AtCCMA Interacts with AtCcmB to Form a Novel Mitochondrial ABC Transporter Involved in Cytochrome c Maturation in Arabidopsis
2007; Elsevier BV; Volume: 282; Issue: 29 Linguagem: Inglês
10.1074/jbc.m704091200
ISSN1083-351X
AutoresNaganand Rayapuram, Jérémie Hagenmuller, Jean-Michel Grienenberger, Philippe Giegé, Géraldine Bonnard,
Tópico(s)Metabolism and Genetic Disorders
ResumoABC transporters make a large and diverse family of proteins found in all phylae. AtCCMA is the nucleotide binding domain of a novel Arabidopsis mitochondrial ABC transporter. It is encoded in the nucleus and imported into mitochondria. Sub-organellar and topology studies find AtCCMA bound to the mitochondrial inner membrane, facing the matrix. AtCCMA exhibits an ATPase activity, and ATP/Mg2+ can facilitate its dissociation from membranes. Blue Native PAGE shows that it is part of a 480-kDa complex. Yeast two-hybrid assays reveal interactions between AtCCMA and domains of CcmB, the mitochondria-encoded transmembrane protein of a conserved ABC transporter. All these properties designate the protein as the ortholog in plant mitochondria of the bacterial CcmA required for cytochrome c maturation. The transporter that involves AtCCMA defines a new category of eukaryotic ABC proteins because its transmembrane and nucleotide binding domains are encoded by separate genomes. ABC transporters make a large and diverse family of proteins found in all phylae. AtCCMA is the nucleotide binding domain of a novel Arabidopsis mitochondrial ABC transporter. It is encoded in the nucleus and imported into mitochondria. Sub-organellar and topology studies find AtCCMA bound to the mitochondrial inner membrane, facing the matrix. AtCCMA exhibits an ATPase activity, and ATP/Mg2+ can facilitate its dissociation from membranes. Blue Native PAGE shows that it is part of a 480-kDa complex. Yeast two-hybrid assays reveal interactions between AtCCMA and domains of CcmB, the mitochondria-encoded transmembrane protein of a conserved ABC transporter. All these properties designate the protein as the ortholog in plant mitochondria of the bacterial CcmA required for cytochrome c maturation. The transporter that involves AtCCMA defines a new category of eukaryotic ABC proteins because its transmembrane and nucleotide binding domains are encoded by separate genomes. ATP binding cassette (ABC) 6The abbreviations used are: ABC, ATP binding cassette; NBD, nucleotide binding domain; TMD, transmembrane domain; NAP, non-intrinsic ABC protein; MBP, maltose binding protein; BN, Blue Native; AD, activation domain; BD, binding domain. transporters belong to a large family of proteins that are found in all prokaryotic and eukaryotic species. ABC proteins are composed of four domains, two nucleotide binding domains (NBD) characterized by a highly conserved ATP binding cassette, and two transmembrane domains (TMD). These four modules may be expressed into a single polypeptide (full transporter), two polypeptides (half-transporter), or four polypeptides. The substrates of these transporters are actively transported using ATP hydrolysis as energy source. Substrates include a wide variety of structurally and chemically different compounds such as ions, sugars, lipids, proteins, antibiotics, and drugs (1Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3375) Google Scholar). Other ABC proteins regulate the activity of channels instead of being transporters themselves (2Higgins C.F. Cell. 1995; 82: 693-696Abstract Full Text PDF PubMed Scopus (340) Google Scholar). The analysis of Arabidopsis thaliana nuclear genome has revealed 129 potential ABC proteins, a large number when compared with other eukaryotes (3Sanchez-Fernandez R. Davies T.G. Coleman J.O. Rea P.A. J. Biol. Chem. 2001; 276: 30231-30244Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar). Most of the Arabidopsis ABC proteins could be assigned to known subfamilies with the exception of a heterogeneous group of ABC proteins lacking transmembrane domains that were termed non-intrinsic ABC protein (NAP). These proteins are reminiscent of ATP binding quarter molecules of prokaryotic ABC transporters. However, up to now none of these NAPs was found to be associated to TMDs, thus reconstituting a complete ABC transporter. During evolution, distinct assembly pathways have arisen to achieve the covalent attachment of the heme prosthetic group to apocytochromes c of bacteria, mitochondria, and chloroplasts (4Kranz R. Lill R. Goldman B. Bonnard G. Merchant S. Mol. Microbiol. 1998; 29: 383-396Crossref PubMed Scopus (237) Google Scholar, 5Thoöny-Meyer L. Biochem. Soc. Trans. 2002; 30: 633-638Crossref PubMed Google Scholar, 6Stevens J.M. Daltrop O. Allen J.W. Ferguson S.J. Acc. Chem. Res. 2004; 37: 999-1007Crossref PubMed Scopus (129) Google Scholar). System I was first described in α and γ proteobacteria, system II in chloroplasts, and Gram-positive bacteria and system III in yeast mitochondria. Although the origin of system III could not be traced back to prokaryotes, mitochondria of plants and of some protists have inherited system I from their endosymbiotic α proteobacteria ancestor. One representative for system I is Escherichia coli, where eight genes named ccm for cytochrome c maturation are essential for the periplasmic assembly of c-type cytochromes (7Thoöny-Meyer L. Fischer F. Kunzler P. Ritz D. Hennecke H. J. Bacteriol. 1995; 177: 4321-4326Crossref PubMed Google Scholar). In system I, CcmE is a central player of heme delivery; this so-called heme chaperon binds heme in a covalent but transient manner before its transfer to CcmF, proposed to catalyze the ligation of heme to apocytochrome c (8Schulz H. Hennecke H. Thoöny-Meyer L. Science. 1998; 281: 1197-1200Crossref PubMed Scopus (156) Google Scholar, 9Lee D. Pervushin K. Bischof D. Braun M. Thoöny-Meyer L. J. Am. Chem. Soc. 2005; 127: 3716-3717Crossref PubMed Scopus (52) Google Scholar). In E. coli, ccm genes are organized in an operon starting with ccmA that encodes the nucleotide binding domain of an ABC transporter. The two following genes which encode CcmB and CcmC both contain six transmembrane helices as found in most TMDs. The exact composition of the ABC transporter and its function have been debated for long (for review, see Ref. 10Goldman B.S. Kranz R.G. Res. Microbiol. 2001; 152: 323-329Crossref PubMed Scopus (37) Google Scholar). Briefly, two models have been proposed. The first model proposed that the ABC transporter is involved in heme export and consists of CcmA2BC, with CcmB and CcmC being the two TMDs (11Goldman B.S. Beckman D.L. Bali A. Monika E.M. Gabbert K.K. Kranz R.G. J. Mol. Biol. 1997; 268: 724-738Crossref PubMed Scopus (70) Google Scholar, 12Goldman B.S. Beckman D.L. Monika E.M. Kranz R.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5003-5008Crossref PubMed Scopus (93) Google Scholar). In the second model CcmC delivers heme to the heme chaperon CcmE (13Schulz H. Fabianek R.A. Pellicioli E.C. Hennecke H. ThoönyMeyer L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6462-6467Crossref PubMed Scopus (103) Google Scholar), and the ABC transporter consisting of CcmA2B2 is not involved in heme export (for review, see Ref. 14Cianciotto N.P. Cornelis P. Baysse C. Mol. Microbiol. 2005; 56: 1408-1415Crossref PubMed Scopus (44) Google Scholar). It was recently shown that the ATPase activity of CCMA is required for the release of holo-CcmE from CcmC (15Feissner R.E. Richard-Fogal C.L. Frawley E.R. Kranz R.G. Mol. Microbiol. 2006; 61: 219-231Crossref PubMed Scopus (64) Google Scholar, 16Christensen O. Harvat E.M. Thony-Meyer L. Ferguson S.J. Stevens J.M. FEBS J. 2007; 274: 2322-2332Crossref PubMed Scopus (43) Google Scholar). Feissner et al. (15Feissner R.E. Richard-Fogal C.L. Frawley E.R. Kranz R.G. Mol. Microbiol. 2006; 61: 219-231Crossref PubMed Scopus (64) Google Scholar) proposed that ATP-driven conformational changes result in the liberation of holo-CcmE from CcmA2BC that would act as a chaperon releaser rather than as true transporter. For Christensen et al. (16Christensen O. Harvat E.M. Thony-Meyer L. Ferguson S.J. Stevens J.M. FEBS J. 2007; 274: 2322-2332Crossref PubMed Scopus (43) Google Scholar) the need of ATPase activity for heme transfer from holo-CcmE to apocytochrome c could also be interpreted as the requirement of a compound supplied by the ABC transporter to form or break with the unusual histidine-heme bond in CCME. Transcription studies of the mitochondrial genome of wheat reveal the existence of the ccmB (17Faivre-Nitschke E. Nazoa P. Gualberto J.M. Grienenberger J.M. Bonnard G. Biochim. Biophys. Acta. 2001; 1519: 199-208Crossref PubMed Scopus (22) Google Scholar), ccmC (18Bonnard G. Grienenberger J.M. Mol. Gen. Genet. 1995; 246: 81-99Crossref Scopus (44) Google Scholar), and ccmF genes (19Gonzalez D.H. Bonnard G. Grienenberger J.M. Curr. Genet. 1993; 21: 248-255Crossref Scopus (61) Google Scholar, 20Giegé P. Rayapuram N. Meyer E.H. Grienenberger J.M. Bonnard G. FEBS Lett. 2004; 563: 165-169Crossref PubMed Scopus (23) Google Scholar). Orthologs of ccmABCF were identified as well on the mitochondrial genomes of Reclinomonas americana, a jakobid flagellate with an ancestral mitochondrial genome (21Lang B.F. Burger G. O'Kelly C.J. Cedergren R. Golding G.B. Lemieux C. Sankoff D. Turmel M. Gray M.W. Nature. 1997; 387: 493-496Crossref PubMed Scopus (487) Google Scholar), and of Cyanidioschyzon merolae, a primitive unicellular red alga (22Ohta N. Sato N. Kuroiwa T. Nucleic Acids Res. 1998; 26: 5190-5298Crossref PubMed Scopus (141) Google Scholar). However, although ccmBCF orthologs are conserved on all mitochondrial genomes of land plant, ccmA could not be found on any of them. The sequence similarity between CcmA and the nuclear encoded NAP10 strongly suggests that ccmA has been transferred to the nucleus as shown in A. thaliana for genes encoding two other plant mitochondrial CCM proteins, the heme chaperone AtCCME (23Spielewoy N. Schulz H. Grienenberger J.M. Thoöny-Meyer L. Bonnard G. J. Biol. Chem. 2001; 276: 5491-5497Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), and the thiol-disulfide oxidoreductase AtCCMH (24Meyer E.H. Giegé P. Gelhaye E. Rayapuram N. Ahuja U. ThoönyMeyer L. Grienenberger J.M. Bonnard G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 16113-16118Crossref PubMed Scopus (70) Google Scholar). In this work we show that NAP10 is a mitochondrial protein, which has kept the topology of its bacterial counterpart CcmA and has characteristics of the NBD of an ABC transporter. Interaction between AtCCMA and domains of AtCcmB were revealed by yeast two-hybrid assays. Therefore, we propose that NAP10 is the CcmA counterpart of the mitochondrial CCM ABC transporter. Contrary to the mitochondrial ABC transporters described so far, which belong to the full or half-transporter types (25Lill R. Kispal G. Res. Microbiol. 2001; 152: 331-340Crossref PubMed Scopus (62) Google Scholar), the plant ABC transporter involved in c-type cytochrome maturation makes a novel class of organellar ABC transporter since its TMDs are encoded on the mitochondrial genome and its NBDs are encoded by the nuclear gene NAP10. Cloning and Expression of AtCCMAs, Production of Antibodies—Total RNA was prepared from the aerial parts of 4-week-old A. thaliana plants. cDNA was synthesized using random hexamers as primers for reverse transcription. The coding region of AtCCMA was amplified by PCR using primers P1 and P2 (supplemental Table S1) and cloned into the HindIII/EcoRI sites of pSK, resulting in pNG22, the plasmid used for in vitro transcription/translation. The coding sequence of AtCCMA was amplified using primer P3 and P4 and cloned in pQE60 (Qiagen), generating pNG31. The recombinant CCMA protein expressed in E. coli BL21D3 possesses an additional Val at position +2 and a His6 tag at the C-terminal end. CCMA-His was expressed after induction with 1 mm isopropyl-1-thio-β-d-galactopyranoside, affinity-purified under denaturing conditions on a nickel-nitrilotriacetic acid-Sepharose column (Novagen), resolved on preparative SDS-PAGE, and electroeluted. The purified protein was used to immunize rabbits. Anti-AtCCMA-His antibodies were purified by immunoaffinity for the overexpressed protein coupled with CNBr-activated Sepharose (Amersham Biosciences). Using pNG31 as a template, the PCR product obtained with P5 and P6 was digested by SmaI and introduced in the EcoRV site of pACYC184, generating pNG34 used in complementation experiments. Similarly, the AtCCMA coding region was amplified with P7 and P8 using pNG31 as template. The PCR fragment obtained was digested by HindIII and EcoRI and cloned in the corresponding sites of pMALc2 (New England Biolabs) generating pNG35. The fusion protein maltose binding protein (MBP)-CCMA was expressed in Roseta strain E. coli cells (Novagen). After induction by isopropyl-1-thio-β-d-galactopyranoside, it was purified by affinity chromatography under native conditions using an amylose resin column. Subcellular Fractionation of A. thaliana Protoplasts and Sub-mitochondrial Fractionation—Cytosolic, chloroplast, and mitochondrial fractions from A. thaliana protoplasts were obtained as described previously (26Laloi C. Rayapuram N. Chartier Y. Grienenberger J.M. Bonnard G. Meyer Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14144-14149Crossref PubMed Scopus (223) Google Scholar). Mitoplasts were prepared as described (27Werhahn W. Niemeyer A. Jansch L. Kruft V.V. Schmitz U.K. Braun H.P. Plant Physiol. 2001; 125: 943-954Crossref PubMed Scopus (156) Google Scholar). Mitoplasts were resuspended at a protein concentration of 1 mg/ml, and trypsin was added at 25 μg/mg of mitochondrial proteins and incubated for 30 min on ice. Trypsin inhibitor was added to stop the reaction, and the mitoplasts were recovered after centrifugation through a 27% (w/v) sucrose cushion in 20 mm Tris-HCl, pH 7.5, 1 mm EDTA pH 8.0, 100 mm K2HPO4, and 1 mg/ml bovine serum albumin) at 15,000 × g for 10 min. Membrane and soluble fractions of mitochondria and mitoplast were prepared as described previously (23Spielewoy N. Schulz H. Grienenberger J.M. Thoöny-Meyer L. Bonnard G. J. Biol. Chem. 2001; 276: 5491-5497Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Extrinsic membrane proteins were solubilized with 0.1 m Na2CO3, pH 11.5, for 30 min at 4 °C. Mitochondrial membranes were treated with varying concentrations of urea (3.6 and 7.3 m) and with 6.6 m urea in the presence of 15 mm ATP and 15 mm MgSO4 on ice for 60 min. Centrifugation at 100,000 × g for 30 min at 4 °C in a Beckman TLA-100 rotor allowed the separation of soluble proteins from insoluble ones. Complementation Experiments—The ΔccmA E. coli strain EC21 (28Throne-Holst M. Thoöny-Meyer L. Hederstedt L. FEBS Lett. 1997; 410: 351-355Crossref PubMed Scopus (40) Google Scholar) and the plasmids pRJ3291 containing Bradyrhizobium japonicum cycA coding for the soluble periplasmic cytochrome c550 (13Schulz H. Fabianek R.A. Pellicioli E.C. Hennecke H. ThoönyMeyer L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6462-6467Crossref PubMed Scopus (103) Google Scholar) and pEC71 (EcccmA cloned in pACYC184) were provided by Prof. L. Thoöny-Meyer (ETH, Zurich). EC21 was transformed with pNG34 or pEC71 containing AtCCMA or EcccmA, respectively, and pRJ3291 containing the reporter cycA gene. E. coli were grown in anaerobic conditions in minimal salt medium in the presence of 5 mm nitrite as the electron acceptor (29Iobbi-Nivol C. Crooke H. Griffiths L. Grove J. Hussain H. Pommier J. Mejean V. Cole J.A. FEMS Microbiol. Lett. 1994; 119: 89-94Crossref PubMed Scopus (83) Google Scholar). Expression of the reporter cytochrome c550 was induced with 0.4% (w/v) arabinose. Preparation of total bacterial and periplasmic protein extracts and heme staining were performed as described (7Thoöny-Meyer L. Fischer F. Kunzler P. Ritz D. Hennecke H. J. Bacteriol. 1995; 177: 4321-4326Crossref PubMed Google Scholar, 30Thoöny-Meyer L. Stax D. Hennecke H. Cell. 1989; 57: 683-697Abstract Full Text PDF PubMed Scopus (84) Google Scholar, 31Schulz H. Pellicioli E.C. Thoöny-Meyer L. Mol. Microbiol. 2000; 37: 1379-1388Crossref PubMed Scopus (58) Google Scholar). Immunodetection Assays—Proteins were resolved by SDS-polyacrylamide gel electrophoresis and transferred on to polyvinylidene difluoride membranes (Immobilon-P, Millipore). Immunodetection with purified anti-AtCCMA antibodies was done at a dilution of 1/1000. Antibodies used as control were directed against the cytosolic Arabidopsis thioredoxin h3, the chloroplastic Chlamydomonas light harvesting complex II, and mitochondrial yeast cytochrome c1 as an intrinsic inner membrane protein, tobacco manganese-superoxide dismutase as a soluble matrix protein, wheat subunit 9 of NADH dehydrogenase as an extrinsic inner membrane protein (32Lamattina L. Gonzalez D. Gualberto J.M. Grienenberger J.M. Eur. J. Biochem. 1993; 217: 831-838Crossref PubMed Scopus (136) Google Scholar), and AtCCME (23Spielewoy N. Schulz H. Grienenberger J.M. Thoöny-Meyer L. Bonnard G. J. Biol. Chem. 2001; 276: 5491-5497Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) for a mitochondrial inner membrane protein oriented toward the intermembrane space. In Vitro Import of Radiolabeled Proteins into Isolated Mitochondria—Mitochondria were purified from cauliflower heads using a juice extractor (33Fey J. Vermel M. Grienenberger J.M. Maréchal-Drouard L. Gualberto J. FEBS Lett. 1999; 458: 124-128Crossref PubMed Scopus (17) Google Scholar). Synthesis of the radiolabeled proteins was done using the coupled reticulocyte transcription/translation system (TNT system, Promega) in the presence of [35S]methionine. In vitro import assays were performed as described previously (26Laloi C. Rayapuram N. Chartier Y. Grienenberger J.M. Bonnard G. Meyer Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14144-14149Crossref PubMed Scopus (223) Google Scholar). Blue Native PAGE and In-gel ATPase Activity—Blue native PAGE and BN-SDS-PAGE of mitochondrial membrane proteins were carried out as described (20Giegé P. Rayapuram N. Meyer E.H. Grienenberger J.M. Bonnard G. FEBS Lett. 2004; 563: 165-169Crossref PubMed Scopus (23) Google Scholar). Total mitochondrial proteins solubilized with digitonin, 5/1 detergent/protein (w/w), and native MBP-CCMA recombinant protein were loaded on a BN-PAGE. The ATPase activity was visualized by histochemical staining based on the precipitation of lead nitrate by the inorganic phosphate produced by ATP hydrolysis. The BN gel was incubated in 50 mm glycine, pH 8.6, 5 mm MgCl2,5 mm ATP, and Pb(NO3)2 reagent was added stepwise to a final concentration of 0.1% (w/v) as described (34Sabar M. Balk J. Leaver C.J. Plant J. 2005; 44: 893-901Crossref PubMed Scopus (86) Google Scholar). Yeast Two-hybrid Assays—cDNA of AtCcmB were cloned in pSK– after reverse transcription-PCR using primers P9 and P10. A fully edited version (pNG36) was selected after sequencing. cDNA fragments coding for the full-length AtCCMA protein and for seven domains of AtCcmB (corresponding to following regions: B1, Met-1—Ile-19; B2, Pro-33—Trp-52; B3, Ser-61—Gln-99; B4, Leu-110—Gly-129; B5, Gly-138—Pro-167; B6, Leu-168—Tyr-188; B7, Phe-186—Asp-206) were amplified with primers listed in supplemental Table S1. The PCR products obtained were amplified with primers attB1 and attB2 to introduce phage λ att recombination sites. Final products were recombined with pDONR207 (Invitrogen) to obtain entry vectors that were recombined with Clontech pGBKT7- and pGADT7-modified vector (35Dieterle M. Thomann A. Renou J.P. Parmentier Y. Cognat V. Lemonnier G. Muller R. Shen W.H. Kretsch T. Genschik P. Plant J. 2005; 41: 386-399Crossref PubMed Scopus (76) Google Scholar) to obtain constructs expressing all the combinations of AtCCMA or part of AtCcmB fused to the activation (AD) or binding (BD) domains of Gal4. Constructs were transformed in yeast strain pJ69-4A by heat shock. Two-hybrid assays were performed according to standard methods (36MacDonald P.N. Two-hybrid Systems: Methods and Protocols: Methods in Molecular Biology Series. 2001; 177 (, Vol. , Humana Press, Totowa, NJ)Google Scholar). Transformation in yeast was controlled by the growth on media lacking leucine and tryptophan. The expression of reporter genes ADE2 and HIS3 was monitored by the growth on media lacking both adenine and histidine. The expression of lacZ was followed by measuring at A420 the accumulation of the product metabolized by β-galactosidase with 2.2 mm 2-nitrophenyl β-d-galactopyranoside (Sigma) as substrate. Identification and Expression of AtCCMA—In A. thaliana, five mitochondrial genes encode proteins showing sequence similarities with bacterial CcmB-C proteins and with three domains of CcmF. No ortholog to the bacterial ccmA was found in land plant mitochondrial genomes. The conservation of genes coding for the TMDs of a potential CCM transporter prompted us to search for a nuclear gene encoding the NBD CcmA ortholog. Because of the high sequence similarity between NBD from non-homologue ABC transporters and the limited information concerning CcmA-specific conserved patterns, it was essential to search in a fully sequenced plant genome. Using the tblastn program, we identified the Arabidopsis At1g63270 gene as the best candidate for AtCCMA. The coding region is 690-bp long and codes for a protein of 229 amino acids showing around 30% identity with its bacterial counterparts. This unique gene was registered as NAP10 in the complete inventory of the A. thaliana ABC protein superfamily (3Sanchez-Fernandez R. Davies T.G. Coleman J.O. Rea P.A. J. Biol. Chem. 2001; 276: 30231-30244Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar). NAP10 belongs to a heterogeneous group of soluble proteins containing a single NBD. AtNAP10 protein sequence is aligned with NAP10 orthologs from other plant species, a subset of bacterial CcmA, and the mitochondria encoded CcmA of the protist Reclinomonas (Fig. 1). AtNAP10 possesses the typical Walker A (P-loop) and Walker B motifs and the ABC signature. In the bacterial CcmA family a conserved charged residue lysine or arginine is found at the 7th position within the ABC signature LSXGQX(K/R)R, whereas a glutamine is found at this position in bacterial importers (11Goldman B.S. Beckman D.L. Bali A. Monika E.M. Gabbert K.K. Kranz R.G. J. Mol. Biol. 1997; 268: 724-738Crossref PubMed Scopus (70) Google Scholar, 37Ames F.-L.G Int. Rev. Cytol. 1992; 137: 1-35Crossref PubMed Scopus (22) Google Scholar). This feature is conserved in AtNAP10 and its plant orthologs. Both bacterial and mitochondrial CcmA subgroups possess a conserved histidine but located at a different position (Fig. 1). The analysis of a full-length AtNAP10 cDNA reveals the presence of an intron in the 5′-untranslated region. No organ-specific expression data were available for AtCCMA. Reverse transcription-PCR analysis revealed the presence of AtCCMA transcripts in A. thaliana stems, leaves, and flowers (data not shown). In Arabidopsis, the protein is detected in cell culture, root, leave, and flower mitochondrial extracts (data not shown). AtCCMA is expressed in all organs of the plant but at low levels, suggesting that this protein has a housekeeping function. Complementation Assay—The bacterial protein CcmA has been shown to be essential for the maturation of c-type cytochromes (28Throne-Holst M. Thoöny-Meyer L. Hederstedt L. FEBS Lett. 1997; 410: 351-355Crossref PubMed Scopus (40) Google Scholar, 38Ramseier T.M. Winteler H.V. Hennecke H. J. Biol. Chem. 1991; 266: 7793-7803Abstract Full Text PDF PubMed Google Scholar, 39Beckman D.L. Trawick D.R. Kranz R.G. Genes Dev. 1992; 6: 268-283Crossref PubMed Scopus (147) Google Scholar). The complementation of ΔccmA E. coli strain EC21 lacking the chromosomal copy of ccmA with AtCCMA for the production of holocytochrome c was tested in anaerobic conditions that induce the expression of the chromosomal ccm operon. ΔccmA E. coli were transformed with a plasmid carrying the reporter cytochrome c550 gene (pRJ3291) and the vector alone pACYC184 or the vector carrying either the bacterial ccmA (pEC71) or AtCCMA (pNG34). Although the bacterial ccmA gene could fully complement the absence of the chromosomal ccmA, resulting in the formation of holocytochrome c550 and two endogenous c-type cytochromes NrfA and NapB, no c-type cytochromes were detected in bacteria transformed with AtCCMA (supplemental Fig. S1). This was not because of the absence of expression of AtCCMA, because the protein is detected in total extract. The inability of AtCCMA to functionally complement ΔccmA E. coli could be due to its failure to associate with the bacterial transmembrane domains to form a functional ABC transporter. AtCCMA Is a Mitochondrial Protein—AtCCMA only possesses a short N-terminal extension of 4–8 amino acids when compared with most bacterial proteins. The amino acids at the N terminus do not form a typical amphiphilic α helix, a characteristic feature of N-terminal mitochondrial targeting sequences. However, the prediction programs unanimously predicted a mitochondrial location for AtCCMA. Mitochondria from cauliflower, a Cruciferae as is A. thaliana, were used for in vitro import assays. The calculated molecular weight of AtCCMA is 25.9 kDa. When AtCCMA is translated in vitro,2 products are obtained with apparent molecular masses of 28 and 23 kDa (Fig. 2A, lane 1). Both proteins could be immunoprecipitated by anti-CCMA antibodies, indicating that these bands correspond to in-frame translation products of AtCCMA (data not shown). After import, a processed band of about 25 kDa was detected (Fig. 2A, lane 2) but disappeared with proteinase K treatment of the mitochondria after import (Fig. 2A, lane 3). Only the 28-kDa protein was resistant to proteinase K (Fig. 2A, compare lanes 2 and 3). This protection to proteinase K was lost when the mitochondrial membranes were solubilized with Triton X-100 (Fig. 2A, lane 4). The import of AtCCMA requires an electrochemical membrane potential as evidenced by the inhibition of import after treatment by the ionophore valinomycin (Fig. 2A, compare lanes 3 and 6). AtTRX-o1, a thioredoxin of the mitochondrial matrix, was used as a positive control (26Laloi C. Rayapuram N. Chartier Y. Grienenberger J.M. Bonnard G. Meyer Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14144-14149Crossref PubMed Scopus (223) Google Scholar) to validate the import competence of the mitochondria and the efficiency of the protease treatment. The precursor form of thioredoxin is still detectable after proteinase K treatment. This band corresponds to an imported protein not processed, as often observed in import assays rather than resulting from an incomplete proteolysis of precursor. In the later case such undigested protein should also be detected in the import assay with valinomycin followed by proteinase K treatment. No signal was observed in this assay for both AtTRX-o1 precursor form and AtCCMA 28-kDa band, indicating that, when import is inhibited, the bound precursors are fully digested by proteinase K treatment. We, therefore, conclude that AtCCMA is imported into mitochondria in a membrane potential-dependent manner without apparent processing. To confirm the localization of AtCCMA, an analysis of subcellular protein fractions of A. thaliana protoplasts was carried out by Western blotting and immunodetection with anti-AtCCMA antibodies (Fig. 2B). A 28-kDa signal was detected in mitochondria, which corresponded to the size of the imported AtCCMA in the in vitro import experiments. This band was absent in both cytosolic and chloroplastic extracts, indicating that AtCCMA is exclusively located in mitochondria. Anti-AtCCMA antibodies also recognized unidentified proteins of 66 and 40 kDa in the mitochondrial protein fraction. In addition, a 42-kDa cytosolic protein and a 40-kDa chloroplastic protein cross-react with anti-AtCCMA antibodies. The results obtained with antibodies directed against a cytosolic protein (AtTRX-h3), a chloroplastic protein (LHCII), and a mitochondrial protein (Cyt c1) show no detectable cross-contamination between the different protein fractions obtained. The in vitro import, the immunoprecipitation assays, and the immunolocalization experiments clearly show that the 28-kDa protein corresponds to AtCCMA and is localized exclusively in mitochondria. AtCCMA Is Associated with the Inner Membrane and Oriented toward the Mitochondrial Matrix—The sub-mitochondrial location of AtCCMA was further investigated by the immunodetection of various sub-mitochondrial protein fractions with anti-AtCCMA antibodies. The 28-kDa AtCCMA protein is detected in the membrane protein fraction prepared from mitoplasts but not in the soluble matrix protein fraction (Fig. 3A). Although it is a hydrophilic protein, AtCCMA is present in or associated with the mitochondrial inner membrane. Mitoplasts were treated with trypsin to digest proteins located on the intermembrane space side of the inner membrane. Anti-AtCCMA antibodies detected AtCCMA in mitoplasts that had undergone this treatment, indicating that it is oriented toward the matrix (Fig. 3B). Antibodies against Nad9, a subunit of the mitochondrial respiratory complex I facing the matrix (32Lamattina L. Gonzalez D. Gualberto J.M. Grienenberger J.M. Eur. J. Biochem. 1993; 217: 831-838Crossref PubMed Scopus (136) Google Scholar), were used as a control for the intactness of the inner membrane. Nad9 is indeed protected against protease treatment. Conversely, the efficiency of the digestion was ascertained by antibodies directed against the hydrophilic domain of AtCCME oriented toward the intermembrane space (23Spielewoy N. Schulz H. Grienenberger J.M. Thoöny-Meyer L. Bonnard G. J. Biol. Chem. 2001; 276: 5491-5497Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). AtCCME is not detected in mitoplasts treated with trypsin. AtCCMA is oriented toward the mitochondrial matrix, a topology analogous to that of its bacterial counterpart. AtCCMA Has Properties of the ATP Binding Domain of an ABC Transporter—The association of AtCCMA with the mitochondrial membranes was further investigated. The mitochondrial m
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