Membrane Topology of the 60-kDa Oxa1p Homologue fromEscherichia coli

We have characterized the membrane topology of a 60-kDa inner membrane protein from Escherichia coli that is homologous to the recently identified Oxa1p protein in Saccharomyces cerevisiae mitochondria implicated in the assembly of mitochondrial inner membrane proteins. Hydrophobicity and alkaline phosphatase fusion analyses suggest a membrane topology with six transmembrane segments, including an N-terminal signal-anchor sequence not present in mitochondrial Oxa1p. In contrast to partial N-terminal fusion protein constructs, the full-length protein folds into a protease-resistant conformation, suggesting that important folding determinants are present in the C-terminal part of the molecule. We have characterized the membrane topology of a 60-kDa inner membrane protein from Escherichia coli that is homologous to the recently identified Oxa1p protein in Saccharomyces cerevisiae mitochondria implicated in the assembly of mitochondrial inner membrane proteins. Hydrophobicity and alkaline phosphatase fusion analyses suggest a membrane topology with six transmembrane segments, including an N-terminal signal-anchor sequence not present in mitochondrial Oxa1p. In contrast to partial N-terminal fusion protein constructs, the full-length protein folds into a protease-resistant conformation, suggesting that important folding determinants are present in the C-terminal part of the molecule. transmembrane 3-[(3-cholamidopropyl)dimethylamonio]-1-propanesulfonate polymerase chain reaction. The nuclear gene OXA1 was first isolated in Saccharomyces cerevisiae (1Bonnefoy N. Chalvet F. Hamel P. Slonimski P.P. Dujardin G. J. Mol. Biol. 1994; 239: 201-212Crossref PubMed Scopus (186) Google Scholar). Recent studies have shown that the Oxa1p protein is localized to mitochondria and is involved in the assembly of mitochondrial inner membrane proteins (2He S. Fox T.D. Mol. Biol. Cell. 1997; 8: 1449-1460Crossref PubMed Scopus (160) Google Scholar, 3Kermorgant M. Bonnefoy N. Dujardin G. Curr. Genet. 1997; 31: 302-307Crossref PubMed Scopus (48) Google Scholar, 4Hell K. Herrmann J.M. Pratje E. Neupert W. Stuart R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2250-2255Crossref PubMed Scopus (186) Google Scholar, 5Altamura N. Capitanio N. Bonnefoy N. Papa S. Dujardin G. FEBS Lett. 1996; 382: 111-115Crossref PubMed Scopus (127) Google Scholar). Both nuclearly and mitochondrially encoded inner membrane proteins depend on Oxa1p for efficient export of their N- and C-terminal tails to the intermembrane space and have been shown to physically interact with Oxa1p (2He S. Fox T.D. Mol. Biol. Cell. 1997; 8: 1449-1460Crossref PubMed Scopus (160) Google Scholar, 4Hell K. Herrmann J.M. Pratje E. Neupert W. Stuart R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2250-2255Crossref PubMed Scopus (186) Google Scholar, 6Hell K. Herrmann J. Pratje E. Neupert W. Stuart R. FEBS Lett. 1997; 418: 367-370Crossref PubMed Scopus (150) Google Scholar). Oxa1p is believed to represent a component of a novel export machinery in the mitochondrial inner membrane that may also be present in bacteria (2He S. Fox T.D. Mol. Biol. Cell. 1997; 8: 1449-1460Crossref PubMed Scopus (160) Google Scholar, 4Hell K. Herrmann J.M. Pratje E. Neupert W. Stuart R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2250-2255Crossref PubMed Scopus (186) Google Scholar). Oxa1p is synthesized as a precursor with an N-terminal presequence. After import into the mitochondrial matrix, the presequence is cleaved off by the mitochondrial processing peptidase, and the 90-amino acid-long N-terminal tail is translocated to the inter-membrane space in a process dependent on pre-existing Oxa1p (4Hell K. Herrmann J.M. Pratje E. Neupert W. Stuart R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2250-2255Crossref PubMed Scopus (186) Google Scholar, 7Herrmann J.M. Neupert W. Stuart R.A. EMBO J. 1997; 16: 2217-2226Crossref PubMed Scopus (135) Google Scholar). Based on hydrophobicity analysis and limited proteolysis data, Oxa1p is thought to span the mitochondrial inner membrane five times with the C terminus in the matrix (7Herrmann J.M. Neupert W. Stuart R.A. EMBO J. 1997; 16: 2217-2226Crossref PubMed Scopus (135) Google Scholar), although this model remains to be experimentally verified. Oxa1p homologues have been found in both Gram-positive and Gram-negative bacteria (1Bonnefoy N. Chalvet F. Hamel P. Slonimski P.P. Dujardin G. J. Mol. Biol. 1994; 239: 201-212Crossref PubMed Scopus (186) Google Scholar, 8Sundberg E. Slagter J. Fridborg I. Cleary S. Robinson C. Coupland G. Plant Cell. 1997; 9: 717-730PubMed Google Scholar). A homologue of Oxa1p, ALB3, is present in chloroplasts and is involved in chloroplast biogenesis (8Sundberg E. Slagter J. Fridborg I. Cleary S. Robinson C. Coupland G. Plant Cell. 1997; 9: 717-730PubMed Google Scholar), and a human homologue has also been cloned (9Bonnefoy N. Kermorgant M. Groudinsky O. Minet M. Slonimski P.P. Dujardin G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11978-11982Crossref PubMed Scopus (91) Google Scholar). To further characterize this potentially important family of proteins, we have determined the membrane topology of the Escherichia coli Oxa1p homologue (Oxa1Ec), a 60-kDa inner membrane protein encoded by the yidC gene (10Burland V. Plunkett G.D. Daniels D.L. Blattner F.R. Genomics. 1993; 16: 551-561Crossref PubMed Scopus (173) Google Scholar). Hydrophobicity and PhoA fusion analyses suggest a topology with six transmembrane (TM)1 segments. In contrast to mitochondrial Oxa1p, Oxa1Ec has an uncleaved, N-terminal signal-anchor sequence. Interestingly, although most Oxa1Ec-PhoA fusions are degraded by periplasmically added proteinase K, only a short N-terminal piece is removed from the wild-type and full-length fusion proteins. C-terminal parts of the protein are thus essential for folding and/or oligomerization into a protease-resistant form. Unless otherwise stated, all enzymes were from Promega. T7 DNA polymerase was from Amersham Pharmacia Biotech. Proteinase K was from Life Technologies, Inc. [35S]Met was from Amersham Pharmacia Biotech. Oligonucleotides were from Kebo Lab (Stockholm, Sweden). PhoA antiserum was from 5 Prime → 3 Prime, Inc. (Boulder, CO). Hen egg white lysozyme, CHAPS (3-[(3-cholamidopropyl)dimethylamonio]-1-propanesulfonate), phenylmethylsulfonyl flouride, and the alkaline phosphatase chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate were from Sigma. Experiments were performed in E. coli strains MC1061 (ΔlacX74, araD139,Δ(ara,leu)7697, galU, galK, hsr, hsm, strA) (11Dalbey R.E. Wickner W. J. Biol. Chem. 1986; 261: 13844-13849Abstract Full Text PDF PubMed Google Scholar), CC118 (Δ(ara-leu)7697 ΔlacX74 ΔphoA20 galE galK thi rpsE rpoB argE(am) recA1) (12Lee E. Manoil C. J. Biol. Chem. 1994; 269: 28822-28828Abstract Full Text PDF PubMed Google Scholar) and TOP10F′ (F′(tetr) (mrr-hsdRMS-mcrBC) lacZΔM15 rpsL (Smr) endA1) (Stratagene). Cloning of the Oxa1Ec gene was performed using the pGEM-T Easy Vector System 1 (Promega). All constructs were expressed in E. colifrom the pING1 plasmid (13Johnston S. Lee J.H. Ray D.S. Gene (Amst.). 1985; 34: 137-145Crossref PubMed Scopus (100) Google Scholar) by induction with arabinose. All plasmid constructs were confirmed by DNA sequencing using T7 DNA polymerase. The yidC gene encoding Oxa1Ec was amplified by PCR from E. coli TOP10F′ chromosomal DNA. The naturally occurring KpnI site was removed by introducing a silent mutation using the "double PCR" approach and a 5′ XhoI and a 3′ KpnI site were introduced in the regions flanking the yidC open reading frame. The PCR product was first cloned into the pGEM-T Easy Vector System 1, excised using XhoI and KpnI, and cloned behind the ara promoter in a XhoI-KpnI restricted plasmid derived from pING1 containing a lep gene with a 5′ XhoI site just upstream of the initiator ATG and aKpnI site in codon 78. Relevant parts of the yidCgene were amplified by PCR from the pING1 plasmid with a 5′SalI and a 3′ KpnI site encoded in the primers. Finally, the PCR SalI-KpnI fragment carrying thelep upstream region and the relevant yidC segment were cloned into a previously constructed plasmid (14Whitley P. Nilsson I. von Heijne G. Nat. Struct. Biol. 1994; 1: 858-862Crossref PubMed Scopus (44) Google Scholar) carrying aphoA gene lacking the 5′ segment coding for the signal sequence and the first 5 residues of the mature protein and immediately preceded by a KpnI site. In all constructs, an 18-amino acid linker (VPDSYTQVASWTEPFPFC) was present between the Oxa1Ecand PhoA moieties. E. coli strain MC1061 transformed with the pING1 vector carrying the relevant constructs under control of the arabinose promoter was grown at 37 °C in M9 minimal medium supplemented with 100 μg/ml ampicillin, 0.5% fructose, 100 μg/ml thiamin, and all amino acids (50 μg/ml each) except methionine. An overnight culture was diluted 1:25 in fresh medium, shaken for 3.5 h at 37 °C, induced with arabinose (0.2%) for 5 min, and labeled with [35S]methionine (75 μCi/ml). After 1 min, nonradioactive methionine was added (final concentration 500 μg/ml) and stopped by chilling on ice. For the experiment in Fig. 4 C, pulse labeling was done for 15 s. Cells were spun in an Eppendorf bench-top centrifuge at 14,000 rpm for 2 min, resuspended in ice-cold buffer (40% w/v sucrose, 33 mm Tris-HCl, pH 8.0), and incubated with lysozyme (5 mg/ml) and 1 mm EDTA for 15 min on ice. Aliquots of the cell suspension were incubated 1 h on ice, either with no additions or with the addition of 400 μg/ml proteinase K (15Rusch S.L. Chen H.F. Izard J.W. Kendall D.A. J. Cell. Biochem. 1994; 55: 209-217Crossref PubMed Scopus (35) Google Scholar). After addition of phenylmethylsulfonyl flouride, samples were acid-precipitated (trichloroacetic acid, 10% final concentration), resuspended in 10 mm Tris, pH 7.5, 2% SDS, immunoprecipitated with antisera to PhoA, Lep, or Oxa1Ec as required, washed, and analyzed by SDS-polyacrylamide gel electrophoresis. Gels were scanned in a FUJIX Bas 1000 phosphoimager and analyzed using the MacBAS software (version 2.31). Alkaline phosphatase activity was measured by growing strain CC118 transformed with the appropriate pING1-derived plasmids in liquid culture in the presence of 0.2% arabinose (16Manoil C. Methods Cell Biol. 1991; 34: 61-75Crossref PubMed Scopus (200) Google Scholar). Mean activity values were obtained from two independent measurements and were normalized by the rate of synthesis of the fusion protein determined by pulse labeling of arabinose-induced CC118 cells for 2 min followed by immunoprecipitation and quantitation by phosphoimager analysis. Normalized activities were calculated as:A = (A 0 ×A 600 × n Met)/counts/min, where A 0 is the measured activity,A 600 is the cell density at the time of pulse labeling, n Met is the number of Met residues in the fusion protein, and counts/min is the intensity of the relevant band measured on the phosphoimager. A rabbit antiserum was raised by Agrisera AB, Umeå, Sweden, against a synthetic peptide comprising the last 17 amino acids of Oxa1Ec(YRGLEKRGLHSREKKKS). The C-terminal membrane domain of Oxa1p is conserved from prokaryotes to eukaryotes (1Bonnefoy N. Chalvet F. Hamel P. Slonimski P.P. Dujardin G. J. Mol. Biol. 1994; 239: 201-212Crossref PubMed Scopus (186) Google Scholar, 8Sundberg E. Slagter J. Fridborg I. Cleary S. Robinson C. Coupland G. Plant Cell. 1997; 9: 717-730PubMed Google Scholar), and is thus very likely to have the same topology in different organisms. Hydrophobicity analysis of Oxa1p and Oxa1Ec does not give a clear-cut prediction of the number of transmembrane (TM) segments, however, and models with five to seven TMs are possible, Fig. 1. In particular, it cannot be determined whether the weakly predicted TM around residue 250 is real and whether the C-terminal hydrophobic region around residue 500 corresponds to one or two TMs (although the Oxa1p hydrophobicity plot would suggest two closely spaced TMs in this region). To experimentally determine the membrane topology of Oxa1Ec, we made a series of Oxa1Ec-PhoA fusions (17Manoil C. Beckwith J. Science. 1986; 233: 1403-1408Crossref PubMed Scopus (368) Google Scholar). As two critical disulfide bonds are necessary for PhoA activity (18Derman A.I. Beckwith J. J. Bacteriol. 1991; 173: 7719-7722Crossref PubMed Scopus (173) Google Scholar), PhoA will only be active when located in the oxidizing environment of the periplasm but not when located in the cytoplasm. To retain topological information in the cytoplasmic and periplasmic loops (19Boyd D. Traxler B. Beckwith J. J. Bacteriol. 1993; 175: 553-556Crossref PubMed Google Scholar), PhoA fusions were made near the C-terminal end of predicted loops in Oxa1Ec. The fusion proteins were expressed in the PhoA− strain CC118, Fig. 2, and alkaline phosphatase activities were measured in liquid culture. Alkaline phosphatase activities and relative expression levels measured for the various fusions are given in Table I. Fusions in the region between the first hydrophobic domain and residue 340 all have high normalized activities, suggesting a periplasmic location. Two additional periplasmic loops are identified by the high activity fusions in positions 458 and 512. Low activity fusions in positions 415, 494, and at the C terminus (positions 548) identify cytoplasmic parts. To confirm the topology in the C-terminal part of the protein, two additional fusions were made in the last periplasmic loop (positions 509 and 515; both have high activity) and in the C-terminal tail (position 538; low activity); these fusion proteins are not shown in Fig. 2.Table IAlkaline phosphatase activity of OxaEc-PhoA fusionsFusion (fusion-joint codon indicated)Alkaline phosphatase activityRate of synthesisNormalized activityunitsarbitrary unitsOxaEc(40)-PhoA75811.466OxaEc(225)-PhoA6907.592OxaEc(290)-PhoA10645.4197OxaEc(340)-PhoA6919.970OxaEc(415)-PhoA6410.76OxaEc(458)-PhoA2673.674OxaEc(494)-PhoA173.76OxaEc(509)-PhoA2821.7160OxaEc(512)-PhoA2545.942OxaEc(515)-PhoA2255.442OxaEc(538)-PhoA849.39OxaEc(548)-PhoA1639.817 Open table in a new tab These data strongly suggest a topology with an N-terminal TM, a large periplasmic domain, and five additional, closely spaced C-terminal TMs. Since TM1 of Oxa1Ec is not present in mitochondrial Oxa1p, we wanted to determine whether it is a cleavable signal peptide or an uncleaved signal-anchor. To this end, the shortest PhoA fusion (at residue 40) was expressed in the absence and presence of sodium azide, an inhibitor of the SecA ATPase activity (20Oliver D.B. Cabelli R.J. Dolan K.M. Jarosik G.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8227-8231Crossref PubMed Scopus (254) Google Scholar) that blocks the translocation of large periplasmic domains in inner membrane proteins and thus also prevents cleavage of signal peptides by the periplasmically exposed leader peptidase enzyme (21Dalbey R.E. Lively M.O. Bron S. Van Dijl J.M. Protein Sci. 1997; 6: 1129-1138Crossref PubMed Scopus (215) Google Scholar). As shown in Fig. 3, there is no difference in size of the protein expressed in the absence (lane 1) or presence (lane 3) of azide, indicating that TM1 is not cleaved by leader peptidase during or after translocation. Proteinase K treatment of spheroplasts confirms that the PhoA domain is translocated to the periplasm in the absence (lane 2) but not in the presence (lane 4) of azide. Note that periplasmic, properly folded PhoA is intrinsically protease-resistant (15Rusch S.L. Chen H.F. Izard J.W. Kendall D.A. J. Cell. Biochem. 1994; 55: 209-217Crossref PubMed Scopus (35) Google Scholar), and that proteinase K thus only removes the TM1 segment from the fusion protein (lane 2). As expected, the Oxa1Ec(40)-PhoA is found in the membrane fraction after sonication of the cells (data not shown). Interestingly, only two of the cytoplasmic Oxa1Ec-PhoA fusions are sensitive to proteinase K treatment of spheroplasts and give rise to protease-protected fragments of sizes expected for cleavage in the N-terminal periplasmic domain and in the periplasmic loop between TM3 and TM4, respectively, Fig. 4 A (lanes 1-4). In contrast, the full-length fusion (at residue 548) is largely resistant to proteinase K (lanes 5 and 6). To confirm the unexpected protease resistance of Oxa1Ec, we carried out further protease protection experiments both on the wild-type protein and on an Oxa1Ec-Lep(P2) fusion where the C-terminal, periplasmic P2 domain of the E. coli inner membrane protein leader peptidase (Lep) has been fused to residue 548 in Oxa1Ec. As shown in Fig. 4 B, only a small N-terminal fragment (roughly corresponding to TM1) is removed from the Oxa1Ec-Lep(P2) fusion (lanes 1 and 2), and the protein is almost completely protease-resistant when expressed in the presence of azide (lanes 3and 4); incidentally, the protease resistance of the Lep P2 domain further supports the cytoplasmic localization of the C terminus of Oxa1Ec, since the P2 domain is readily digested by proteinase K when located in the periplasm (22Wolfe P.B. Wickner W. Goodman J.M. J. Biol. Chem. 1983; 258: 12073-12080Abstract Full Text PDF PubMed Google Scholar). Similarly, only a small N-terminal fragment of ∼7 kDa (corresponding to ∼65 residues) is removed from the 59-kDa wild-type protein upon proteinase K treatment of spheroplasts, Fig. 4 C (note that the antibody used was raised against a C-terminal Oxa1Ec peptide). Obviously, the periplasmically exposed parts of Oxa1Ec fold into a protease-resistant conformation, but only in the full-length protein. Possibly, this reflects assembly into an oligomeric complex, as has been suggested to occur for mitochondrial Oxa1p (4Hell K. Herrmann J.M. Pratje E. Neupert W. Stuart R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2250-2255Crossref PubMed Scopus (186) Google Scholar). Mitochondrial Oxa1p has been implicated in the translocation of N- and C-terminal tails of mitochondrial inner membrane proteins from the matrix to the intermembrane space (2He S. Fox T.D. Mol. Biol. Cell. 1997; 8: 1449-1460Crossref PubMed Scopus (160) Google Scholar, 4Hell K. Herrmann J.M. Pratje E. Neupert W. Stuart R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2250-2255Crossref PubMed Scopus (186) Google Scholar, 6Hell K. Herrmann J. Pratje E. Neupert W. Stuart R. FEBS Lett. 1997; 418: 367-370Crossref PubMed Scopus (150) Google Scholar). Since S. cerevisiae mitochondria do not contain a Sec-type protein translocation machinery (23Glick B.S. von Heijne G. Protein Sci. 1996; 5: 2651-2652Crossref PubMed Scopus (69) Google Scholar), Oxa1p is thought to be a component of a new protein translocation pathway present also in bacteria, possibly specialized in the assembly of inner membrane proteins. We have determined the membrane topology of the 60-kDa Oxa1p homologue present in E. coli (Oxa1Ec). Hydrophobicity and PhoA fusion analyses support a Ncyt-Ccyttopology with six transmembrane segments, Fig. 5. The distribution of positively charged residues (Arg, Lys) conforms to the "positive inside" rule (24von Heijne G. J. Mol. Biol. 1992; 225: 487-494Crossref PubMed Scopus (1424) Google Scholar), lending further support to the proposed topology. Mitochondrial Oxa1p lacks a TM domain at the N terminus but otherwise has a similar hydrophobicity profile as Oxa1Ec (Fig. 1). In agreement with the topology model proposed here for Oxa1Ec, previous work has shown that the large N-terminal domain and the loop between the second and third TM in Oxa1p is exposed to the intermembrane space (7Herrmann J.M. Neupert W. Stuart R.A. EMBO J. 1997; 16: 2217-2226Crossref PubMed Scopus (135) Google Scholar). It is thus likely that Oxa1p and Oxa1Ec have the same topology beyond the N-terminal TM present only in OxaEc. Interestingly, despite having most of its mass exposed to the periplasm, Oxa1Ec folds into a protease-resistant conformation where only the most N-terminal TM can be removed by proteinase K treatment of spheroplasts. Protease resistance is only seen in the full-length protein, suggesting that important folding determinants are present in the C-terminal ∼50 residues and that full-length Oxa1Ec might be part of an oligomeric complex, as has been proposed for the mitochondrial Oxa1p (4Hell K. Herrmann J.M. Pratje E. Neupert W. Stuart R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2250-2255Crossref PubMed Scopus (186) Google Scholar). We thank Petra Bäverbäck and Anders Welander for technical assistance.