Structure, Functioning, and Assembly of the ATP Synthase in Cells from Patients with the T8993G Mitochondrial DNA Mutation

The structure and functioning of the ATP synthase of human fibroblast cell lines with 91 and 100%, respectively, of the T8993G mutation have been studied, with MRC5 human fibroblasts and Rho0 cells derived from this cell line as controls. ATP hydrolysis was normal but ATP synthesis was reduced by 60% in the 100% mutants. Both activities were highly oligomycin-sensitive. The levels of F1F0 were close to normal, and the enzyme was stable. It is concluded that the loss of ATP synthesis is because of disruption of the proton translocation step within the F0 part. This is supported by membrane potential measurements using the dye JC-1. Cells with a 91% mutation load grew well and showed only a 25% loss in ATP synthesis. This much reduced effect for only a 9% difference in mutation load mirrors the reduced pathogenicity in patients. F1F0 has been purified for the first time from human cell lines. A partial complex was obtained from Rho0 cells containing the F1subunits associated with several stalk, as well as F0subunits, including oligomycin sensitivity conferring protein,b, and c subunits. This partial complex no longer binds inhibitor protein. The structure and functioning of the ATP synthase of human fibroblast cell lines with 91 and 100%, respectively, of the T8993G mutation have been studied, with MRC5 human fibroblasts and Rho0 cells derived from this cell line as controls. ATP hydrolysis was normal but ATP synthesis was reduced by 60% in the 100% mutants. Both activities were highly oligomycin-sensitive. The levels of F1F0 were close to normal, and the enzyme was stable. It is concluded that the loss of ATP synthesis is because of disruption of the proton translocation step within the F0 part. This is supported by membrane potential measurements using the dye JC-1. Cells with a 91% mutation load grew well and showed only a 25% loss in ATP synthesis. This much reduced effect for only a 9% difference in mutation load mirrors the reduced pathogenicity in patients. F1F0 has been purified for the first time from human cell lines. A partial complex was obtained from Rho0 cells containing the F1subunits associated with several stalk, as well as F0subunits, including oligomycin sensitivity conferring protein,b, and c subunits. This partial complex no longer binds inhibitor protein. 2-(N-morpholino)ethanesulfonic acid N,N′,dicyclohexylcarbodiimide oligomycin sensitivity conferring protein An F1F0 type ATP synthase is involved in oxidative and photosynthetic ATP synthesis in prokaryotes and eukaryotes. Recent electron microscopy (1.Gogol E.P. Johnston E. Aggeler R. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9585-9589Crossref PubMed Scopus (100) Google Scholar), crystallography (2.Abrahams J.P. Leslie A.G. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2754) Google Scholar), fluorescence (3.Sabbert D. Engelbrecht S. Junge W. Nature. 1996; 381: 623-625Crossref PubMed Scopus (464) Google Scholar), cross-linking (4.Aggeler R. Ogilvie I. Capaldi R.A. J. Biol. Chem. 1997; 272: 19621-19624Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 5.Zhou Y. Duncan T.M. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10583-10587Crossref PubMed Scopus (102) Google Scholar), and single molecule video microscopy studies (6.Noji H. Yasuda R. Yoshida M. Kinosita Jr., K. Nature. 1997; 386: 299-302Crossref PubMed Scopus (1966) Google Scholar) show that the F1F0complex is a molecular motor. In the emerging view of the structure and function of the enzyme, it is thought that the well-known central stalk connecting F1 to F0 is a rotor that moves in relation to a stator holding the catalytic α/β pairs (reviewed in Refs. 7.Engelbrecht S. Junge W. FEBS Lett. 1997; 414: 485-491Crossref PubMed Scopus (118) Google Scholar and 8.Ogilvie I. Wilkens S. Rodgers A.J. Aggeler R. Capaldi R.A. Acta Physiol. Scand. Suppl. 1998; A 643: 169-175PubMed Google Scholar). The stator is probably the second stalk observed recently at the periphery of the F1F0 complex (9.Wilkens S. Capaldi R.A. Nature. 1998; 393: 29Crossref PubMed Scopus (135) Google Scholar). Key subunits in the rotor are the γ and ε subunits of F1 and the c 12 subunit ring of F0 (10.Watts S.D. Tang C. Capaldi R.A. J. Biol. Chem. 1996; 271: 28341-28347Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The stator contains the a andb 2 subunits of F0 and the δ subunit (Escherichia coli nomenclature) attached at the top of the three α/β pairs (11.Lill H. Hensel F. Junge W. Engelbrecht S. J. Biol. Chem. 1996; 271: 32737-32742Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 12.Ogilvie I. Aggeler R. Capaldi R.A. J. Biol. Chem. 1997; 272: 16652-16656Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Eukaryotic F1F0-ATPases have up to eight additional subunits when compared with the bacterial enzyme, most of which contribute to the stalks or F0 parts (13.Collinson I.R. Runswick M.J. Buchanan S.K. Fearnley I.M. Skehel J.M. van Raaij M.J. Griffiths D.E. Walker J.E. Biochemistry. 1994; 33: 7971-7978Crossref PubMed Scopus (166) Google Scholar, 14.Collinson I.R. van Raaij M.J. Runswick M.J. Fearnley I.M. Skehel J.M. Orriss G.L. Miroux B. Walker J.E. J. Mol. Biol. 1994; 242: 408-421PubMed Google Scholar). The biogenesis and assembly of eukaryotic F1F0is complicated because of the presence of polypeptides encoded in two separate genomes with synthesis of these occurring in two compartments, the cytosol and the mitochondrion. In mammals, subunit 6 (ora in E. coli) and A6L are encoded in the mitochondrial genome. In yeast, subunit 9 (also called subunitc) is mitochondrially encoded (15.Tzagoloff A. Meaher P. J. Biol. Chem. 1972; 247: 594-603Abstract Full Text PDF PubMed Google Scholar, 16.Sierra M.F. Tzagoloff A. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 3155-3159Crossref PubMed Scopus (68) Google Scholar). Assembly of the F1 part occurs in the absence of some or all of the F0 subunits, as revealed by studies of Rho0cells in Saccharomyces cerevisiae (17.Orian J.M. Hadikusumo R.G. Marzuki S. Linnane A.W. J. Bioenerg. Biomembr. 1984; 16: 561-581Crossref PubMed Scopus (15) Google Scholar), Kluyveromyces lactis (18.Chen X.J. Hansbro P.M. Clark-Walker G.D. Mol. Gen. Genet. 1998; 259: 457-467Crossref PubMed Scopus (17) Google Scholar), and human cell lines (19.Nijtmans L.G. Klement P. Houstek J. van den Bogert C. Biochim. Biophys. Acta. 1995; 1272: 190-198Crossref PubMed Scopus (68) Google Scholar). A growing number of human diseases are now known that are because of a deficiency of mitochondrial function, and many are the result of mutations of mtDNA. A particularly interesting mutation of subunit 6 is the human T8993G mutation, by which Leu-156 is changed to Arg. Depending on the percentage of mtDNA molecules with this mutation, patients present with neurogenic muscle weakness, atraxia, and retinitis pigmentosa or Leigh's Syndrome (above 95% mutation) (20.Tatuch Y. Christodoulou J. Feigenbaum A. Clarke J.T. Wherret J. Smith C Rudd N. Petrova-Benedict R Robinson B.H. Am. J. Hum. Genet. 1992; 50: 852-858PubMed Google Scholar). Studies of the functioning of the ATP synthase in cell lines from patients with the T8993G mutation have established that ATP synthesis is decreased (21.Robinson B.H. J. Bioenerg. Biomemb. 1994; 26: 311-316Crossref PubMed Scopus (17) Google Scholar, 22.Vázquez-Memije M.E. Shanske S. Santorelli F.M. Kranz-Eble P. Davidson E. DeVivo D.C. DiMauro S. J. Inherit. Metab. Dis. 1996; 19: 43-50Crossref PubMed Scopus (55) Google Scholar, 23.Vázquez-Memije M.E. Shanske S. Santorelli F.M. Kranz-Eble P. DeVivo D.C. DiMauro S. J. Inherit. Metab. Dis. 1998; 21: 829-836Crossref PubMed Scopus (44) Google Scholar, 24.Majander A. Lamminen T. Juvonen V. Aula P. Nikoskelainen E. Savontaus M.L. Wikström M. FEBS Lett. 1997; 412: 351-354Crossref PubMed Scopus (21) Google Scholar), but an unambiguous distinction between effects on assembly and on enzyme turnover has not been made. Here, we have examined the ATP synthase in mitochondria from fibroblasts of patients with 91 and 100%, respectively, of the T8993G mutation. Activity measurements are reported and the levels of assembled enzyme in mutant and Rho0 ATP synthase have been measured. The ATP synthase has been isolated from cell cultures for the first time. Finally, the role of the inhibitor protein in determining the properties of mutant and Rho0 enzymes is described. MRC5 fibroblasts were obtained from the American Type Culture Collection. The population doubling of the cells was in the range of 30–45 before harvesting to isolated mitochondria. To grow Rho0 cells, MRC5 cells (population doubling = 28–30) were cultured continuously for a further 14–20 population doublings in the presence of 50 ng/ml ethidium bromide. The T8993G mutant-containing fibroblast cell lines were from two patients. Both were diagnosed with Leigh's Disease in the clinic of the Hospital for Sick Children, Toronto, Canada. The levels of mutation were established by standard protocols (20.Tatuch Y. Christodoulou J. Feigenbaum A. Clarke J.T. Wherret J. Smith C Rudd N. Petrova-Benedict R Robinson B.H. Am. J. Hum. Genet. 1992; 50: 852-858PubMed Google Scholar). All cells were grown as described before (25.Marusich M.F. Robinson B.H. Taanman J.W. Kim S.J. Schillace R. Smith J.L. Capaldi R.A. Biochim. Biophys. Acta. 1997; 1362: 145-159Crossref PubMed Scopus (98) Google Scholar) in with 50 and to were isolated from cells to standard D. D. a Scholar) with the described Cells were three with of and in of containing and This was with up and in a and at for at This was three and the were and at to A mitochondrial was by at for 12 at This was in of containing and in of the for at in to The of E. N. J. Bioenerg. Biomembr. 1995; PubMed Scopus Google Scholar) was to F1F0 from fibroblast cell lines as A was as to of F1 by the step to in the were by the isolated mitochondria with in This of was to membrane but not F1 in by with were in containing in to as This mitochondrial a of that was at for at The membrane were in and with protein was to subunit of it was at a of and for before the of the A was by at for and the was to the in 2 The was with of the and enzyme with were to the were and the protein with at for activities were with an ATP M.E. A. E. J. Biol. Chem. Full Text PDF PubMed Google Scholar) at in the presence of and to of was to the of the of the enzyme Biochemistry. 1990; PubMed Scopus Google Scholar). to was by the inhibitor to the sensitivity was by the for with at before the of of by the inhibitor protein was by the before for 2 at in a containing 2 ATP synthase was as of mitochondria were in to type including were at with in for were with 50 and at with by to were to for and to protein. In the which is was by in the presence of of was at ATP was for of which was by known of ATP to the in the absence of The of ATP synthesis reported are at to to was using the of was first to a P. PubMed Scopus Google Scholar) to of of the with the was to Nature. PubMed Scopus Google Scholar) in a were to for at V. were with of to the of the were and in this and described inhibitor protein was and purified as described by and Biophys. PubMed Scopus Google Scholar). fibroblast cell lines containing the T8993G mutation were examined in this with 91% of mutant mtDNA and the second with 100%, for the mutation. MRC5 fibroblasts were as Rho0 cells from this MRC5 cell line were to the effect of a absence of the ATP synthase subunit 6 and All cell lines were grown in and these the population doubling of the MRC5 fibroblasts was that of the 91% T8993G mutant was Rho0 MRC5 cells were that of the 100% T8993G mutant was The activities of mitochondria isolated from cell lines are in A. The 91% mutant showed and the 100% mutant of ATP hydrolysis MRC5 This in mutant cell lines was highly oligomycin-sensitive. In the of Rho0 cells was the and was no oligomycin sensitivity of the ATP synthesis of mitochondria from of the cell lines are in B. The T8993G mutation in 91% of mtDNA a of 25% in the of ATP synthesis. 100% of the mutation, ATP synthesis were only of controls. was ATP synthesis of mitochondria from Rho0 ATP synthesis in the T8993G was highly oligomycin-sensitive. In of the of the fibroblast cell lines and the of mitochondria to a membrane potential was by fluorescence microscopy the dye This dye is in mitochondria in to the membrane potential of the of membrane potential show up as of membrane potential are M. C A. M. 1995; PubMed Scopus Google Scholar for the properties of It that the of cells for the T8993G mutation is from that of MRC5 cells or the 91% mutant or Rho0 cells The 100% cells were much were and to the cell much normal fibroblast cell lines. In MRC5 cells, were of membrane potential revealed by the of The of oligomycin the number and of these as ATP synthesis the of and associated by the In the 100% mutant not in the 91% were of and oligomycin not these The mitochondria of Rho0 cells showed of membrane which were in type or T8993G mutants. The of the of was much in the 100% mutant and not the of oligomycin not The in levels of between the T8993G cells and MRC5 cells is that a F1F0 is assembled in normal in the mutant cell lines. To the of the subunit was compared with that of the subunits of and for cell line a range of protein using in and the levels of the F1subunits in the T8993G 91% cell lines were to of the levels in the 100% mutant and lines in the MRC5 To the of the assembled enzyme, of F1 was by the presence of subunit in the of the was no of subunit in the 91 or 100% mutants. In F1 was in from Rho0 cells by this show that F1 is to F0 in the T8993G mutant cells but from This was in of subunit from cell was purified by an first by and M. A. Biophys. PubMed Scopus Google Scholar). of purified mitochondria was for the the and of the 100% mutant cell line of the enzyme from this of of the cell lines were to to of The of F1F0 was from the and T8993G 91% but only from was for studies of subunit and a of the subunit of F1F0 isolated from MRC5 cells, the 91% and Rho0 cell lines. from the mutant the as that isolated from MRC5 as the properties of the In was only a partial assembly of the F1F0 in the Rho0 cells, with the on the subunit subunits and γ are present in Rho0 mitochondria with but at levels in the or mutant cell lines. are on the at the of subunits and but are in to the subunits. subunit a was The presence of in the by To the presence of subunit the of using and Rho0 mitochondria first with as described and The enzyme was with acid before to c subunit as described by E. B. J. PubMed Scopus Google Scholar). this F1F0 with several of the but a single in the acid The was obtained with and the of c subunit was only partial in the human the presence of in of by and of human Rho0 and purified F1F0 were an mitochondria were with and the F1F0 complex was purified as described and The enzyme was to and was before and acid the as in was to human mitochondria to the c was to and T8993G 91% T8993G 100% and Rho0 and mitochondrial or of were The of the was in and purified in E. coli as described and In the ATP hydrolysis and ATP synthesis of F1F0 are the of a the inhibitor or a A. M. Biophys. Chem. PubMed Scopus Google Scholar). The functioning of this protein by the of the enzyme at an effect with that a in at a which the of the and the 91 and 100% mutant was and that this inhibitor in the mutant as well as in MRC5 mitochondria. In the of Rho0 mitochondria was only effects were observed when purified F1F0 was examined in this the of was the at because of partial of the F1 of protein the of Rho0 mitochondria at was to that of mitochondria that been to the effect of This that the inhibitor was no longer to the assembled F1F0 of Rho0 This was using and the human protein. In line with the it was that as well as T8993G 91 and 100% showed levels of when by Rho0 showed or levels of the to The result was obtained with purified but with these that the and by are in the T8993G mutant but not in This the effect of the T8993G mutation on the structure and functioning of the ATP synthase using fibroblast cell lines with 91 and 100%, respectively, of the mutation. MRC5 fibroblasts were as The properties of the ATP synthase in the mutant were compared with of enzyme in Rho0 Rho0 cells mtDNA not subunits 6 and a a ATP synthase is not Rho0 cells are of the of the ATP synthase in cells mitochondrial (25.Marusich M.F. Robinson B.H. Taanman J.W. Kim S.J. Schillace R. Smith J.L. Capaldi R.A. Biochim. Biophys. Acta. 1997; 1362: 145-159Crossref PubMed Scopus (98) Google Scholar), a recently described in studies of cell lines and the T8993G mutation have that the of for at in the of subunit 6 ATP synthesis by (20.Tatuch Y. Christodoulou J. Feigenbaum A. Clarke J.T. Wherret J. Smith C Rudd N. Petrova-Benedict R Robinson B.H. Am. J. Hum. Genet. 1992; 50: 852-858PubMed Google Scholar, B.H. J. Bioenerg. Biomemb. 1994; 26: 311-316Crossref PubMed Scopus (17) Google Scholar, 22.Vázquez-Memije M.E. Shanske S. Santorelli F.M. Kranz-Eble P. Davidson E. DeVivo D.C. DiMauro S. J. Inherit. Metab. Dis. 1996; 19: 43-50Crossref PubMed Scopus (55) Google Scholar, 23.Vázquez-Memije M.E. Shanske S. Santorelli F.M. Kranz-Eble P. DeVivo D.C. DiMauro S. J. Inherit. Metab. Dis. 1998; 21: 829-836Crossref PubMed Scopus (44) Google Scholar). ATP hydrolysis was examined in of these and the of the loss of functioning was because of reduced assembly enzyme has been only Houstek J. Klement P. J. H. H. den Bogert C. J. Biochim. Acta. 1995; PubMed Scopus Google Scholar) several of a for the T8993G mutation and that the ATP synthase was and on concluded that assembly was the of the pathogenicity of the mutation. Here, we have a fibroblast cell line in which the T8993G mutation is In this mutant cell ATP synthesis was of a fibroblast ATP hydrolysis was Both ATP synthesis and hydrolysis were highly oligomycin are with studies with fibroblast mitochondria M.E. Shanske S. Santorelli F.M. Kranz-Eble P. Davidson E. DeVivo D.C. DiMauro S. J. Inherit. Metab. Dis. 1996; 19: 43-50Crossref PubMed Scopus (55) Google Scholar, 23.Vázquez-Memije M.E. Shanske S. Santorelli F.M. Kranz-Eble P. DeVivo D.C. DiMauro S. J. Inherit. Metab. Dis. 1998; 21: 829-836Crossref PubMed Scopus (44) Google Scholar). To the levels of assembly of ATP synthase in the and was with an with the measurements levels of F1 were in T8993G and Rho0 mitochondria the activities of the ATP hydrolysis are in the and An F1 was assembled in Rho0 cells that subunit 6 and a ATP in Rho0 cells, this was not oligomycin-sensitive. the we that a F1F0 is assembled in normal in the T8993G this assembled enzyme is stable. F1 is not by of mitochondria from the T8993G This is in to of F1 by of Rho0 mitochondria the F1F0 is not an F1F0 isolated from the 91% mutant by of mitochondria using to the was the F1 part. A enzyme to In that cells with the T8993G mutation normal of a that the ATP synthesis observed are by catalytic functioning of the An as by Houstek J. Klement P. J. H. H. den Bogert C. J. Biochim. Acta. 1995; PubMed Scopus Google Scholar) in the from have from enzyme assembly or by the mutation. to studies of the E. H. R. K. J. 1998; PubMed Scopus Google Scholar, S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), the mutation T8993G a in the of subunit 6 with an Arg. in E. coli W. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar, M.E. W. F. J. Acta Physiol. Scand. Suppl. 1998; 643: Google Scholar, W. Biochim. Biophys. Acta. 1998; PubMed Scopus Google Scholar) that the of this is close to the between subunit 6 and the ring of c subunits in a for proton The mutation proton by the to by or by the of the proton translocation step by the of ring to subunit The membrane potential measurements in 2 at the F0 because of the mutation. The of of membrane potential the mitochondrial in the 100% mutant cell line is that of MRC5 cells, of of and in mutant In the T8993G cells, the of the mitochondrial with that of cells in which of ATP synthesis is by the mutation of subunit 6 to proton It is interesting to that M.E. Shanske S. Santorelli F.M. Kranz-Eble P. Davidson E. DeVivo D.C. DiMauro S. J. Inherit. Metab. Dis. 1996; 19: 43-50Crossref PubMed Scopus (55) Google Scholar, 23.Vázquez-Memije M.E. Shanske S. Santorelli F.M. Kranz-Eble P. DeVivo D.C. DiMauro S. J. Inherit. Metab. Dis. 1998; 21: 829-836Crossref PubMed Scopus (44) Google Scholar) have recently that F1F0 from the T8993G mutant has sensitivity to oligomycin compared with cell lines. this inhibitor to the mutant load of cultured cells the T8993G mutation N. M.E. J.E. A. D. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is an of the T8993G mutation ATP synthesis. In ATP synthesis is by or inhibitor of the human ATP synthesis the of ATP is to that mutation of subunit 6 inhibitor and J.L. Biophys. PubMed Scopus Google Scholar) have recently that mutations of subunit a in the E. the of the inhibitor with the F1 Here, we have the role of in the of the T8993G mutation for the first time. The functioning of as by of was to the in the mutant as in the MRC5 cells, that of this has been to hydrolysis and ATP synthesis. Studies of Rho0 cells are a In the absence of subunit 6 in these cells, is no effect of the on It that no longer binds to the enzyme the mitochondrially encoded subunits This loss of effect Rho0cells by as the of ATP the cell of mitochondria in a to protein the Recent studies that this is by of the with an from ATP to the K. C. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, D. J. PubMed Scopus (144) Google Scholar). of the F1 by this 91% of T8993G mutation, cells grew was a and ATP synthesis was reduced only 100% of mutation, the cells grew and ATP synthesis was reduced This difference in and in relation to only a difference in of mutant the of the T8993G mutation in patients. 100%, the effect is a and known as Leigh's patients have the called neurogenic muscle weakness, atraxia, and retinitis pigmentosa (20.Tatuch Y. Christodoulou J. Feigenbaum A. Clarke J.T. Wherret J. Smith C Rudd N. Petrova-Benedict R Robinson B.H. Am. J. Hum. Genet. 1992; 50: 852-858PubMed Google Scholar). The is that a 25% loss of ATP but are by a 60% effects have been for many mutations in mtDNA. The studies described show that is ATP synthesis in the 91% mutant for by 91% of mutant enzyme molecules at and 9% molecules functioning at on the in this an ATP synthesis of compared with of ATP that 9% of This the levels of subunit 6 normal and are not for assembly of the F1F0 and the of subunit 6 is the mutant by the enzyme An is that the mutant subunit 6 is well or to a of subunit for the of mutant to mtDNA interesting of the present to the assembly of It been reported that human cells grown in the presence of to mitochondrial protein assembled a which was associated to the membrane subunit c (19.Nijtmans L.G. Klement P. Houstek J. van den Bogert C. Biochim. Biophys. Acta. 1995; 1272: 190-198Crossref PubMed Scopus (68) Google Scholar). we show that in the absence of subunits 6 and the rotor subunits, to including and the of the second stalk, subunits are The these subunits the F1 attached to the mitochondrial in cell lines. This is in line with in E. coli and subunit a is not for assembly of subunits and c or for of F1 to the membrane (17.Orian J.M. Hadikusumo R.G. Marzuki S. Linnane A.W. J. Bioenerg. Biomembr. 1984; 16: 561-581Crossref PubMed Scopus (15) Google Scholar, Hadikusumo R.G. Marzuki S. Biochim. Biophys. Acta. PubMed Scopus (21) Google Scholar, R.G. S. M.J. Linnane A.W. Marzuki S. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). subunits and c and subunit of are for assembly of subunit a J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, M.F. J. M. B. J. PubMed Scopus Google Scholar, B. J. J. 1992; PubMed Scopus Google Scholar). subunit a at the in assembly of This proton the F0 the assembly that assembly of the inhibitor protein F1F0 or A6L for of the as is when subunits a and A6L are not The inhibitor protein further to the F1F0 complex H. J.L. Biophys. 1998; PubMed Scopus Google Scholar). is in of the for purified inhibitor protein from and for for the of purified and for coli the inhibitor protein. The of F. to the inhibitor protein is for in the cell and for in the fluorescence

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