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• W2021336642 abstract "The first cytoplasmic loop of subunita of the Escherichia coli ATP synthase has been analyzed by cysteine substitution mutagenesis. 13 of the 26 residues tested were found to be accessible to the reaction with 3-(N-maleimidylpropionyl)-biocytin. The other 13 residues predominantly found in the central region of the polypeptide chain between the two transmembrane spans were more resistant to labeling by 3-(N-maleimidylpropionyl)-biocytin while in membrane vesicle preparations. This region of subunit acontains a conserved residue Glu-80, which when mutated to lysine resulted in a significant loss of ATP-driven proton translocation. Other substitutions including glutamine, alanine, and leucine were much less detrimental to function. Cross-linking studies with a photoactive cross-linking reagent were carried out. One mutant, K74C, was found to generate distinct cross-links to subunitb, and the cross-linking had little effect on proton translocation. The results indicate that the first transmembrane span (residues 40–64) of subunit a is probably near one or both of the b subunits and that a less accessible region of the first cytoplasmic loop (residues 75–90) is probably near the cytoplasmic surface, perhaps in contact with bsubunits. The first cytoplasmic loop of subunita of the Escherichia coli ATP synthase has been analyzed by cysteine substitution mutagenesis. 13 of the 26 residues tested were found to be accessible to the reaction with 3-(N-maleimidylpropionyl)-biocytin. The other 13 residues predominantly found in the central region of the polypeptide chain between the two transmembrane spans were more resistant to labeling by 3-(N-maleimidylpropionyl)-biocytin while in membrane vesicle preparations. This region of subunit acontains a conserved residue Glu-80, which when mutated to lysine resulted in a significant loss of ATP-driven proton translocation. Other substitutions including glutamine, alanine, and leucine were much less detrimental to function. Cross-linking studies with a photoactive cross-linking reagent were carried out. One mutant, K74C, was found to generate distinct cross-links to subunitb, and the cross-linking had little effect on proton translocation. The results indicate that the first transmembrane span (residues 40–64) of subunit a is probably near one or both of the b subunits and that a less accessible region of the first cytoplasmic loop (residues 75–90) is probably near the cytoplasmic surface, perhaps in contact with bsubunits. 3-(N-maleimidylpropionyl)-biocytin 9-amino-6-chloro-2-methoxyacridine hemagglutinin N,N′-dicyclohexylcarbodiimide lauryl dimethylamine oxide 3-(N-morpholino)propanesulfonic acid nickel-nitriloacetic acid N-(4-azido-2,3,5,6-tetrafluorobenzyl)-3-maleimidopropionamide The ATP synthase from Escherichia coli is typical of the ATP synthases found in mitochondria, chloroplasts, and many other bacteria (1Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1595) Google Scholar). It contains an F1 sector with subunits for nucleotide binding and catalysis and an F0 sector, which conducts protons across the membrane. Five different subunits are found in the E. coli F1 (α, β, γ, δ, and ε) with a stoichiometry of 3:3:1:1:1. Three different subunits are found in the E. coli F0(a, b, and c) with a stoichiometry of 1:2:9–12 (2Foster D.L. Fillingame R.H. J. Biol. Chem. 1982; 257: 2009-2015Abstract Full Text PDF PubMed Google Scholar). The mechanism by which an electrochemical proton gradient across the membrane drives ATP synthesis involves the rotation of the subunitc oligomer of F0, which drives the rotation of γ and ε as a rotor with subunits a and bfunctioning as the stator along with δ, α, and β. The crystallization of F1 from bovine mitochondria led to a high resolution structure of the α3β3 hexamer plus the parts of γ in the central core (3Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2753) Google Scholar). This structural information led to the numerous studies that have provided evidence supporting the hypothesis of rotation (4Noji H. Yasuda R. Yoshida M. Kinosita K., Jr. Nature. 1997; 386: 299-302Crossref PubMed Scopus (1966) Google Scholar, 5Duncan T.M. Bulygin V.V. Zhou Y. Hutcheon M.L. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10964-10968Crossref PubMed Scopus (459) Google Scholar, 6Sabbert D. Engelbrecht S. Junge W. Nature. 1996; 381: 623-625Crossref PubMed Scopus (464) Google Scholar, 7Sambongi Y. Iko Y. Tanabe M. Omote H. Iwamoto-Kihara A. Ueda I. Yanagida T. Wada Y. Futai M. Science. 1999; 286: 1722-1724Crossref PubMed Scopus (420) Google Scholar, 8Tsunoda S.P. Aggeler R. Yoshida M. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 898-902Crossref PubMed Scopus (138) Google Scholar). The mechanism of proton translocation in F0 remains to be understood. The information regarding the tertiary and quarternary structures of the F0 subunits is needed to understand how protons move through F0. Subunit b seems to be embedded in the membrane via a span of hydrophobic amino acids at its N terminus. A truncated soluble form of subunit b has been shown to be extended and dimeric (9Dunn S.D. J. Biol. Chem. 1992; 267: 7630-7636Abstract Full Text PDF PubMed Google Scholar). NMR studies have shown a α-helical hairpin structure for subunit c with two transmembrane spans connected by a polar loop (10Girvin M.E. Rastogi V.K. Abildgaard F. Markley J.L. Fillingame R.H. Biochemistry. 1998; 37: 8817-8824Crossref PubMed Scopus (272) Google Scholar, 11Girvin M.E. Fillingame R.H. Biochemistry. 1993; 32: 12167-12177Crossref PubMed Scopus (77) Google Scholar). The number of c subunits that make up the oligomer in F0 of E. coli is still uncertain, but atomic force microscopy (12Seelert H. Poetsch A. Dencher N.A. Engel A. Stahlberg H. Müller D.J. Nature. 2000; 405: 418-419Crossref PubMed Scopus (414) Google Scholar, 13Stahlberg H. Müller D.J. Suda K. Fotiadis D. Engel A. Meier T. Matthey U. Dimroth P. EMBO J. 2001; 2: 229-233Crossref Scopus (168) Google Scholar) has provided evidence for a ring of c subunits. Recent work (14Jiang W. Hermolin J. Fillingame R.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4966-4971Crossref PubMed Scopus (216) Google Scholar) indicates that the probable stoichiometric number for subunitc in E. coli is 10. Electron spectroscopic imaging has provided evidence for a ring of c subunits next to a and b subunits (15Birkenhäger R. Hoppert M. Deckers-Hebestreit G. Mayer F. Altendorf K. Eur. J. Biochem. 1995; 230: 58-67Crossref PubMed Scopus (129) Google Scholar). A 3.9-Å crystal structure of the ATP synthase from Saccharomyces cerevisiaerevealed the c subunit to be an oligomer of 10 subunits (16Stock D. Leslie A.G. Walker J.E. Science. 1999; 286: 1700-1705Crossref PubMed Scopus (1087) Google Scholar). This structure is in agreement with a model proposed for theE. coli c oligomer with the exception that theE. coli oligomer is predicted to contain 12 csubunits (17Dmitriev O.Y. Jones P.C. Fillingame R.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7785-7790Crossref PubMed Scopus (107) Google Scholar). This would place the conserved carboxylate (cD61 in E. coli), believed to be the proton binding site, in approximately the middle of the bilayer. Recent cross-linking experiments have demonstrated the Na+-binding site in a homologous enzyme from Propionigenium modestum to be found within the lipid bilayer (18von Ballmoos C. Appoldt Y. Brunner J. Granier T. Vasella A. Dimroth P. J. Biol. Chem. 2002; 277: 3504-3510Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Lacking structural information of subunit a by x-ray diffraction or NMR, other lower resolution methods have been employed to understand its arrangement in the membrane and its relationship to the other subunits of the F1F0-ATP synthase. Previously, the surface labeling of uniquely engineered cysteine residues was used to establish the number of transmembrane spans and locate the termini of subunit a (19Long J.C. Wang S. Vik S.B. J. Biol. Chem. 1998; 273: 16235-16240Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 20Valiyaveetil F.I. Fillingame R.H. J. Biol. Chem. 1998; 273: 16241-16247Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 21Wada T. Long J.C. Zhang D. Vik S.B. J. Biol. Chem. 1999; 274: 17353-17357Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Five transmembrane spans were identified with the N terminus in the periplasm and the C terminus in the cytoplasm as shown in Fig.1. Substitution mutagenesis has been used to determine that residues important for function reside in transmembrane spans 4 and 5 (22Cain B.D. Simoni R.D. J. Biol. Chem. 1986; 261: 10043-10050Abstract Full Text PDF PubMed Google Scholar, 23Cain B.D. Simoni R.D. J. Biol. Chem. 1988; 263: 6606-6612Abstract Full Text PDF PubMed Google Scholar, 24Cain B.D. Simoni R.D. J. Biol. Chem. 1989; 264: 3292-3300Abstract Full Text PDF PubMed Google Scholar, 25Lightowlers R.N. Howitt S. Hatch L. Gibson F. Cox G. Biochim. Biophys. Acta. 1988; 933: 241-248Crossref PubMed Scopus (49) Google Scholar, 26Lightowlers R.N. Howitt S.M. Hatch L. Gibson F. Cox G.B. Biochim. Biophys. Acta. 1987; 894: 399-406Crossref PubMed Scopus (86) Google Scholar, 27Valiyaveetil F.I. Fillingame R.H. J. Biol. Chem. 1997; 272: 32635-32641Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Cross-linking studies have shown that a surface of transmembrane span 4 between residues 207 and 225 of subunit a appears to be in contact with subunit c(28Jiang W. Fillingame R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6607-6612Crossref PubMed Scopus (150) Google Scholar). This region includes aR210, which is strictly conserved among all known species, and in E. coli, all of the known mutations at this position result in the inability to grow on succinate minimal medium and in a loss of ATP-driven proton translocation. Only an alanine substitution at this position allows for passive proton permeability (27Valiyaveetil F.I. Fillingame R.H. J. Biol. Chem. 1997; 272: 32635-32641Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 29Hatch L.P. Cox G.B. Howitt S.M. J. Biol. Chem. 1995; 270: 29407-29412Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). It is probable thataR210 interacts with the essential residue cD61 during coupled proton translocation. It is not clear exactly how subunit a contributes to the proton conducting the path of the F1F0-ATP synthase, but it does play an essential role in this function. Models have been proposed in which subunit a contributes amino acids that make up two half-channels, one opening to the periplasm and one to the cytoplasm, that allow access to the proton binding site on subunit c(30Junge W. Lill H. Engelbrecht S. Trends Biochem. Sci. 1997; 22: 420-423Abstract Full Text PDF PubMed Scopus (439) Google Scholar, 31Vik S.B. Patterson A.R. Antonio B.J. J. Biol. Chem. 1998; 273: 16229-16234Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 32Elston T. Wang H.Y. Oster G. Nature. 1998; 391: 510-513Crossref PubMed Scopus (446) Google Scholar). This study is the first in a series of studies designed to elucidate details regarding the secondary, tertiary, and quarternary structures of subunit a to establish what structural features are important to its role in the function of the F1F0 ATP synthase. We began with the first cytoplasmic loop and a highly conserved glutamic acid (Glu-80 inE. coli) found there. Surface-labeling techniques were used to determine the accessibility of the residues found between the first and second transmembrane spans, substitution mutagenesis was used to study the role of the conserved Glu-80, and cross-linking was used to show the proximity of this loop to the other subunits of F0. Restriction endonucleases and T4 DNA ligase were obtained from New England Biolabs. Plasmid mini-prep kits were obtained from Promega. The synthetic oligonucleotides were obtained from Operon Technologies. MPB,1 ACMA, and TFPAM-3 were obtained from Molecular Probes. Ni-NTA resin was obtained from Qiagen. Octyl glucoside, deoxycholate, cholate, andn-dodecylmaltoside were obtained from Sigma or Anatrace. Polyclonal anti-a rabbit antibody was a gift from Karlheinz Altendorf (Universität Osnabrück), and monoclonal anti-b mouse antibody was a gift from Roderick A. Capaldi (University of Oregon). Immunoblotting reagents and a protein assay kit were obtained from Bio-Rad. Automated DNA sequencing was done by Lone Star Laboratories (Houston TX). Plasmids pLN6-HisHA (21Wada T. Long J.C. Zhang D. Vik S.B. J. Biol. Chem. 1999; 274: 17353-17357Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) and pLN7-HisHA (see below) were used for the construction of mutants. These plasmids produce versions of subunita that include both a His6 tag and a HA epitope at the C terminus of the protein. pLN7-HisHA was constructed by first eliminating the AseI site in pLN6-HisHA by digestion, filling in the ends using the Klenow fragment of DNA polymerase I, and religating. The modified pLN6-HisHA was digested with DraIII and PvuI, and the 2.96-kb fragment was isolated. A similar digest was carried out on pSBV17 (33Vik S.B. Antonio B.J. J. Biol. Chem. 1994; 269: 30364-30369Abstract Full Text PDF PubMed Google Scholar), and the 0.37-kb fragment was isolated and then ligated to the 2.96-kb fragment to make pLN7-HisHA. The plasmid pSBV17 is identical to pSBV16 (33Vik S.B. Antonio B.J. J. Biol. Chem. 1994; 269: 30364-30369Abstract Full Text PDF PubMed Google Scholar) with the exception that the region between the two BamHI sites in uncB is wild type. An amino acid substitution was carried out using cassette mutagenesis and pairs of synthetic oligonucleotides as described previously (34Vik S.B. Cain B.D. Chun K.T. Simoni R.D. J. Biol. Chem. 1988; 263: 6599-6605Abstract Full Text PDF PubMed Google Scholar). For expression, E. coli RH305 (uncB205, recA56,srl::Tn10, bgl R,thi-1, rel-1, HfrPO1) (35Humbert R. Brusilow W.S. Gunsalus R.P. Klionsky D.J. Simoni R.D. J. Bacteriol. 1983; 153: 416-422Crossref PubMed Google Scholar) was used as the host strain for pLN6-HisHA and pLN7-HisHA. RH305 produces a form of subunit a, which is truncated near Pro-240 and cannot be detected in cells (36Hartzog P.E. Cain B.D. J. Bacteriol. 1993; 175: 1337-1343Crossref PubMed Google Scholar). It can be complemented by plasmids containing a wild type uncB gene. The subcloning and growth of cultures were carried out as described previously (19Long J.C. Wang S. Vik S.B. J. Biol. Chem. 1998; 273: 16235-16240Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Inside-out membrane vesicles were prepared as described previously (19Long J.C. Wang S. Vik S.B. J. Biol. Chem. 1998; 273: 16235-16240Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The membrane vesicles were labeled as described previously (19Long J.C. Wang S. Vik S.B. J. Biol. Chem. 1998; 273: 16235-16240Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) using the conditions of 120 μm MPB at room temperature for 15 min. After labeling with MPB, subunita was purified using Ni-NTA as described previously (19Long J.C. Wang S. Vik S.B. J. Biol. Chem. 1998; 273: 16235-16240Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Protein concentrations were determined by a detergent-compatible protein assay using bovine serum albumin as standard. Fluorescence assays and measurement of ATP hydrolysis were performed as described previously (31Vik S.B. Patterson A.R. Antonio B.J. J. Biol. Chem. 1998; 273: 16229-16234Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Membrane vesicles were incubated with 350 μm TFPAM-3 for 1 h at room temperature. After the quenching of excess reagent with 15 mm cysteine, photolysis was carried out for 2 h at room temperature in a microcentrifuge tube (1.5 ml) at a distance of 3 cm from a 6-watt, 365-nm UV lamp (model UVL-56 Blak-Ray lamp, UVP, Inc.) according to a previously published procedure (37Aggeler R. Chicas-Cruz K. Cai S.X. Keana J.F. Capaldi R.A. Biochemistry. 1992; 31: 2956-2961Crossref PubMed Scopus (95) Google Scholar). The purification of subunit a following procedures for labeling or cross-linking was performed according to methods described previously (19Long J.C. Wang S. Vik S.B. J. Biol. Chem. 1998; 273: 16235-16240Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 21Wada T. Long J.C. Zhang D. Vik S.B. J. Biol. Chem. 1999; 274: 17353-17357Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). A series of unique cysteine mutants was constructed in the first cytoplasmic loop of subunit a at residues 73–98. Each of these mutants was found to grow normally on succinate minimal medium. Each mutant was tested individually in membrane vesicles for accessibility of the unique cysteine to the surface label MPB, and the results are shown in Figs. 2 and3. In each figure, panel A shows the reaction for each residue with MPB, whereaspanel B is the corresponding immunoblot for subunit a. In Fig. 2B, the samples were probed with an antibody to subunit a, whereas in Fig. 3Ban anti-HA antibody was used. In Fig. 2, residues 67 and 69 are positive controls from previous studies (19Long J.C. Wang S. Vik S.B. J. Biol. Chem. 1998; 273: 16235-16240Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 21Wada T. Long J.C. Zhang D. Vik S.B. J. Biol. Chem. 1999; 274: 17353-17357Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Labeling is also apparent for residues 73 and 74, and, to a less extent, for residue 76, whereas residues 75, 77, and 78 show at most a trace of labeling. In Fig. 3, residues 84, 87, 88, and 91–98 are all readily labeled. Residues 80, 81, 85, 86, and 90 show only a trace of labeling, whereas 79, 82, 83, and 89 are not labeled. All of the mutants that were not accessible to MPB in membrane vesicles were subjected to labeling after solubilization in detergent (1.5% octylglucoside, 0.1% deoxycholate, and 0.5% cholate) to confirm the presence of the cysteine substitutions. All of the mutants tested for labeling with MPB reacted with the reagent in detergent solution to a similar extent (results not shown).Figure 3MPB labeling of subunit ausing anti-HA antibodies for detection. Residues indicated were mutated to cysteine and probed in membrane vesicle preparations with MPB as described under “Experimental Procedures.”Lanes at each end show the protein standards of 28 and 36 kDa. Residues tested are as indicated: I79C, E80C, L81C, V82C, I83C, G84C, F85C, V86C, N87C, G88C, S89C, V90C, K91C, D92C, M93C, Y94C, H95C, G96C, K97C, and S98C. For each residue, MPB labeling is shown inA with a > symbol at the left to indicate the location of subunit a. An immunoblot using anti-HA antibodies is shown in B.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Residue Glu-80 in E. coli subunit a is a conserved residue among all known bacteria and chloroplasts. To probe its possible functional properties, four substitution mutations were made at the aE80 position: alanine, glutamine, leucine, and lysine. All of the mutants were grown on succinate minimal medium to confirm ATP synthase activity. All showed normal growth after 48 h with the exception of E80K, which showed marginal growth. The aE80A, aE80K,aE80L, and aE80Q mutants were further characterized in a quantitative fashion using minimal A medium and a limiting amount of glucose. The results of the growth yield measurements expressed relative to that of the wild type (pLN7HisHA) were aE80A (89%), aE80K (59%), aE80L (92%), aE80Q (86%), and RH305 (50%). The level of subunita present in the membranes of these four mutants was tested by immunoblotting using the anti-a antibody, and the results are shown in Fig. 4. The level of subunita in membranes from the aE80K mutant was clearly somewhat lower than in the wild type or other mutants. Membrane vesicles from all of the four mutants and wild type were prepared and assayed for ATP hydrolysis with the results shown in TableI. The specific activity of theaE80K mutant is only half of the wild type level, andaE80A and aE80L are also somewhat lower. When ATP hydrolysis was carried out in the presence of LDAO, which releases an uninhibited ATPase from the membranes (38Lötscher H.R. deJong C. Capaldi R.A. Biochemistry. 1984; 23: 4140-4143Crossref PubMed Scopus (99) Google Scholar), the results showed that all of the mutants have within 85% of the total ATPase activity of the wild type. ATP hydrolysis was also measured after treatment with the inhibitory reagent DCCD under conditions in which it is specific for the reaction with the F0 sector. Three of the mutants have at least 80% of the wild type sensitivity to DCCD, andaE80K is somewhat lower at ∼65%.Table IATP hydrolysis by membrane vesicles containing aE80 mutationsMutationSpecific activity1-aSpecific activities reported are the average of at least four determinations from at least two preparations and did not differ by >10%.Specific activity1-bSpecific activities were measured in the presence of 1.5% LDAO. The fold stimulation is shown in parentheses. + LDAOSensitivity to DCCD1-cATP hydrolysis was measured after an incubation with 50 μm DCCD for 15 min at 35UC in 50 mm MOPS, 10 mm MgCl2, pH 7.3.μmol mg−1 min−1-Fold stimulation%wild type0.561.76 (3.0)71aE80A0.411.50 (3.7)72aE80K0.261.50 (5.8)46aE80L0.391.54 (4.0)63aE80Q0.561.93 (3.5)591-a Specific activities reported are the average of at least four determinations from at least two preparations and did not differ by >10%.1-b Specific activities were measured in the presence of 1.5% LDAO. The fold stimulation is shown in parentheses.1-c ATP hydrolysis was measured after an incubation with 50 μm DCCD for 15 min at 35UC in 50 mm MOPS, 10 mm MgCl2, pH 7.3. Open table in a new tab Proton translocation by the aE80 mutants was studied by observing the ability of membrane vesicle preparations to generate a proton gradient as indicated by quenching the fluorescence of the dye ACMA. This experiment was done in three ways, and the results presented are typical of traces from at least three different experiments using at least two different membrane preparations. First, as shown in Fig.5A, a proton gradient was generated by the addition of NADH, which drives proton translocation via the electron transport chain. This serves to verify the integrity of the membrane vesicles. The four mutants, the wild type (pLN7-HisHA), and the negative control (RH305) all quench the fluorescence of ACMA at approximately the same rate. Second, the proton translocation was driven by the addition of ATP as shown in Fig. 5B. This measures the ability of the ATP synthase to pump protons. Proton translocation by aE80K is much slower than that of the wild type (pLN7-HisHA). aE80A and aE80L show some impairment, and aE80Q quenching is approximately the same as the wild type. Third, the proton permeability of F0 alone was measured, and the results are shown in Fig. 3B. The membranes first are stripped of F1, and then proton translocation is driven by NADH. Since F1 is absent, the F0 channel is “open” to passive proton movement and therefore, little quenching is seen in the wild type andaE80Q. At the other extreme, the negative control (RH305) quenches to the same level as seen in the unstripped membranes (Fig. 5A), because it lacks a functional subunita and the F0 channel cannot form.aE80K quenches nearly as much as RH305, whereasaE80A and aE80L are again intermediate. Eight of the accessible cysteine mutants near the conserved aE80 were subjected to cross-linking experiments using the bifunctional UV-activated cross-linker, TFPAM-3. At the maleimidyl end, TFPAM-3 can react with the free sulfhydryl group of an available cysteine, and when activated by UV light, its nitrene can insert into nearby C–H bonds. The results are shown in Fig. 6. The immunoblots were incubated with antibodies against subunit a(panel A) or b (panel B) as indicated. A distinct band is present in both blots for mutant aK74C that migrates to approximately 47 kDa, a position consistent with an a/b cross-link (subunita, 30.2 kDa; subunit b, 17.2 kDa). A similar-sized band seems to be present for aK91C, although its intensity is much less. The cross-linking appeared to have little effect on proton translocation as seen in Fig. 7. The ATP driven proton translocation was not significantly affected by the cross-linking procedure for both the wild type (pLN7-HisHA) and theaK74C mutant as shown in Fig. 7. The proton permeability of F0 in stripped membranes was also not affected significantly (data not shown). In addition, the residues in the second cytoplasmic loop found in a previous study (19Long J.C. Wang S. Vik S.B. J. Biol. Chem. 1998; 273: 16235-16240Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) to be accessible to MPB were subjected to cross-linking with TFPAM-3. No cross-links were detected for residues 172, 176, or 196 (data not shown). The transmembrane topology of subunit a has been investigated previously with a chemical-labeling approach using mutants with substitutions of unique cysteine residues (19Long J.C. Wang S. Vik S.B. J. Biol. Chem. 1998; 273: 16235-16240Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 20Valiyaveetil F.I. Fillingame R.H. J. Biol. Chem. 1998; 273: 16241-16247Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 21Wada T. Long J.C. Zhang D. Vik S.B. J. Biol. Chem. 1999; 274: 17353-17357Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In that approach, relatively small numbers of residues in each exposed region of the protein were probed with maleimide reagents. The results indicated a model with five transmembrane spans, the N terminus located in the periplasm, and the C terminus located in the cytoplasm as shown in Fig. 1. In addition, two long loops of 35–40 residues were placed on the cytoplasmic side, and two shorter loops of 3–11 residues were placed on the periplasmic side. In this study, we expanded the analysis to nearly all of the residues in the first cytoplasmic loop between transmembrane spans 1 and 2. This loop is characterized by two lysines (residues 65 and 66) at its N-terminal region near transmembrane span 1 and two additional lysines (residues 97 and 99) at its C-terminal region near transmembrane span 2. This region is shown schematically in Fig.8. The overall hydrophobicity of the intervening region is moderately hydrophobic but less than that of a typical membrane-spanning region (39Vik S.B. Dao N.N. Biochim. Biophys. Acta. 1992; 1140: 199-207Crossref PubMed Scopus (24) Google Scholar). The most hydrophobic segment, FQTAIELVIGF (residues 75–85), lies just between two predicted turns, VPGK and VNGSV. Previous labeling studies had found that after mutagenesis to cysteine, residues 67–71 were readily labeled by maleimide reagents (19Long J.C. Wang S. Vik S.B. J. Biol. Chem. 1998; 273: 16235-16240Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 20Valiyaveetil F.I. Fillingame R.H. J. Biol. Chem. 1998; 273: 16241-16247Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 21Wada T. Long J.C. Zhang D. Vik S.B. J. Biol. Chem. 1999; 274: 17353-17357Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Here it was shown thataG73C and aK74C, the last two residues in the predicted turn, are also readily labeled. Among the 16 residues fromaF75C to aV90C, only three positions were readily labeled (aG84C, aI87C, and aG88C), and seven more residues showed a trace of labeling. In contrast, all of the residues from 91 to 98 (KDMYHGKS) could be labeled to a significant extent. Overall, the labeling pattern of the residues between the two pairs of lysines can be described as follows. The eight residues closest to the lysines near the membrane interface can be labeled, whereas most of the intervening 16 residues are rather resistant to labeling. The labeling results indicate that the regions of the polypeptide that extend out from the transmembrane spans are highly exposed. This would be consistent with a peripheral location of these transmembrane spans relative to spans 3–5 of subunita, the two transmembrane spans of subunit b, and the oligomer of subunit c. Such an arrangement would allow access of the bulky MPB to five or more consecutive residues in the polypeptide chain. The labeling pattern also suggests a relatively fixed conformation for the central region of this loop in which the more hydrophobic residues are shielded. Such shielding could be from lipids, other regions of subunit a, or from other subunits. The lipids would seem to be the least likely source for the shielding of a loop based on the observation of the loops of membrane proteins of known three-dimensional structures. The interaction with other regions of subunit a or with subunit b is more probable based on our results of cross-linking and analysis of Glu-80 mutants. Glu-80 is a conserved residue that has been found in all of the bacterial and chloroplast sequences examined so far. It is found in the middle of a generally hydrophobic stretch of seven residues, and it always follows a conserved glutamine, Gln-76, in E. coli. The mutagenesis of Glu-80 had only minor effects, except when the substitution was lysine. Although immunoblots showed that the level of protein in the membranes from the aE80K strain is somewhat reduced relative to wild type, it is not probable that this is the primary explanation for the altered properties seen. For example, it has been shown that only ∼10–15% of the normal level of ATP synthase is sufficient for growth on a minimal succinate medium (40Boogerd F.C. Boe L. Michelsen O. Jensen P.R. J. Bacteriol. 1998; 180: 5855-5859Crossref PubMed Google Scholar). Here, the analysis of ATP hydrolysis indicates that a normal level of F1-ATPase was present in membranes from all of the four mutants and that even the aE80K mutant showed substantial sensitivity of ATP hydrolysis to inhibition by DCCD. In addition, whereas aE80Q is essentially normal with respect to ATP-driven proton translocation, the other mutants and especiallyaE80K showed a diminished ability to generate a proton gradient. Finally, the fluorescence traces of stripped membranes indicated that all of the four mutants have diminished rates of passive proton movement through F0 with aE80K showing the lowest permeability and aE80Q showing a nearly wild type rate. These results suggest that aE80K disrupts to some degree the assembly of the F0 sector, resulting in a reduced level of subunit a and in somewhat altered properties of the assembled complex. The rather low specific activity of the aE80K membranes might be explained by the enhanced inhibition of ATP hydrolysis by ε as is seen in free F1-ATPase. Upon the addition of LDAO, which releases F1 from F0 and from inhibition by ε, the rate was stimulated almost 6-fold compared with ∼3-fold for the wild type. The results also indicate that in the aE80K mutant, some F1 is bound loosely to the membrane. Glu-80 shares some similarities with Glu-196 (34Vik S.B. Cain B.D. Chun K.T. Simoni R.D. J. Biol. Chem. 1988; 263: 6599-6605Abstract Full Text PDF PubMed Google Scholar) as a conserved residue on the cytoplasmic side of subunit a that may be involved in shuttling protons at least indirectly to and from the subunit c oligomer. Because of the partial (aE196Q) or substantial (aE80Q) growth yields and rates of proton translocation, these residues cannot be considered as obligatory sites of protonation/deprotonation. Rather, the strength of the effect of the mutations might reflect the relative proximity of these residues to protonation sites in subunit c as well as the nature of the substitution. Whether there is a proton access channel or half-channel from the cytoplasmic surface through subunita to a Asp-61 site in subunit c, the evidence so far indicates that the two cytoplasmic loops rather than transmembrane spans are key elements. In this regard, the two access channels (31Vik S.B. Patterson A.R. Antonio B.J. J. Biol. Chem. 1998; 273: 16229-16234Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) would be quite different, because evidence indicates that on the periplasmic side, residues within the transmembrane spans are important for proton translocation. The cross-linking results presented here are the first in which a photoactivated cross-linker was attached to a specific site in subunita and was found to attach covalently to subunitb. The converse reaction from bR36C to subunita using the bifunctional cross-linker benzophenone-4-maleimide was reported by McLachlin et al.(41McLachlin D.T. Coveny A.M. Clark S.M. Dunn S.D. J. Biol. Chem. 2000; 275: 17571-17577Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), indicating that these subunits have interactions outside the lipid bilayer. Much earlier studies had addressed the role of interactions between subunits a and b. Chemical cross-linking studies had identified the close proximity of these subunits (42Aris J.P. Simoni R.D. J. Biol. Chem. 1983; 258: 14599-14609Abstract Full Text PDF PubMed Google Scholar, 43Hermolin J. Gallant J. Fillingame R.H. J. Biol. Chem. 1983; 258: 14550-14555Abstract Full Text PDF PubMed Google Scholar). The studies by Kumamoto and Simoni (44Kumamoto C.A. Simoni R.D. J. Biol. Chem. 1986; 261: 10037-10042Abstract Full Text PDF PubMed Google Scholar) showed thataP240A or aP240L could partially suppress the deleterious effects of bG9D, a site that is most probably within the lipid bilayer. The demonstration that antibodies against F1 subunits could co-immunoprecipitate subunitsa and b in stoichiometric complexes (45Vik S.B. Simoni R.D. J. Biol. Chem. 1987; 262: 8340-8346Abstract Full Text PDF PubMed Google Scholar) suggested strong interactions between the two subunits. The results presented here support the recent findings of McLachlin et al. (41McLachlin D.T. Coveny A.M. Clark S.M. Dunn S.D. J. Biol. Chem. 2000; 275: 17571-17577Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) that that subunits a and b interact within the cytoplasm and demonstrate that the first cytoplasmic loop of subunit a is involved in those interactions. The role of ana·b2 complex as a stator (30Junge W. Lill H. Engelbrecht S. Trends Biochem. Sci. 1997; 22: 420-423Abstract Full Text PDF PubMed Scopus (439) Google Scholar) during ATP synthesis or hydrolysis is supported by the finding that proton translocation is not significantly affected by the cross-linking of the two subunits. In summary, the cross-linking and labeling results indicate that this cytoplasmic loop of subunit a is in close proximity to one or both b subunits. An analysis of aE80 mutants is consistent with a role for this residue in interactions with theb subunits. The conservation pattern of aE80 is also consistent with such a role in that this glutamatic acid is conserved among bacteria and chloroplasts, both of which have twob subunits, although it is not conserved among mitochondria ATP synthases that have a single b subunit (47Collinson I.R. Skehel J.M. Fearnley I.M. Runswick M.J. Walker J.E. Biochemistry. 1996; 35: 12640-12646Crossref PubMed Scopus (72) Google Scholar). Furthermore, bacterial and chloroplast b subunits contain a conserved arginine (48Caviston T.L. Ketchum C.J. Sorgen P.L. Nakamoto R.K. Cain B.D. FEBS Lett. 1998; 429: 201-206Crossref PubMed Scopus (37) Google Scholar) that has been shown to serve as a site for cross-linking to subunit a (41McLachlin D.T. Coveny A.M. Clark S.M. Dunn S.D. J. Biol. Chem. 2000; 275: 17571-17577Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). We thank Dr. K. Altendorf (Universität Osnabrück, Germany) and Dr. R. Capaldi (University of Oregon) for generously providing antibodies and Lata Narawane for constructing several plasmids." @default.
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• W2021336642 title "Characterization of the First Cytoplasmic Loop of Subunit a of the Escherichia coli ATP Synthase by Surface Labeling, Cross-linking, and Mutagenesis" @default.
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