The Vacuolar H+-ATPase of Lemon Fruits Is Regulated by Variable H+/ATP Coupling and Slip

Abstract

Lemon fruit tonoplasts, unlike those of seedling epicotyls, contain nitrate-insensitive H+-ATPase activity (Müller, M. L., Irkens-Kiesecker, U., Rubinstein, B., and Taiz, L. (1996) J. Biol. Chem. 271, 1916–1924). However, the degree of nitrate-insensitivity fluctuates during the course of the year with a seasonal frequency. Nitrate uncouples H+ pumping from ATP hydrolysis both in epicotyls and in nitrate-sensitive fruit V-ATPases. Neither bafilomycin nor oxidation cause uncoupling. The initial rate H+/ATP coupling ratios of epicotyl and the nitrate-sensitive fruit proton pumping activities are the same. However, the H+/ATP coupling ratio of the nitrate-insensitive fruit H+ pumping activity is lower than that of nitrate-sensitive and epicotyl V-ATPases. Several properties of the nitrate-insensitive H+-ATPase of the fruit indicate that it is a modified V-ATPase rather than a P-ATPase: 1) insensitivity to low concentrations of vanadate; 2) it is initially strongly uncoupled by nitrate, but regains coupling as catalysis proceeds; 3) both the nitrate-sensitive and nitrate-insensitive fruit H+-pumps have identicalK m values for MgATP, and show similar pH-dependent slip and proton leakage rates. We conclude that the ability of the juice sac V-ATPase to build up steep pH gradients involves three factors: variable coupling,i.e. the ability to regain coupling under conditions that initially induce uncoupling; a low pH-dependent slip rate; the low proton permeability of the membrane. Lemon fruit tonoplasts, unlike those of seedling epicotyls, contain nitrate-insensitive H+-ATPase activity (Müller, M. L., Irkens-Kiesecker, U., Rubinstein, B., and Taiz, L. (1996) J. Biol. Chem. 271, 1916–1924). However, the degree of nitrate-insensitivity fluctuates during the course of the year with a seasonal frequency. Nitrate uncouples H+ pumping from ATP hydrolysis both in epicotyls and in nitrate-sensitive fruit V-ATPases. Neither bafilomycin nor oxidation cause uncoupling. The initial rate H+/ATP coupling ratios of epicotyl and the nitrate-sensitive fruit proton pumping activities are the same. However, the H+/ATP coupling ratio of the nitrate-insensitive fruit H+ pumping activity is lower than that of nitrate-sensitive and epicotyl V-ATPases. Several properties of the nitrate-insensitive H+-ATPase of the fruit indicate that it is a modified V-ATPase rather than a P-ATPase: 1) insensitivity to low concentrations of vanadate; 2) it is initially strongly uncoupled by nitrate, but regains coupling as catalysis proceeds; 3) both the nitrate-sensitive and nitrate-insensitive fruit H+-pumps have identicalK m values for MgATP, and show similar pH-dependent slip and proton leakage rates. We conclude that the ability of the juice sac V-ATPase to build up steep pH gradients involves three factors: variable coupling,i.e. the ability to regain coupling under conditions that initially induce uncoupling; a low pH-dependent slip rate; the low proton permeability of the membrane. The vacuolar H+-ATPases (V-ATPases) 1The abbreviations used are: V-ATPase, vacuolar H+-ATPase; a.u., arbitrary units; BTP, 1,3-bis-(tris(hydroxymethyl)methylamino)pro-pane; DTT, dithiothreitol; Mes, 2-(N-morpholino)ethanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; NEM, N-ethylmaleimide; PAGE, polyacrylamide gel electrophoresis; RB, resuspension buffer; ΔμH+, proton electrochemical gradient; Δψ , membrane potential.1The abbreviations used are: V-ATPase, vacuolar H+-ATPase; a.u., arbitrary units; BTP, 1,3-bis-(tris(hydroxymethyl)methylamino)pro-pane; DTT, dithiothreitol; Mes, 2-(N-morpholino)ethanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; NEM, N-ethylmaleimide; PAGE, polyacrylamide gel electrophoresis; RB, resuspension buffer; ΔμH+, proton electrochemical gradient; Δψ , membrane potential. of eukaryotic cells are large, multimeric proton pumps composed of 10 to 13 different subunits organized into a hydrophilic catalytic complex, V1, and a hydrophobic transmembrane H+ channel, Vo (1Finbow M.E. Harrison M.A. Biochem. J. 1997; 324: 697-712Crossref PubMed Scopus (229) Google Scholar). V-ATPases are structurally related to the ATP synthases, or F-ATPases, of mitochondria and chloroplasts. F- and V-ATPases exhibit many structural and functional similarities at the molecular level, including several homologous subunits which play central roles in catalysis and proton transport (2Taiz L. Nelson N. Baltscheffsky H. Origin and Evolution of Biological Energy Conversion. VCH Verlagsgesellschaft, Germany1996: 291-305Google Scholar, 3Bowman B.J. Bowman E.J. Brambl R. Marzluf G.A. The Mycota III. A Comprehensive Treatise on Fungi as Experimental Systems for Basic and Applied Research: Biochemistry and Molecular Biology. Springer-Verlag, Berlin1996: 57-83Google Scholar). Because of these similarities in structure and catalytic mechanism, F- and V-ATPases are also thought to exhibit similar mechanochemical properties (4Oster G. Wang H. Encyclopedia of Molecular Biology. 1999; (in press)Google Scholar). When operating in ATP synthesis mode, F-ATPases convert the proton-motive force present across the internal membrane of chloroplasts and mitochondria into a rotary torque used to drive the synthesis of ATP by the catalytic F1 sector (5Junge W. Lill H. Engelbrecht S. Trends Biochem. Sci. 1997; 22: 420-423Abstract Full Text PDF PubMed Scopus (430) Google Scholar, 6Yasuda R. Noji H. Kinosita K. Motojima F. Yoshida M. J. Bioenerg. Biomembr. 1997; 29: 207-209Crossref PubMed Scopus (25) Google Scholar). Conversely, when F-ATPases operate in proton pumping mode, they hydrolyze ATP at their catalytic site and convert the liberated energy into a rotary torque utilized to drive proton transport across the membrane (7Noji H. Yasuda R. Yoshida M. Kinosita K. Nature. 1997; 386: 299-302Crossref PubMed Scopus (1924) Google Scholar). V-ATPases are also thought to operate via a rotary mechanism, even though direct experimental evidence for rotation is lacking (1Finbow M.E. Harrison M.A. Biochem. J. 1997; 324: 697-712Crossref PubMed Scopus (229) Google Scholar). In V- and F-ATPases, the efficiency of coupling between ATP hydrolysis and proton transport represents a critical, rate-limiting factor. Both pumps have been proposed to undergo slip, or intrinsic uncoupling, under specific conditions (8Groth G. Junge W. Biochemistry. 1993; 32: 8103-8111Crossref PubMed Scopus (54) Google Scholar, 9Evron Y. Avron M. Biochim. Biophys. Acta. 1990; 1019: 115-120Crossref Scopus (27) Google Scholar, 10Moriyama Y. Nelson N. Stein W.D. The Ion Pumps: Structure, Function, and Regulation. Alan R. Liss, Inc., New York1988: 387-394Google Scholar). During slip, ATP is hydrolyzed at the catalytic site without being coupled to H+ transport. Since typical V-ATPases are known to operate far from thermodynamic equilibrium, slip may represent one of the enzyme's regulatory mechanisms. In lemon fruit juice sac cells, the vacuolar pH can reach as low as 2.2, about 3 pH units lower than in typical plant vacuoles. In fact, the trans-tonoplast pH gradient in lemon fruits is close to the for a V-ATPase operating at thermodynamic to pH H+/ATP of Biochem. Biophys. 1993; PubMed Scopus Google Scholar, Scopus Google Scholar). We have that proton pumping by juice sac is to the V-ATPase nitrate, bafilomycin and to concentrations to L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). 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PubMed Scopus Google Scholar). pumps have the H+/ATP but in their coupling the with the H+/ATP coupling ratio to a than the with the lower coupling the coupling ratio of the lemon fruit V-ATPase than that of the epicotyl V-ATPase even the pumps the have to the coupling ratios of the fruit and epicotyl V-ATPases under initial rate the activity of the fruit is to also the of on the coupling ratio of the juice sac proton show in to pH-dependent slip, the H+/ATP coupling ratios of the juice sac and epicotyl proton under initial rate are the same. with the of the epicotyl V-ATPase, a of the fruit H+ pumping activity uncoupled under However, the properties of the juice sac proton pumps to those in the strongly that in juice sac the proton pumping activity to a of rather than to a different of proton A of the of from juice that the of activity during the course of the the of uncoupled the H+/ATP coupling ratio in juice sac The of of variable H+/ATP coupling in lemon fruits is but the activity may represent a of V-ATPases under is to build the across the of juice Lemon L. for seedling epicotyls by Inc., Lemon fruits from a on the of the of from from or from lemon fruit juice and epicotyls as L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus (54) Google Scholar). juice in fruit m pH DTT, and and epicotyls in epicotyl m pH DTT, and a of the for at the to for at and the on a gradient for at The with pH and and for at The in at a of of membrane up to of in of and or on for and at The membrane in of and used for activity and The in of the and of the used for being with with and by pumping and ATP hydrolysis by by the of 1990; PubMed Scopus Google Scholar). In proton transport into the by the of at ATP hydrolysis by coupling the of to the oxidation of and by the at The of 10 pH and of a of and also present in the The with and the values at and in with a with a the L. and for the into a and When activity are the of the initial of proton pumping and ATP hydrolysis and in arbitrary units The H+/ATP coupling ratios of the of membrane used in the We to in the of of membrane When proton pumping in the of in the for the of to on proton pumping the conditions as L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). H+/ATP coupling ratios to in the that H+ pumping and ATP hydrolysis activities in the Because the initial rate of H+ pumping from the activity rate in the of nitrate, the for activity in the of The of from epicotyls and juice by to Nature. PubMed Scopus Google in polyacrylamide and the to as L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The with a to the of the V-ATPase and by a L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of concentrations with N. M. H. Biol. Google Scholar). that proton pumping by fruit juice sac to by and bafilomycin L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google that in the insensitivity of the lemon fruit V-ATPase from one membrane to 3 A the of fruit from a of fruits at the of from a on the of the of We the of the the of the proton pumping activity by a from to in the to in the of the V-ATPases to and bafilomycin proton pumping activities of from juice and seedling epicotyls in the of the typical V-ATPase and bafilomycin used to proton transport. in a of the of juice sac used to the of fruit to a of fruits at the of from the membrane the H+ pumping activity in the and of and the activity of the as a of the is initial of of a juice sac to with The proton pumping activities of from juice and seedling epicotyls in the of the typical V-ATPase and bafilomycin used to proton transport. In to the seasonal in a also in the of in 3 even in a juice sac H+ pumping to initially by However, the activity a to reach a rate to that of the in the of the rate of proton pumping under experimental conditions used in the of and ATP present or However, that the ATP to the of the In to H+ ATP hydrolysis show a similar that in the of the fruit proton pumps initially but coupled as catalysis The lemon fruit V-ATPase to to concentrations of L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus (54) Google Scholar). We the of on the initial of proton pumping A and and ATP hydrolysis and and on the coupling ratio and of from juice and of juice sac are a one which by and a which by the of in A and proton pumping and ATP hydrolysis by of epicotyls to in the the coupling ratio of the epicotyl V-ATPase between 10 and The of of that activity is a in of the coupling ratio of the epicotyl V-ATPase concentrations of proton pumping to ATP hydrolysis to under the However, to to the properties of at the used in the coupled In to the with from epicotyls, ATP hydrolysis by lemon fruit by by However, proton pumping by low concentrations of in both the nitrate-sensitive and the nitrate-insensitive that a of membrane in the of of a may present in the fruit Both ATP hydrolysis and proton pumping by to of their activity in the of The nitrate-sensitive fruit about as in proton pumping as the nitrate-insensitive a the coupling ratio for the nitrate-sensitive is about than that of the nitrate-insensitive and is to the coupling ratio of the epicotyl V-ATPase We that in the of the of catalytic subunits from fruit without the H+ pumping activity to L. 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Since the of H+ pumping activity to arbitrary the to have the H+ pumping activity into a the H+/ATP coupling ratio of the nitrate-sensitive which in the in the of to present or a on the nitrate-sensitive H+ pumping activity of juice sac and coupling ratios of fruit for proton pumping and activities of a nitrate-sensitive juice sac under the conditions in the to and coupling ratios are in arbitrary units and the by is in arbitrary units and as a of the activity of the with in a The proton pumping and activities of a nitrate-sensitive juice sac under the conditions in the to and coupling ratios are in arbitrary units and the by is in arbitrary units and as a of the activity of the with A of and coupling ratios also under and conditions for a nitrate-sensitive fruit In the proton pumping by ATP hydrolysis by by the and the proton pumping activity by and The H+/ATP coupling which in the in the of to the coupling ratio of the that of the concentrations of have on the coupling ratios of the the Since the H+-ATPase activity from the activity in of both activities with m and in a fruit to the in it is that the ATP activity by up to a of the of the from the of activity the of the between and m of ATP and the in the or of In to ATP of the proton pumping activity by A and However, the m of ATP for the to that of the that the of as In to the with have a of V-ATPases with a different coupling ratio than the V-ATPase, the initial of pumping of and and their pH gradient at The up by to from that of The pH-dependent slip and leakage rate of and and for the proton pumping rate at during the build in the pH-dependent slip and leakage in and proton pumping by fruit for in the or of juice sac as in the to for similar initial of H+ of membrane for the and of the to reach present in the the pH gradient with the pH-dependent slip and leakage rate as L. 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Chem. 1997; Full Text Full Text PDF PubMed Scopus (54) Google and for the of proton pumping by at by the of a the pumping in A pumping to the epicotyl V-ATPase and the nitrate-sensitive of the fruit V-ATPase the of by Because to pH gradients in the of PubMed Google used to proton transport in the in A the H+ pumping and ATP hydrolysis activities of both the epicotyl and the fruit the for epicotyl The coupling ratios of fruit and epicotyl in the of concentrations are in 10 that the activity The in the H+/ATP coupling ratio between and that in fruit and epicotyl proton pumping to a than ATP and uncouples the the nitrate-sensitive of the activity is of the fruit V-ATPase at low concentrations of at concentrations the epicotyl is strongly uncoupled by than the In the of the H+/ATP coupling ratio of the fruit about that of the epicotyl to the of bafilomycin on the coupling ratios of fruit and epicotyl membrane A and In it that fruit that nitrate-sensitive to bafilomycin as A and show the of bafilomycin on the H+ pumping and ATP hydrolysis activities of epicotyl and a fruit The H+/ATP coupling ratios between and bafilomycin are in on the coupling ratios of the epicotyl or the fruit V-ATPases. 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In the present used initial activity to the H+/ATP coupling ratios of the proton pumps in fruit and epicotyl A of nitrate-sensitive nitrate-insensitive fruit that nitrate-sensitive have H+/ATP coupling ratios than nitrate-insensitive Since the coupling ratio of the nitrate-sensitive is to that of epicotyl and that the epicotyl is by a V-ATPase the nitrate-sensitive juice sac are by one of proton pumping the it a of and with H+/ATP of and Biochem. Biophys. 1993; PubMed Scopus Google Scholar, PubMed Google Scholar, J. Y. PubMed Scopus Google Scholar, PubMed Scopus Google one the coupling ratio of the fruit to lower than that of the though the coupling ratio of the nitrate-insensitive juice sac lower than that of from epicotyls, that these also a V-ATPase rather than of 1) during the initial of H+ pumping in the of nitrate, the nitrate-insensitive fruit are The of coupling is on the of and may of or a 2) juice sac show properties and similar to the nitrate-sensitive juice sac In the as the H+ pumping activity is thought to to a V-ATPase, on m and the ATP hydrolysis even though the coupling ratio is lower than in epicotyl V-ATPases. and Nelson Y. Nelson N. Stein W.D. The Ion Pumps: Structure, Function, and Regulation. Alan R. Liss, Inc., New York1988: 387-394Google have proposed that of proton transport from ATP hydrolysis in V-ATPases. In from lemon juice and epicotyls, When the nitrate-sensitive of H+ pumping and ATP hydrolysis as the of the activities by juice sac and epicotyl under with to the H+/ATP coupling ratio of epicotyl V-ATPases from initial the fruit of their initial coupling a of nitrate-sensitive V-ATPases for to by to the H+ pumping activity of fruit to in is in the to Since H+-ATPases are by to 10 it is that the of the lemon fruit H+ pumping activity involves the of a L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus (54) Google Scholar). 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However, for the of proton pumping The from the in that a of V-ATPases as a of The in a of a molecular that the fruit V-ATPase to to in the of is with in which that a with with the juice sac V-ATPase during by gel and L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus (54) Google Scholar). to fruit V-ATPases by their activity is modified as to the coupling ratio is to of of V-ATPases. The of may in during of the in nitrate-insensitive juice sac proton pumps and a low H+/ATP coupling one is by the that m values for in and in for strongly that the of H+-ATPase and with The that the m values in the to lower than the for the juice sac activity L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google is to the of ATP in the present which even of into a of the the by with nitrate, and the pH-dependent slip and leakage rate to that of the a V-ATPase with a different on the and with the one the seasonal in the H+/ATP coupling ratio as a in the of the V-ATPase, rather than a in the activity of a of proton on the been that of the V-ATPase by the of Biochim. Biophys. Acta. Scopus Google Scholar, H. L. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). the properties of the of the sector from Vo Biochim. Biophys. Acta. Scopus Google proposed to a different of H. L. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). present indicate that is and on the lemon as from to the of to the of from Vo which with the of the sector from the membrane. that even of epicotyl with of the to the even though proton pumping is of the from the membrane is for of proton but may for of ATP and Bowman Bowman B.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google proposed that the low of to properties and the of in and of from the membrane. the of proton pumping that in epicotyl may have been to at the catalytic site L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus (54) Google Scholar, Y. M. J. Biol. Chem. Full Text PDF PubMed Google Y. M. J. Biol. Chem. Full Text PDF PubMed Google but also have of the H. R. M. H. J. 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