Acidic Residues Necessary for Pyrophosphate-energized Pumping and Inhibition of the Vacuolar H+-pyrophosphatase byN,N′-Dicyclohexylcarbodiimide

Abstract

On the basis of a revised topological model of the vacuolar H+-pyrophosphatase (V-PPase; EC 3.6.1.1) derived from the analysis of four published sequences using two structure-predicting programs, TopPred II and MEMSAT, eight acidic amino acid residues located near or within transmembrane α-helices were identified. The codons specifying these amino acids in the cDNA encoding the V-PPase from Arabidopsis thalianawere singly mutated to examine their involvement in pyrophosphate (PPi) hydrolysis and PPi-dependent H+ translocation and the functional significance of the similarities between the sequences encompassing Glu229(227–245) of the V-PPase and theN,N′-dicyclohexylcarbodiimide (DCCD)-binding transmembrane α-helix of the c-subunits of F-ATPases (Nyren, P., Sakai-Nore, Y., and Strid, A. (1993) Plant Cell Physiol. 34, 375–378). Three functional classes were identified after heterologous expression of mutated enzyme in Saccharomyces cerevisiae. Class I (E119Q, E229Q, D573N, E667Q, and E751Q) mutants exhibited PPi hydrolytic and H+ translocation activities and DCCD sensitivities similar to wild type. The one class II mutant obtained (E427Q) was preferentially impaired for H+translocation over PPi hydrolysis but retained sensitivity to DCCD. Class III (E305Q and D504N) mutants exhibited a near complete abolition of both PPi hydrolysis and H+translocation and residual activities with decreased DCCD sensitivity. In none of the mutants was diminished insertion of the V-PPase into the membrane or an increase in the background conductance of the membrane to H+ evident. The decoupled character of E427Q mutants and the enhancement of H+ pumping in E427D mutants by comparison with wild type, in conjunction with the retention of DCCD inhibitability in both E427Q and E427D mutants, implicate a role for Glu427 in DCCD-insensitive H+ translocation by the V-PPase. The proportionate diminution of PPi hydrolytic and H+ translocation activity and conservation of wild type DCCD sensitivity in E229Q mutants refute the notion that Glu229 is the residue whose covalent modification by DCCD is responsible for the abolition of PPi-dependent H+ translocation. Instead, the diminished sensitivity of the residual activities of E305Q and D504N mutants, but not E305D or D504E mutants, to inhibition by DCCD is consistent with the involvement of acidic residues at these positions in inhibitory DCCD binding. The results are discussed with regard to the possible involvement of Glu427 in coupling PPi hydrolysis with transmembrane H+translocation and earlier interpretations of the susceptibility of the V-PPase to inhibition by carbodiimides. On the basis of a revised topological model of the vacuolar H+-pyrophosphatase (V-PPase; EC 3.6.1.1) derived from the analysis of four published sequences using two structure-predicting programs, TopPred II and MEMSAT, eight acidic amino acid residues located near or within transmembrane α-helices were identified. The codons specifying these amino acids in the cDNA encoding the V-PPase from Arabidopsis thalianawere singly mutated to examine their involvement in pyrophosphate (PPi) hydrolysis and PPi-dependent H+ translocation and the functional significance of the similarities between the sequences encompassing Glu229(227–245) of the V-PPase and theN,N′-dicyclohexylcarbodiimide (DCCD)-binding transmembrane α-helix of the c-subunits of F-ATPases (Nyren, P., Sakai-Nore, Y., and Strid, A. (1993) Plant Cell Physiol. 34, 375–378). Three functional classes were identified after heterologous expression of mutated enzyme in Saccharomyces cerevisiae. Class I (E119Q, E229Q, D573N, E667Q, and E751Q) mutants exhibited PPi hydrolytic and H+ translocation activities and DCCD sensitivities similar to wild type. The one class II mutant obtained (E427Q) was preferentially impaired for H+translocation over PPi hydrolysis but retained sensitivity to DCCD. Class III (E305Q and D504N) mutants exhibited a near complete abolition of both PPi hydrolysis and H+translocation and residual activities with decreased DCCD sensitivity. In none of the mutants was diminished insertion of the V-PPase into the membrane or an increase in the background conductance of the membrane to H+ evident. The decoupled character of E427Q mutants and the enhancement of H+ pumping in E427D mutants by comparison with wild type, in conjunction with the retention of DCCD inhibitability in both E427Q and E427D mutants, implicate a role for Glu427 in DCCD-insensitive H+ translocation by the V-PPase. The proportionate diminution of PPi hydrolytic and H+ translocation activity and conservation of wild type DCCD sensitivity in E229Q mutants refute the notion that Glu229 is the residue whose covalent modification by DCCD is responsible for the abolition of PPi-dependent H+ translocation. Instead, the diminished sensitivity of the residual activities of E305Q and D504N mutants, but not E305D or D504E mutants, to inhibition by DCCD is consistent with the involvement of acidic residues at these positions in inhibitory DCCD binding. The results are discussed with regard to the possible involvement of Glu427 in coupling PPi hydrolysis with transmembrane H+translocation and earlier interpretations of the susceptibility of the V-PPase to inhibition by carbodiimides. The membranes constituting the vacuolysosomal complex of plant cells are unusual in possessing an H+ translocating inorganic pyrophosphatase (V-PPase 1The abbreviations used are: V-PPase, vacuolar H+-pyrophosphatase; DCCD,N,N′-dicyclohexylcarbodiimide; Mes, 4-morpholineethanesulfonic acid; NEM, N-ethylmaleimide; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; V-, F-, and P-ATPases, vacuolar-, mitochondrial-, and plasma membrane-type ATPases, respectively.1The abbreviations used are: V-PPase, vacuolar H+-pyrophosphatase; DCCD,N,N′-dicyclohexylcarbodiimide; Mes, 4-morpholineethanesulfonic acid; NEM, N-ethylmaleimide; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; V-, F-, and P-ATPases, vacuolar-, mitochondrial-, and plasma membrane-type ATPases, respectively.; EC 3.6.1.1) (2Rea P.A. Poole R.J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1993; 44: 157-180Crossref Scopus (264) Google Scholar). The V-PPase bears no systematic resemblance to soluble PPases at the sequence level (3Cooperman B.S. Baykov A.A. Lahti R. Trends Biochem. Sci. 1992; 17: 262-266Abstract Full Text PDF PubMed Scopus (181) Google Scholar, 4Rea P.A. Kim Y. Sarafian V. Poole R.J. Davies J.M. Sanders D. Trends Biochem. Sci. 1992; 17: 348-353Abstract Full Text PDF PubMed Scopus (124) Google Scholar) and is considered to belong to a fourth class of H+-phosphohydrolase distinct from the F-, P- and V-ATPases (4Rea P.A. Kim Y. Sarafian V. Poole R.J. Davies J.M. Sanders D. Trends Biochem. Sci. 1992; 17: 348-353Abstract Full Text PDF PubMed Scopus (124) Google Scholar). Moreover, unlike the V-ATPase, which is ubiquitous in the membranes bounding the acidic intracellular compartments of all eukaryotic cells, the V-PPase appears to be restricted to plants and a few species of phototrophic bacteria (2Rea P.A. Poole R.J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1993; 44: 157-180Crossref Scopus (264) Google Scholar, 5Baltscheffsky M. Baltscheffsky H. Ernster L. Molecular Mechanisms in Bioenergetics. Elsevier Science Publishers B.V., Amsterdam1993: 331-348Google Scholar). Notwithstanding the intrinsic evolutionary interest of this phenomenon, it poses a problem: the lack of sequence-divergent homologs from phylogenically remote organisms. Because all published V-PPase sequences are from the same group of organisms, vascular plants, and exhibit greater sequence at the amino acid level Kim P.A. Scopus Google to amino acid residues of significance by sequence the of for the expression of functional in the Saccharomyces P.A. Sci. A. PubMed Scopus Google Scholar, P.A. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). of the the of the cDNA V. Kim Y. Poole R.J. P.A. Sci. A. 1992; PubMed Scopus Google Scholar) encoding of the V-PPase at but from amino acid sequence is to this the and are used Moreover, it is that this is the species constituting the it is to the hydrolysis and covalent modification were the to of the V-PPase at but from amino acid sequence is to this the and are used Moreover, it is that this is the species constituting the it is to the hydrolysis and covalent modification were the to of the V-PPase are to enzyme in PPi-dependent H+ translocation is P.A. Sci. A. PubMed Scopus Google Scholar). the is from the plant the of for the of V-PPase and expression of are to of the membrane and of the V-PPase. the of and a for a residue at for inhibition of the V-PPase by and the of all for P.A. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Kim P.A. Biol. Full Text PDF PubMed Google Scholar). of the V-PPase are at the involvement of acidic residues located near or within in H+ translocation. of these acidic The was the to into the and of acidic residues with the for of and within the of the On the basis of of H+ and acidic residues with transmembrane be to in H+ and by the V-PPase. The was the of Y. A. Plant Cell Physiol. 1993; Google that the sequences by positions of the V-PPase a resemblance to the of the c-subunits of The sequence Glu229 in is and similar and to III and the the of the of the at an acidic residue located in the of the of the two transmembrane of this to Y. A. Plant Cell Physiol. 1993; Google Scholar) that the sequence Glu229 of the V-PPase an in of the sensitivity of the V-PPase to inhibition by DCCD M. Biol. Full Text PDF PubMed Google it that Glu229 is the residue whose covalent modification by this is responsible for the inhibition of PPi-dependent H+ translocation. a revised topological model of the V-PPase, unlike V. Kim Y. Poole R.J. P.A. Sci. A. 1992; PubMed Scopus Google Scholar, Y. M. M. Biochem. 1993; is derived from the of to the sequences of the by the from and the of of the and residues to be located near or within transmembrane the basis of this topological The results of are consistent with a for the V-PPase transmembrane the results of that Glu229 is to a role in H+ translocation or inhibition of the V-PPase by DCCD. Instead, the of the mutants, with the of the V-PPase, are by a in which residues and to DCCD which is located at the between a transmembrane and is for coupling PPi hydrolysis with H+ translocation. The cDNA encoding the V-PPase from A. V. Kim Y. Poole R.J. P.A. Sci. A. 1992; PubMed Scopus Google Scholar) was in vacuolar P.A. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). of with the of between the and sequences P.A. Sci. A. PubMed Scopus Google of the and of the cells for the of membranes were P.A. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). and were for the of and the of for was P.A. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). In all the were to singly or with an or the basis of the cDNA sequence of V. Kim Y. Poole R.J. P.A. Sci. A. 1992; PubMed Scopus Google Scholar). The sequences of the eight of or codons in type and positions of in Glu427 four positions and mutants in which the codons were by codons or were The sequences of the four used for this Glu229 Glu427 was from and were by from the using mutant Sci. A. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). In all was by the In a of V-PPase was the sequence of the of the of was after of the from the vacuolar were P.A. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). The for with DCCD the of PPi and membrane was by the of DCCD in and the were at for the the by the of the were for V-PPase were in an after the of of DCCD were activity was the of of from PPi at in and P.A. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). unlike the V-PPase, is to inhibition by A.A. P.A. 1993; PubMed Scopus Google of in the the of the to hydrolysis P.A. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). and H+translocation was using transmembrane in vacuolar and was by the of to in the of H+ translocation or by the of to in the of The in was at and of and P.A. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). The of H+ translocation and were and in P.A. Plant Biochem. Google Scholar). of the of H+ pumping to the of PPi was of PPi was by a modification of the of Biochem. PubMed Scopus Google Scholar). of the V-PPase, membrane were by with P.A. Biol. 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Biochem. 1993; Scholar) were in using both revised topological model of the V-PPase was derived from the sequences of the by four from A. V. Kim Y. Poole R.J. P.A. Sci. A. 1992; PubMed Scopus Google and from Y. Kim P.A. Plant Physiol. PubMed Scopus Google and from H. Y. M. M. Biochem. 1993; Scholar). The model in was the one of the by the TopPred II and of and and J.M. PubMed Scopus Google Scholar) of a for both the and the the from the of H. Plant PubMed Scopus Google Scholar) and the results of and P.A. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Kim P.A. Biol. Full Text PDF PubMed Google Scholar). of the of the of the V-PPase by TopPred II of The was the of using a Mol. Biol. 1992; PubMed Scopus Google Scholar). the and of the and the and for the were or The was of the in of amino acid residues between the two of the membrane and of the of model to the with the in of and residues in with a in membrane PubMed Google Scholar). The was of the Sci. A. Scopus Google the between the and the residues the transmembrane is is with the of residues in the of the membrane The to the is with to the in membrane possessing a Sci. A. Scopus Google Scholar). of the analysis of of the sequence of the V-PPase in of their of located within a topological analysis of the of amino acids in membrane the amino acids to their for with of of topological two classes of and and classes of transmembrane and J.M. PubMed Scopus Google Scholar). The consistent with the from both programs, the of and the was a model a and a model with the encompassing residues two and this In the the two transmembrane in the model and were the of the and and the In the the transmembrane in the model was the from the to the of the membrane the of all of the were consistent with a for P.A. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Kim P.A. Biol. Full Text PDF PubMed Google but the was with the that of with the of a vacuolar of in A. plants H. Plant PubMed Scopus Google Scholar). that the of with the the of the V-PPase, these the to the of the membrane and the and is the between the from TopPred II and TopPred II the of transmembrane at a amino acid residues in this and is the that all are to the the within a of and amino acids residues in this in of one of the of of the by the two by no amino acid residues and the of the by a of for TopPred were the by MEMSAT, and were the amino acids to the but in both the by TopPred II all of these the were to increase the from to the of the V-PPase was and were The transmembrane not identified in the model V. Kim Y. Poole R.J. P.A. Sci. A. 1992; PubMed Scopus Google Scholar) were of the of in the and the of to to of and TopPred by of the transmembrane by the of a amino acid of the transmembrane in the and were the in of their and and were the of but the of for the transmembrane the of these were to and residue of which was to be by positions from the of the membrane to MEMSAT, and for both were retained in the The of the was from the Sci. A. Scopus Google Scholar). of the residues to the transmembrane no residues and two residues a of The of Y. Kim P.A. Plant Physiol. PubMed Scopus Google and Y. M. M. Biochem. 1993; Scholar) the same to in all in and in and the amino acid residues the of the of two were and one was a of and and one and and no in this of at for the residues the for the residues after the for for and for in all four was consistent with a for the The of the model for a greater of and residues the the of the residues located in are of in with the PubMed Google Scholar) and with the that the of be the of the membrane responsible for and binding. to the model a of eight acidic amino acid residues and were identified near or within transmembrane examine their involvement in PPi and DCCD these residues were singly In all acidic residues were with their in acid an V-PPase enzyme or was Three classes of V-PPase mutant were the basis of their hydrolytic and pumping activities after heterologous expression in of PPi hydrolysis and PPi-dependent H+ translocation similar to wild type, of H+ and of both PPi hydrolysis and PPi-dependent H+ translocation I and of of and residues activity of PPi not not were from in which the codons specifying the amino acids and in no in are not not in a were from in which the codons specifying the amino acids and in no in are In all class I mutants one PPi hydrolytic activity and the and of PPi-dependent H+ translocation were diminished at positions and enzyme with at of wild type PPi hydrolytic and PPi-dependent H+ pumping activity and coupling of PPi similar to wild type In the of an in an increase in coupling the of PPi hydrolysis was was to an increase in the of PPi-dependent H+ translocation I and Glu229 was with PPi hydrolytic activity and PPi-dependent H+ translocation were to and wild type I and The one class II mutant retained of wild type hydrolytic activity and pumping activity and exhibited a coupling of of wild type at this wild type PPi hydrolytic activity and enzyme with a greater wild type I and The two acid mutants, E305Q and into class were in PPi-dependent H+ translocation and PPi hydrolysis at of the wild type I and In to was with class I and class II mutants, and at positions and not PPi-dependent H+ translocation I and of the E305Q with an E305D PPi hydrolytic activity from to of wild type, the to of at the activity from to of wild type In none of the mutants was an increase in membrane H+ conductance of to for the of these of the V-PPase mutants similar and of H+ translocation by the with this membranes wild type V-PPase that the background conductance of the membrane to H+ was by of the V-PPase. Moreover, the of a increase in with the decoupled was by the that the of of membranes this of the enzyme was the same of or not PPi was to the the decreased PPi hydrolytic H+ pumping activities of of the mutants were not in of a in the of V-PPase in expression or expression of with decreased from cells wild type or mutated V-PPase similar of of the type or of the and in none of the membrane was an of a in the of the and analysis of wild type and mutated V-PPase after heterologous expression in vacuolar of membrane from cells were to and with V-PPase of the at are the PPi hydrolytic activities of the membrane DCCD the V-PPase with a group located in a transmembrane of acid at these positions in the enzyme be to decreased sensitivity to this a to the for with the for inhibition were the was the one that the susceptibility of the wild type enzyme to inhibition by DCCD In to the for of the V-PPase from inhibition by P.A. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Kim P.A. Biol. Full Text PDF PubMed Google PPi and not the inhibitory of and not greater The for inhibition by DCCD were and greater for membranes in the of and in the of and The of inhibition of wild type V-PPase by DCCD were consistent with a in which the modification of two the enzyme is for and by to a The of inhibition by DCCD was by the that a of the of V-PPase activity at a of is activity at and is the decreased the for by DCCD a of to an of of all eight acid mutants for inhibition by DCCD that two (E305Q and D504N) were to DCCD and acid at positions and or no DCCD inhibitability wild type, the at positions and diminished the inhibitability of the residual activity by and In none of the mutants, with the of which an was In with a for acidic residues at positions and for inhibition by of the E305Q and D504N with E305D and D504E wild type DCCD inhibitability and of wild type and mutated V-PPase to inhibition by were with the of DCCD in with and for and activity was The are activities a of the activity of membrane with DCCD. wild type. in a were with the of DCCD in with and for and activity was The are activities a of the activity of membrane with DCCD. wild type. of the to the of the V-PPase, TopPred II and MEMSAT, a model two transmembrane in to the V. Kim Y. Poole R.J. P.A. Sci. A. 1992; PubMed Scopus Google Scholar) and an for the and of the of the model the results of and it a over The from both were similar for all four of the sequences with in the and of a few of the transmembrane The results of both were with the the of P.A. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Kim P.A. Biol. Full Text PDF PubMed Google Scholar) and the of H. Plant PubMed Scopus Google Scholar). the model of the V-PPase by Y. M. M. Biochem. 1993; the basis of of the sequence of the the of the membrane and a transmembrane in their amino acid was by of the revised topological model of the V-PPase that the eight acidic residues located within or near transmembrane were identified for acidic residues with a similar were from the model these were not in all four of the sequences or located in and considered to in transmembrane H+ translocation DCCD binding. Three discussed from the results of these acidic E229Q mutated V-PPase of PPi hydrolysis and H+translocation both are diminished in the diminution of coupling is Moreover, in to the role of Glu229 in inhibition by V-PPase is no to inhibition by DCCD wild type or mutated that an acidic residue at this is not for inhibition by this On this and in to the in Y. A. Plant Cell Physiol. 1993; Google Scholar, it is that the between the V-PPase sequences to Glu229 and the transmembrane of the of F-ATPases a functional and residues and not to be for PPi-dependent or inhibition by DCCD. of these residues by their or no PPi hydrolytic H+ coupling or DCCD E305Q and mutants exhibit wild type PPi hydrolytic no PPi-dependent and residual activities to inhibition by DCCD that of wild type with the of DCCD inhibitability by E305D and D504E mutants, are consistent with the involvement of acidic residues at these positions in inhibition by DCCD. is that and are the residues in of the V-PPase by this mutated enzyme acid at both of these positions is no mutant to the of residues and The that or a in hydrolytic activity in the of and a increase in activity in the of that the for are for DCCD binding. a of one group in the is to DCCD inhibitability appears to the V-PPase from inhibition by DCCD by with a In with the results from earlier the of DCCD inhibition are consistent with an for of The of hydrolysis by the V-PPase a in which the of two in to with the pyrophosphate is for a with a of and a with a of Sanders D. Plant Physiol. 1992; PubMed Scopus Google Scholar, A.A. P.A. Biochem. 1993; PubMed Scopus Google Scholar). of the V-PPase from inhibition by the an consistent with for of R. Plant Physiol. PubMed Scopus Google Scholar). their it is that these results a between and the residues or that covalent modification in all the notion of is consistent with the that of the V-PPase not to the DCCD and EDAC, but NEM, a and a that preferentially with A.A. P.A. Biochem. 1993; PubMed Scopus Google Scholar, R. Plant Physiol. PubMed Scopus Google Scholar) and the that one that between transmembrane and the residue responsible for inhibition of the V-PPase by P.A. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Kim P.A. Biol. Full Text PDF PubMed Google and it not two to inhibition by DCCD is EDAC, NEM, and the enzyme with R. Plant Physiol. PubMed Scopus Google Scholar) and A. it for the diminution of DCCD inhibitability and of which are located in this with their that coupling of the residues that be to DCCD not with the of inhibition of the V-PPase by DCCD is that in DCCD or that or but not the by the or the of these acid at one or both of these positions the of and the sensitivity of the V-PPase to inhibition by DCCD in the of at the same E427Q mutants, in PPi H+ translocation at of the wild type to an diminished coupling it be to that these results a role for Glu427 in H+ by a that enzyme the of wild type H+ the increase in PPi hydrolytic activity by a an role for an acidic residue at this for H+translocation the for PPi hydrolysis is by the E427Q is with an E427D the of H+ translocation is by Glu427 in H+ two or V-PPase is to DCCD wild type inhibition by DCCD not a with the results of of and the DCCD inhibitability of the V-PPase in of the of a residue in transmembrane M. Biol. Full Text PDF PubMed Google Scholar). the results and the of a is not that of and modification of a is the of or from the of 1993; PubMed Scopus Google inhibition by DCCD that activity is or from it not that the residue in the same lack of of an acidic residue with is the for not it from involvement in to the revised topological model of the V-PPase, Glu427 a this residue is in H+ it that it of an responsible for the of H+ at the of the is that the E427Q to the group that be at this of the residues of the that the residues in the transmembrane into their at a of a increase in H+ activity to a in H+ from this in E427Q mutants, the and the coupling decoupled mutants for D. 1993; PubMed Scopus Google A. M. 1993; PubMed Scopus Google Scholar) and A. Sci. A. PubMed Scopus Google Scholar). In four residues are for H+ translocation Sci. A. PubMed Scopus Google Scholar) and of one of these to H+ translocation the D. 1993; PubMed Scopus Google Scholar). In I of of an residue in a H+translocation from A. M. 1993; PubMed Scopus Google Scholar). In R. of an residue at a to that of a similar A. Sci. A. PubMed Scopus Google Scholar). The results an of the of one for the by the V-PPase and of the of of DCCD in this but to be of their insertion into the at a in the H+ of Glu427 that mutated at these unlike mutated at is in both H+ translocation and PPi is not acidic residues in H+ translocation. for the of and for this