31755Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 30Rangan V.S. Smith S. J. Biol. Chem. 1997; 272: 11975-11978Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). On the basis of this analysis, we identified expressed sequence tag cDNA clones encoding the putative human mitochondrial MT protein for further study. A similar approach identified ACP sequences in animals, fungi, and plants that have a high probability of containing putative mitochondrial targeting sequences (see Supplemental Fig. 1B). The coding sequence for the putative mitochondrial MT is located on human chromosome 22 (22q13.31), and that for the mitochondrial ACP is located on chromosomes 16 (16p12.3) in the human genome sequence data base. The coding sequences for both proteins are interrupted by three introns. Verification That the Putative N-terminal Mitochondrial Recognition Elements Direct the Mitochondrial FAS Proteins into Mitochondria—Vectors encoding mitochondrial ACP and MT sequences fused to the N terminus of GFP were engineered and expressed in HeLa cells. Confocal fluorescence microscopic analysis revealed that both the ACPmit-GFP and MTmit-GFP chimeras colocalized with mitochondrial markers when the putative N-terminal mitochondrial targeting sequence was present (Fig. 1, A and B, left column, ACPmit1-GFP and MTmit1-GFP, respectively). In contrast, GFP expressed throughout the cytoplasm when either the entire mitochondrial ACP or MT coding sequence or the putative mitochondrial targeting sequence was omitted from the cDNA constructs (Fig. 1, A and B, center and right columns, GFP, ACPmit68-GFP, and MTmit22-GFP, respectively). These results confirmed that the sequences identified do indeed encode bona fide mitochondrially targeted proteins. Expression of Mitochondrial Malonyltransferase in Sf9 Cells: Subcellular Localization of Recombinant Protein—Because the mitochondrial MT could not be expressed as a soluble protein in E. coli (see "Experimental Procedures" for details), we explored the possibility of using the insect Sf9/baculoviral host/vector system. Cells infected with recombinant baculoviruses encoding either the full-length (MTmit1) or the putative processed form (MTmit22) were homogenized, and mitochondrial, microsomal, and cytosolic fractions were prepared for analysis by SDS-PAGE and Western blotting using anti-His antibodies as the probe (Fig. 2). Cells expressing the full-length form produced immunoreactive recombinant protein mainly in the mitochondrial fraction. Two distinct molecular species were observed with different electrophoretic mobilities. A smaller amount of immunoreactive protein was found associated with the microsomal fraction that had a mobility identical to that of the slower moving species present in the mitochondrial fraction. Only a trace of immunoreactive protein was found in the cytosolic fraction. Cells expressing the putative N-terminally processed form of the enzyme produced, in all three subcellular fractions, a single molecular mass immunoreactive species. The electrophoretic mobility of this species was identical to that of the faster moving species found associated with the mitochondrial fraction in cells infected with baculovirus encoding the full-length protein. These results indicated that most of the full-length MTmit1 species was taken up and partially processed by insect cell mitochondria but that a small portion became associated with the microsomal fraction and remained
Referência(s)