The ATP/Substrate Stoichiometry of the ATP-binding Cassette (ABC) Transporter OpuA

ATP-binding cassette (ABC) transport proteins catalyze the translocation of substrates at the expense of hydrolysis of ATP, but the actual ATP/substrate stoichiometry is still controversial. In the osmoregulated ABC transporter (OpuA) from Lactococcus lactis, ATP hydrolysis and substrate translocation are tightly coupled, and the activity of right-side-in and inside-out reconstituted OpuA can be determined accurately. Although the ATP/substrate stoichiometry determined from the uptake of glycine betaine and intravesicular ATP hydrolysis tends to increase with decreasing average size of the liposomes, the data from inside-out reconstituted OpuA indicate that the mechanistic stoichiometry is 2. Moreover, the two orientations of OpuA in proteoliposomes allowed possible contributions from substrate (glycine betaine) inhibition on the trans-side of the membrane and inhibition by ADP to be determined. Here we show that OpuA is not inhibited by up to 400 mm glycine betaine on the trans-side of the membrane. ADP is an inhibitor, but accumulation of ADP was negligible in the assays with inside-out-oriented OpuA, and potential effects of the ATP/ADP ratio on the ATP/substrate stoichiometry determinations could be eliminated. ATP-binding cassette (ABC) transport proteins catalyze the translocation of substrates at the expense of hydrolysis of ATP, but the actual ATP/substrate stoichiometry is still controversial. In the osmoregulated ABC transporter (OpuA) from Lactococcus lactis, ATP hydrolysis and substrate translocation are tightly coupled, and the activity of right-side-in and inside-out reconstituted OpuA can be determined accurately. Although the ATP/substrate stoichiometry determined from the uptake of glycine betaine and intravesicular ATP hydrolysis tends to increase with decreasing average size of the liposomes, the data from inside-out reconstituted OpuA indicate that the mechanistic stoichiometry is 2. Moreover, the two orientations of OpuA in proteoliposomes allowed possible contributions from substrate (glycine betaine) inhibition on the trans-side of the membrane and inhibition by ADP to be determined. Here we show that OpuA is not inhibited by up to 400 mm glycine betaine on the trans-side of the membrane. ADP is an inhibitor, but accumulation of ADP was negligible in the assays with inside-out-oriented OpuA, and potential effects of the ATP/ADP ratio on the ATP/substrate stoichiometry determinations could be eliminated. ATP-binding cassette (ABC) 1The abbreviations used are: ABC, ATP-binding cassette; DOPG, dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol)-sodium salt; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; AMP-PNP, 5′-adenylyl-β,γ-imidodiphosphate. transporters, found in both prokaryotes and eukaryotes, play an important role in various physiological processes ranging from uptake of nutrients, multidrug resistance, secretion of signal molecules or toxins, cell volume regulation, and other processes (reviewed in Refs. 1Hyde S.C. Emsley P. Hartshorn M.J. Mimmack M.M. Gileadi U. Pearch S.R. Gallagher M.P. Gill D.R. Hubbard R.E. Higgins C.F. Nature. 1990; 346: 362-365Crossref PubMed Scopus (957) Google Scholar, 2Davidson A.L. J. Bacteriol. 2002; 184: 1225-1233Crossref PubMed Scopus (94) Google Scholar, 3Geourjon C. Orelle C. Steinfels E. Blanchet C. Deléage G. Di Pietro A. Jault J.-M. Trends Biochem. Sci. 2001; 26: 539-544Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 4Ames G.F. Mimura C.S. Shyamala V. FEMS Microbiol. Rev. 1990; 75: 429-446Crossref Google Scholar). Despite the diversity of functions and substrates of the ABC transporters, all members fuel the translocation process by ATP hydrolysis. Also, the basic architecture of ABC transporters is remarkably similar among members in the superfamily. Always present are two cytoplasmic-exposed nucleotide-binding domains, the ATP-binding cassettes, and two hydrophobic domains that are predicted to span the membrane multiple times in an α-helical conformation (2Davidson A.L. J. Bacteriol. 2002; 184: 1225-1233Crossref PubMed Scopus (94) Google Scholar). The conserved assembly and arrangement of these domains have been clearly demonstrated in the recently determined structure of BtuCD, the ABC transporter that mediates uptake of vitamin B12 in Escherichia coli (5Locher K.P. Lee A.T. Rees D.C. Science. 2002; 296: 1038-1040Crossref PubMed Scopus (932) Google Scholar). Prokaryotic ABC transporters involved in solute uptake use an additional substrate-binding protein that delivers the substrate to the translocator. The ABC transporter, OpuA, from Lactococcus lactis belongs to a subfamily of the OTCN family of which members have the substrate-binding domain fused to the translocator (6Heide van der T. Poolman B. EMBO Rep. 2002; 3: 938-943Crossref PubMed Scopus (141) Google Scholar). In the dimeric complex and presumably functional state, two of these chimeric substrate-binding/translocator proteins and two ATPase subunits are present. Extensive characterization of OpuA has revealed that this protein responds to osmotic stress, which is sensed at the cytoplasmic face as a change in ionic strength (7Heide van der T. Poolman B. J. Bacteriol. 2000; 182: 203-206Crossref PubMed Scopus (57) Google Scholar, 8Heide van der T. Poolman B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7102-7106Crossref PubMed Scopus (150) Google Scholar, 9Heide van der T. Stuart M.C. Poolman B. EMBO J. 2001; 20: 7022-7032Crossref PubMed Scopus (126) Google Scholar). The issue of ATP/substrate stoichiometry for members of the ABC transport family is still a subject of debate. ATP/substrate stoichiometries for a variety of ABC transporters range from 1 to 50 (10Ambudkar S.V. Cardelli C.O. Pashinsky I. Stein W.D. J. Biol. Chem. 1997; 34: 21160-21166Abstract Full Text Full Text PDF Scopus (173) Google Scholar, 11Shapiro A.B. Ling V. Eur. J. Biochem. 1998; 254: 189-193Crossref PubMed Scopus (89) Google Scholar, 12Davidson A.L. Nikaido H. J. Biol. Chem. 1990; 265: 4254-4260Abstract Full Text PDF PubMed Google Scholar, 13Eytan G.D. Regev R. Assaraf Y.G. J. Biol. Chem. 1996; 271: 3172-3178Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 14Sauna Z.E. Ambudkar S.V. J. Biol. Chem. 2001; 276: 11653-11661Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 15Sauna Z.E. Ambudkar S.V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2515-2520Crossref PubMed Scopus (201) Google Scholar, 16Mimmack M.L. Gallagher M.P. Pearce S.R. Hyde S.C. Booth I.R. Higgins C.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8257-8261Crossref PubMed Scopus (81) Google Scholar, 17Bishop L. Agbayani Jr., R. Ambudkar S.V. Maloney P.C. Ames G.F.-L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6953-6957Crossref PubMed Scopus (125) Google Scholar, 18Muir M. Williams L. Ferenci T. J. Bacteriol. 1985; 163: 1237-1242Crossref PubMed Google Scholar, 19Liu C.E. Liu P.Q. Ames G.F.-L. J. Biol. Chem. 1997; 272: 21883-21891Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 20Shapiro A.B. Ling V. J. Biol. Chem. 1995; 270: 16167-16175Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Given the similarity in architecture between different members of the ABC superfamily, it is likely that a universal mechanistic stoichiometry is applicable to all members. The large variations observed are most likely the result of experimental flaws, differences in the setup of the experiments, and/or the need to correct for uncoupled ATP hydrolysis and/or substrate leak pathways. The OpuA system affords optimal conditions for determination of ATP/substrate stoichiometries because ATP hydrolysis is tightly coupled to the osmotic signal and the presence of substrate. In this report, we perform stoichiometry measurements on proteoliposomes in which OpuA has been functionally reconstituted. We have performed two independent assays to derive the stoichiometries and conclude that 2 molecules of ATP are hydrolyzed per molecule of glycine betaine translocated. Materials—M17 broth was purchased from Difco, nickel-nitrilotriacetic acid resin was obtained from Qiagen Inc., Biobeads SM-2 were from Bio-Rad, n-dodecyl-β-d-maltoside was purchased from Anatrace, Triton X-100 was obtained from Roche Applied Science. 1,2-Dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol)-sodium salt (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were obtained from Avanti Polar Lipids. Radiolabeled N-methyl-14Ccholine chloride (55 mCi/mmol) and N-methyl-3Hcholine chloride (80 Ci/mmol) were purchased from Amersham Biosciences and used as precursors to synthesize N-methyl-14Cglycine betaine and N-methyl-3Hglycine betaine, respectively, as described (21Landfald B. Strom A.R. J. Bacteriol. 1986; 165: 849-855Crossref PubMed Scopus (281) Google Scholar). Unlabeled glycine betaine was purchased from Sigma, and its purity was confirmed by proton-NMR spectroscopy. Creatine kinase, mono-phosphate creatine-disodium salt, tetracaine, ATP-disodium salt, and carnitine were reagent grade and obtained from commercial sources. ATP and Pi commercial standards were from ATPlite™-M (PerkinElmer Life Sciences) and Sigma, respectively. Isolation of Membrane Vesicles and OpuA Purification—L. lactis strain NZ9000 (22De Ruyter P.G. Kuipers O.P. de Vos W.M. Appl. Environ. Microbiol. 1996; 62: 3662-3667Crossref PubMed Google Scholar) was grown semi-anaerobically at 30 °C, and large scale isolation of membranes was performed as described (9Heide van der T. Stuart M.C. Poolman B. EMBO J. 2001; 20: 7022-7032Crossref PubMed Scopus (126) Google Scholar, 23Poolman B. Konings W.N. Robillard G.T. Eur. J. Biochem. 1983; 135: 41-46Crossref PubMed Scopus (31) Google Scholar). OpuA was purified from membrane vesicles as described previously (9Heide van der T. Stuart M.C. Poolman B. EMBO J. 2001; 20: 7022-7032Crossref PubMed Scopus (126) Google Scholar). The resulting protein fractions were assayed for purity and concentration via SDS-PAGE and A 280 UV-visible spectroscopy, respectively. Membrane Reconstitution of OpuA—OpuA was incorporated into liposomes consisting of synthetic DOPG and DOPE at a 1:1 ratio. Equal amounts of DOPG and DOPE in chloroform were mixed, dried in a rotary evaporator, and, subsequently, resuspended to 20 mg/ml in 50 mm phosphate, pH 7.0. For optimal hydration, lipids were sonicated with a point tip sonicator (S PubMed Scopus Google Scholar). this n-dodecyl-β-d-maltoside and purified OpuA was to the lipids at or protein to ratio and the was for 30 at with a protein to ratio of to 1 to on a The proteoliposomes were by of by the of the of protein and mg/ml were in a of 15 at 15 at 4 °C, 30 at 4 °C, at 4 °C, and at 4 The were by and the was with 50 mm phosphate, pH which the proteoliposomes were by at for The proteoliposomes were and resuspended to 20 mg/ml and in liquid in uptake or mm or an system mg/ml kinase, mm mm was in the proteoliposomes three cycles of freezing in liquid nitrogen and thawing at 4 were via of various size or the proteoliposomes were with 50 mm phosphate, pH and by at for to In experiments, a 20 was used to proteoliposomes or to for the proteoliposomes were by at for and resuspended to 20 mg/ml proteoliposomes were into of to and betaine concentration at 30 various a of the was with at 4 °C, and size The use of allowed of the proteoliposomes on the for The proteoliposomes were with 2 of 4 to The on the was determined by liquid from of was by with mm to an osmotic to uptake of betaine as described a of glycine betaine was the proteoliposomes were into a volume of of similar mm which inside-out-oriented OpuA and of were and as described of ATP to of glycine betaine uptake and ATP hydrolysis was by the in concentration of ATP or by the in concentration of Biochem. PubMed Scopus Google Scholar). For of ATP the were assayed for betaine uptake at 30 the of proteoliposomes were in a that ATP hydrolysis. with the Life Sciences) for the was for and with an ATP The ADP were determined from the in ATP concentration of ADP into ATP, mm and 50 kinase, and the actual ATP the ATP hydrolysis the of to the of the was and the were performed in The activity of OpuA was in and not the the was of the proteoliposomes on A of the was with the and the was with the of The of was determined by the at and with a were performed with liposomes not OpuA to correct for of and were by freezing point with an concentration determinations were with the of M.M. Biochem. PubMed Scopus Google Scholar) as a UV-visible measurements of protein concentration and of lipids were a and ATP by and of OpuA activity were for right-side-in and inside-out reconstituted molecules A and of glycine betaine was performed with proteoliposomes or the system In the of glycine betaine uptake was observed not The system allowed a of uptake because the ATP the ADP for a with or glycine betaine uptake was observed OpuA was incorporated into liposomes of The transporter is in the these conditions (7Heide van der T. Poolman B. J. Bacteriol. 2000; 182: 203-206Crossref PubMed Scopus (57) Google Scholar, 8Heide van der T. Poolman B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7102-7106Crossref PubMed Scopus (150) Google Scholar). the OpuA transporter, and the of glycine betaine uptake was of betaine was observed the of to the proteoliposomes that glycine betaine for from the of the inside-out-oriented OpuA with the osmotic by ionic strength at the cytoplasmic face of the membrane (9Heide van der T. Stuart M.C. Poolman B. EMBO J. 2001; 20: 7022-7032Crossref PubMed Scopus (126) Google of glycine betaine with of or and not with or not in the of or in the presence of the ATP not The of glycine betaine by inside-out-oriented OpuA in the presence of amounts of ATP was the of uptake by OpuA ATP or an system the which the of the molecules with this J. Poolman B. 1998; PubMed Scopus Google Scholar). different of we observed that the of OpuA is in the range of The in ATP and ADP as a result of glycine betaine uptake are in The data indicate that ATP hydrolysis the of the clearly that both glycine betaine and ionic are for ATP that ATP hydrolysis and substrate translocation are tightly not observed for other ABC transporters, a of for of stoichiometry of OpuA of glycine betaine uptake A and a of mm ATP is most likely to of ATP in proteoliposomes with OpuA and a of liposomes OpuA and a ATP the inhibition of uptake could have a that it could be a of the accumulation of ADP and/or glycine ADP is to the ATPase activity of ABC transporters, and inhibition by of on the trans-side of the has been observed for glycine betaine uptake in and E. Konings W.N. Poolman B. J. Bacteriol. 1998; PubMed Google Scholar, A. E. Poolman B. T. J. Bacteriol. 1997; PubMed Google Scholar). is to the effects of inhibition by ADP or glycine betaine with OpuA because it of the we possible effects of ADP and substrate on the activity of the inside-out-oriented OpuA In we the of ATP and different ADP on the of of glycine betaine from the the of ATP, of glycine betaine was conditions of with mm the of was The for ATP of was mm not was a of glycine betaine was with the for ADP inhibition of was mm not ADP has a on the and of glycine betaine with a in ATP the uptake with possible effects by betaine was with up to 400 mm glycine betaine on the In we show that of glycine betaine on the trans-side of the membrane not glycine betaine Also, a substrate of glycine betaine, not the of glycine betaine by is not a substrate of OpuA but has been to effects on the glycine transporter of L. A. E. Poolman B. T. J. Bacteriol. 1997; PubMed Google Scholar). these indicate that by substrate is not a that the uptake as the glycine A of proteoliposomes OpuA, with the inhibition by be the for the inhibition of glycine betaine uptake the presence of an mm ATP in the of the of glycine betaine that a of the proteoliposomes that has glycine betaine not have inside-out OpuA to the substrate. stoichiometries can be obtained from of the and of ATP hydrolysis and glycine betaine uptake or In we show the of ATP hydrolysis and glycine betaine uptake measurements performed on proteoliposomes of different average and by of and size in the presence of mm We used proteoliposomes of different size with the that the volume present a on the of ATP that can be the of glycine betaine that can be the of ATP in the vesicles with the size of the which the proteoliposomes were Moreover, the ratio of ATP hydrolysis and glycine betaine that the ATP/substrate to be in proteoliposomes of and volume In these proteoliposomes with a volume to the concentration of ATP as a result of for stoichiometry not by a in ATP or accumulation of of ATP hydrolysis and of glycine In this the concentration of ATP was and the ATP hydrolysis by inside-out-oriented OpuA was determined from the of for of phosphate, as a of ATP A of experiments, OpuA activity in differences in of ATP hydrolysis coupled to glycine betaine or ionic not was hydrolysis of ATP glycine betaine was not present in the or the translocator was not In we show the and glycine betaine from proteoliposomes of and The ratio of both measurements is in and that the ATP betaine transport stoichiometry is 2 and is independent of the and The data in the average of three independent experiments, with three independent protein In this report, we have the of substrate translocation inhibition and ATP/substrate stoichiometry for the osmoregulated ABC transporter The inhibition of substrate translocation is of for the of glycine betaine the in L. OpuA as glycine betaine to (7Heide van der T. Poolman B. J. Bacteriol. 2000; 182: 203-206Crossref PubMed Scopus (57) Google Scholar, 8Heide van der T. Poolman B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7102-7106Crossref PubMed Scopus (150) Google Scholar). the hydrolysis of 1 or 2 ATP molecules per molecule of substrate be to a concentration of to cytoplasmic The between the and observed of substrate translocation can be by inhibition of the transport possible of the transporter by previously substrate. on transporters, a acid transporter, and a transport system for have that the transporters are inhibited by with inhibition in the range A. T. A. J. PubMed Scopus Google Scholar, Life Sci. 2001; PubMed Scopus Google Scholar, C. S. L. J. J. 1989; PubMed Scopus Google Scholar). by substrate has been observed for glycine betaine uptake in the transport from L. and L. E. Konings W.N. Poolman B. J. Bacteriol. 1998; PubMed Google Scholar, A. E. Poolman B. T. J. Bacteriol. 1997; PubMed Google Scholar). clearly as a that substrate betaine present at at the cytoplasmic face of OpuA has on the transport that accumulation of ADP a role in the inhibition of substrate at in ADP or the ATP/ADP ratio a role in the osmotic of the glycine betaine in to be We the that cytoplasmic ionic strength is the of OpuA which the glycine betaine accumulation (9Heide van der T. Stuart M.C. Poolman B. EMBO J. 2001; 20: 7022-7032Crossref PubMed Scopus (126) Google Scholar). is on the ATP/substrate stoichiometry for members of the ABC transport the is with variations in stoichiometries ranging from 1 to 50 on the ABC transporter (10Ambudkar S.V. Cardelli C.O. Pashinsky I. Stein W.D. J. Biol. Chem. 1997; 34: 21160-21166Abstract Full Text Full Text PDF Scopus (173) Google Scholar, 11Shapiro A.B. Ling V. Eur. J. Biochem. 1998; 254: 189-193Crossref PubMed Scopus (89) Google Scholar, 12Davidson A.L. Nikaido H. J. Biol. Chem. 1990; 265: 4254-4260Abstract Full Text PDF PubMed Google Scholar, 13Eytan G.D. Regev R. Assaraf Y.G. J. Biol. Chem. 1996; 271: 3172-3178Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 14Sauna Z.E. Ambudkar S.V. J. Biol. Chem. 2001; 276: 11653-11661Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 15Sauna Z.E. Ambudkar S.V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2515-2520Crossref PubMed Scopus (201) Google Scholar, 16Mimmack M.L. Gallagher M.P. Pearce S.R. Hyde S.C. Booth I.R. Higgins C.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8257-8261Crossref PubMed Scopus (81) Google Scholar, 17Bishop L. Agbayani Jr., R. Ambudkar S.V. Maloney P.C. Ames G.F.-L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6953-6957Crossref PubMed Scopus (125) Google Scholar, 18Muir M. Williams L. Ferenci T. J. Bacteriol. 1985; 163: 1237-1242Crossref PubMed Google Scholar, 19Liu C.E. Liu P.Q. Ames G.F.-L. J. Biol. Chem. 1997; 272: 21883-21891Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 20Shapiro A.B. Ling V. J. Biol. Chem. 1995; 270: 16167-16175Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). the determined or variations of stoichiometries the members of the ABC transporter the members of the ABC superfamily, are differences in the and of substrate and variations in the of ATP are the that different members of the ABC have different ATP/substrate the of of it is likely that the large variations in stoichiometry are to experimental We have possible of experimental in of ATP/substrate stoichiometries for ABC the of on different and this that ATP was per or M. Williams L. Ferenci T. J. Bacteriol. 1985; 163: 1237-1242Crossref PubMed Google Scholar). The of this in to the multiple processes that are to and potential to of ATP hydrolysis and substrate ATP hydrolysis translocation of substrate result in can correct for by the for ATP hydrolysis in the of substrate from the in the presence of but this could to an of the ATP/substrate The hydrolysis of ATP in the and presence of substrate is not the the hydrolysis of ATP is in the presence of substrate because the of a complex the of the complex and the hydrolysis of in in of 50 of to both a of ATP hydrolysis and of between the and A.B. Ling V. J. Biol. Chem. 1995; 270: 16167-16175Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). ATP hydrolysis with and substrate was in uptake on the of and the stoichiometry of was to of ATP hydrolysis to substrate translocation L. Agbayani Jr., R. Ambudkar S.V. Maloney P.C. Ames G.F.-L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6953-6957Crossref PubMed Scopus (125) Google Scholar). For the transport system from E. ATP/substrate stoichiometries up to were to differences in protein to variations in the of that to of the ATPase A.L. Nikaido H. J. Biol. Chem. 1990; 265: 4254-4260Abstract Full Text PDF PubMed Google Scholar). The of the substrate additional in the stoichiometry A hydrophobic substrate of a resulting in Moreover, the hydrophobic of the substrates has been to result in to the membrane of to of the ATP/substrate stoichiometry M.L. Gallagher M.P. Pearce S.R. Hyde S.C. Booth I.R. Higgins C.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8257-8261Crossref PubMed Scopus (81) Google Scholar). In this we could the by of the of ATP hydrolysis to substrate translocation in the OpuA system and osmotic are for ATP and the of glycine betaine on the scale of the We that OpuA with its and activity the ABC system to ATP/substrate stoichiometry In stoichiometry measurements performed with OpuA, we observed variations in stoichiometries in different of that were the proteoliposomes and we could a increase in stoichiometry with decreasing size of the of stoichiometries on the of inside-out-oriented OpuA that the size of the not the In we observed an ATP/substrate stoichiometry of 2 with with to OpuA, the ATP/substrate stoichiometry was determined from the in ATP and the ATP concentration ATP/ADP in the of the OpuA, the ATP is present on the and could be at and this a the ATP and the ADP Although the glycine betaine in the the concentration of the for glycine betaine is we the that the of the proteoliposomes the of OpuA, resulting in the stoichiometry on are with a of two translocated. G.D. Regev R. Assaraf Y.G. J. Biol. Chem. 1996; 271: 3172-3178Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) have the transport of an complex in which the by the of most the ATPase and transport of conditions and observed molecule and Ambudkar Z.E. Ambudkar S.V. J. Biol. Chem. 2001; 276: 11653-11661Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 15Sauna Z.E. Ambudkar S.V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2515-2520Crossref PubMed Scopus (201) Google the conformation of the effects of ATP and hydrolysis on substrate the of these and it was that two ATP hydrolysis in a for substrate translocation and for the protein for the Z.E. Ambudkar S.V. J. Biol. Chem. 2001; 276: 11653-11661Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 15Sauna Z.E. Ambudkar S.V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2515-2520Crossref PubMed Scopus (201) Google Scholar). a with the translocation of substrate to an ATP/substrate stoichiometry of 2. the be that 2 molecules of substrate are per of a stoichiometry of two stoichiometry the of a for the of the OpuA transporter and is most likely for ABC transporters in A of glycine betaine to the substrate domains by a of ATP to the ATPase ATP be hydrolyzed to the OpuA from the to the substrate to be at the cytoplasmic The ATP be used to the that to OpuA from the to state, resulting in of the translocation