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• W1990337490 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 than 85% sequence identity at the amino acid level (6Zhen R.-G. Kim E.J. Rea P.A. Adv. Bot. Res. 1997; 25: 297-337Crossref Scopus (65) Google Scholar), most attempts to identify conserved amino acid residues of potential mechanistic significance by sequence alignment procedures have been unproductive. Crucial, therefore, has been the development of methods for the expression of functional pump in the yeast, Saccharomyces cerevisiae (7Kim E.J. Zhen R.-G. Rea P.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6128-6132Crossref PubMed Scopus (99) Google Scholar, 8Kim E.J. Zhen R.-G. Rea P.A. J. Biol. Chem. 1995; 270: 2630-2635Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). When constructs of the yeast-Escherichia coli shuttle vector pYES2, containing the entire open reading frame of the cDNA (AVP; Ref. 9Sarafian V. Kim Y. Poole R.J. Rea P.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1775-1779Crossref PubMed Scopus (170) Google Scholar) encoding theMr 66,000 substrate-binding subunit 2The substrate-binding subunit of the V-PPase migrates at Mr 66,000 (64,500–72,000) on SDS gels, but its probable mass deduced from its amino acid sequence is 79–81 kDa. Thus, when referring to this polypeptide the terms “Mr 66,000 subunit” and “81-kDa subunit” are used interchangeably. Moreover, although it is now known that this subunit is probably the sole polypeptide species constituting the enzyme, it is referred to as the “substrate-binding subunit” because substrate hydrolysis and substrate-protectable covalent modification were the first functions assigned to it.2The substrate-binding subunit of the V-PPase migrates at Mr 66,000 (64,500–72,000) on SDS gels, but its probable mass deduced from its amino acid sequence is 79–81 kDa. Thus, when referring to this polypeptide the terms “Mr 66,000 subunit” and “81-kDa subunit” are used interchangeably. Moreover, although it is now known that this subunit is probably the sole polypeptide species constituting the enzyme, it is referred to as the “substrate-binding subunit” because substrate hydrolysis and substrate-protectable covalent modification were the first functions assigned to it. of the V-PPase fromArabidopsis thaliana are employed to transform S. cerevisiae, endomembrane-associated enzyme active in PPi-dependent H+ translocation is generated (7Kim E.J. Zhen R.-G. Rea P.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6128-6132Crossref PubMed Scopus (99) Google Scholar). Since the heterologously expressed pump is indistinguishable from the native plant enzyme, thereby establishing the sufficiency of AVP for the elaboration of active V-PPase inS. cerevisiae, approaches based on site-directed mutagenesis, epitope tagging, and expression of fusion proteins are now applicable to investigations of the membrane organization and catalytic mechanism of the V-PPase. By the parallel application of mutational and protein chemical methods, we have demonstrated a specific requirement for a cytosolically oriented Cys residue at position 634 for inhibition of the V-PPase by maleimides and the dispensability of all conserved Cys residues, including Cys634, for catalysis (8Kim E.J. Zhen R.-G. Rea P.A. J. Biol. Chem. 1995; 270: 2630-2635Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 10Zhen R.-G. Kim E.J. Rea P.A. J. Biol. Chem. 1994; 269: 23342-23350Abstract Full Text PDF PubMed Google Scholar). Our current studies of the V-PPase are directed at elucidating the involvement of acidic (Asp, Glu) residues located near or within hydrophobic spans in substrate turnover and/or H+ translocation. Two factors prompted investigation of these acidic residues. The first was the need to gain insight into the identity and location of acidic residues with the potential for undergoing cycles of protonation and deprotonation within the hydrophobic core of the membrane. On the basis of analyses of other H+ pumps and H+-coupled transporters, acidic residues associated with transmembrane spans might be expected to directly participate in H+ uptake, translocation, and release by the V-PPase. The second factor was the observations of Nyren et al. (1Nyren P. Sakainore Y. Strid A. Plant Cell Physiol. 1993; 34: 375-378PubMed Google Scholar), who noted that the sequences encompassed by positions 227–245 of the V-PPase fromArabidopsis bear a resemblance to the C-terminal regions of the c-subunits of F-ATPases. The C-terminal sequence flanking Glu229 in AVP is 71, 65, and 67% similar (35, 47, and 39% identical) to Rhodospirillum rubrum c-subunit (positions 58–74), Pisum sativum chloroplast subunit III (positions 61–77), and P. sativum mitochondrial subunit 9 (positions 55–72) (Fig. 1). Since the c-peptide, the most highly conserved subunit of the H+-conductive Fo sector of F-ATPases, binds the hydrophobic carboxyl reagent, N,N′-dicyclohexylcarbodiimide (DCCD) at an acidic residue located in the middle of the second of the two transmembrane α helices of this polypeptide, to abolish H+translocation, Nyren et al. (1Nyren P. Sakainore Y. Strid A. Plant Cell Physiol. 1993; 34: 375-378PubMed Google Scholar) have proposed that the sequence flanking Glu229 of the V-PPase may assume an analogous function. Specifically, in view of the sensitivity of the V-PPase to inhibition by DCCD (11Maeshima M. Yoshida S. J. Biol. Chem. 1989; 264: 20068-20073Abstract Full Text PDF PubMed Google Scholar), it has been suggested that Glu229 is the residue whose covalent modification by this carbodiimide is responsible for the inhibition of PPi-dependent H+ translocation. Here we present (i) a revised topological model of the V-PPase, which, unlike those reported previously (9Sarafian V. Kim Y. Poole R.J. Rea P.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1775-1779Crossref PubMed Scopus (170) Google Scholar, 12Tanaka Y. Chiba K. Maeda M. Maeshima M. Biochem. Biophys. Res. Commun. 1993; 190: 962-967Google Scholar), is derived from the concerted application of multiple computer-based structural criteria to the deduced sequences of the polypeptides specified by the cDNAs from several independent sources; and (ii) the single substitution of most of the conserved Asp and Glu residues inferred to be located near or within transmembrane spans on the basis of this topological model. The results of modeling are consistent with a structure for the V-PPase incorporating 15 transmembrane spans, while the results of mutagenesis demonstrate that Glu229 is unlikely to play a role in H+ translocation or inhibition of the V-PPase by DCCD. Instead, the characteristics of the mutants, combined with the inferred topology of the V-PPase, are better accommodated by a scheme in which membrane-embedded residues Glu305 and Asp504contribute to DCCD binding, whereas Glu427, which is located at the interface between a transmembrane span and its adjoining cytosolic loop, is required for coupling PPi hydrolysis with H+ translocation. The cDNA encoding the V-PPase from A. thaliana(AVP; Ref. 9Sarafian V. Kim Y. Poole R.J. Rea P.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1775-1779Crossref PubMed Scopus (170) Google Scholar) was heterologously expressed in vacuolar protease-deficient S. cerevisiae haploid strain BJ5459 (MATa, ura3–52, trp1, lys2–801, leu2Δ1, his3-Δ200, pep4::HIS3, prbΔ1.6R, can1, GAL) (8Kim E.J. Zhen R.-G. Rea P.A. J. Biol. Chem. 1995; 270: 2630-2635Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 13Jones E.W. Methods Enzymol. 1991; 194: 428-453Crossref PubMed Scopus (363) Google Scholar). Transformation of BJ5459 with yeast-E. coli shuttle vector pYES2, containing the entire open reading frame of AVP inserted between the GAL1 promoter and CYC1 termination sequences (pYES2-AVP; Ref. 7Kim E.J. Zhen R.-G. Rea P.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6128-6132Crossref PubMed Scopus (99) Google Scholar), isolation of the Ura+ transformants and growth of the cells for the preparation of membranes were performed as described (8Kim E.J. Zhen R.-G. Rea P.A. J. Biol. Chem. 1995; 270: 2630-2635Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). E. coli DH5α and CJ236 (dut − ung −) were employed for the amplification of pYES2-AVP and the generation of single-stranded, uracilated template for site-directed mutagenesis, respectively. Mutagenesis was performed directly on pYES2-AVP vector (8Kim E.J. Zhen R.-G. Rea P.A. J. Biol. Chem. 1995; 270: 2630-2635Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). In all cases the mutagenic oligonucleotides were designed to singly substitute each conserved Asp or Glu codon with an Asn or Gln codon on the basis of the cDNA sequence of AVP (9Sarafian V. Kim Y. Poole R.J. Rea P.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1775-1779Crossref PubMed Scopus (170) Google Scholar). The sequences of the eight oligonucleotides (positions of conserved Asp or Glu codons shown in bold type and positions of degeneracy shown in brackets) were: Glu119 → Gln, CGGCTCTGTT[C]AGGGATTCAGCAC; Glu229→ Gln, TCTTTTT[C]AGGCTATTACTGG; Glu305 → Gln, GGATCATATGCT[C]AAGCATCATGCGC; Glu427 → Gln, GTTTCGTCA-CT[C]AGTACTACACTAG; Asp504 → Asn, GGCAATT[A]ATGCTTATGGTCCC; Asp573→ Asn, CCACACCGTA[A]ATGTTTTGACC; Glu667 → Gln, CTTTGGAGTT[C]AGACCCTC-TCTGG; Glu751→ Gln, CATGGCTGTT[C]AGTCTCTTGTC. At four positions (Glu229, Glu305, Glu427, and Asp504), mutants in which the Asp codons were replaced by Glu codons or vice versa were also generated. The sequences of the four oligonucleotides used for this purpose were: Glu229 → Asp, TCTTTTTGA[C]GCTATTACTGG; Glu305 → Asp, GGATCATATGC-TGA[T/C]GCATCATGCGC; Glu427 → Asp, GTTTCGTCACTGA[C]TACTACACTAG; Asp504→ Glu, GGCAATTGA[G]GCTTATGGTCCC. Uracilated single-stranded template DNA was isolated from pYES2-AVP-transformed E. coli CJ236, and site-directed mutations were introduced by second strand synthesis from the template using mutant oligonucleotides (14Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4880) Google Scholar, 15Kunkel T.A. Roberts J.D. Zabour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4540) Google Scholar). In all cases, mutagenesis was confirmed by sequencing the target region before yeast transformation. In selected cases, when a pronounced alteration of V-PPase function was observed, the sequence of the target region of the AVPinsert of pYES2-AVP was determined after extraction of the vector from the yeast transformants. Yeast vacuolar membrane-enriched vesicles were prepared as described (8Kim E.J. Zhen R.-G. Rea P.A. J. Biol. Chem. 1995; 270: 2630-2635Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The standard mixture for reaction with DCCD contained 30 mmTris-Mes buffer (pH 8.0), the indicated concentrations of ligands (Mg2+ as MgSO4, K+ as KCl, PPi as Tris-PPi) and membrane protein (9.7–10.7 μg/ml). Reaction was initiated by the addition of DCCD (0–500 μm dissolved in ethanol), and the samples were incubated at 37 °C for the times indicated. After terminating the reaction by the addition of Mg2+ (1.3 mm), the samples were cooled on ice before assaying aliquots for V-PPase activity. Control samples were treated in an identical manner after the addition of equal volumes of ethanol. All stock DCCD solutions were prepared fresh daily. PPihydrolytic activity was measured as the rate of liberation of Pi from PPi at 37 °C in reaction media containing 0.3 mm Tris-PPi, 1.3 mmMgSO4, 100 mm KCl, 1 mm NaF, 5 μm gramicidin-D, 1 mm Tris-EGTA, and 30 mm Tris-Mes (pH 8.0) (8Kim E.J. Zhen R.-G. Rea P.A. J. Biol. Chem. 1995; 270: 2630-2635Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Since yeast-soluble PPase, unlike the V-PPase, is exquisitely sensitive to inhibition by fluoride ( Kiapp (soluble PPase) = 20 μm; Kiapp (V-PPase) = 3.4 mm) (16Baykov A.A. Dubnova E.B. Bakuleva N.P. Evtushenko O.A. Zhen R.-G. Rea P.A. FEBS Lett. 1993; 327: 199-202Crossref PubMed Scopus (41) Google Scholar), inclusion of 1 mm NaF in the assay media effectively abolishes the contribution of the former to total hydrolysis (8Kim E.J. Zhen R.-G. Rea P.A. J. Biol. Chem. 1995; 270: 2630-2635Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). PPi- and ATP-dependent H+translocation was assayed fluorimetrically using acridine orange (2.5 μm) as transmembrane pH difference indicator in assay media containing vacuolar membrane-enriched vesicles (200 μg), 100 mm KCl, 0.4 m glycerol, 1 mmTris-EGTA, and 5 mm Tris-HCl (pH 8.0). Reaction was initiated by the addition of Tris-PPi (1.0 mm) to media containing MgSO4 (1.3 mm) in the case of V-PPase-mediated H+ translocation or by the addition of MgSO4 (3 mm) to media containing Tris-ATP (3 mm) in the case of V-ATPase-mediated H+translocation. The decrease in fluorescence was measured at excitation and emission wavelengths of 495 and 540 nm, respectively (8Kim E.J. Zhen R.-G. Rea P.A. J. Biol. Chem. 1995; 270: 2630-2635Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The initial rate of H+ translocation and steady state pH gradient were estimated as ΔF%/mg/min (at time zero) and ΔF%/mg (after 5–10 min), where ΔF% = percentage decrease in fluorescence as described (17Rea P.A. Turner J.C. Methods Plant Biochem. 1990; 3: 385-405Crossref Google Scholar). Coupling ratio (the ratio of the rate of H+ pumping to the rate of PPi hydrolysis) was estimated as (ΔF%/min)/(μmol of PPi hydrolyzed/min). Protein was estimated by a modification of the method of Peterson (18Peterson G.L. Anal. Biochem. 1977; 83: 346-356Crossref PubMed Scopus (7066) Google Scholar). For Western analyses of the heterologously expressed V-PPase, membrane samples were delipidated by extraction with acetone:ethanol (1:1; −20 °C) (19Parry R.V. Turner J.C. Rea P.A. J. Biol. Chem. 1989; 264: 20025-20032Abstract Full Text PDF PubMed Google Scholar), dissolved in denaturation buffer, and subjected to one-dimensional SDS-polyacrylamide gel electrophoresis on 11% (w/v) slab gels in a Bio-Rad minigel apparatus (7Kim E.J. Zhen R.-G. Rea P.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6128-6132Crossref PubMed Scopus (99) Google Scholar). The electrophoresed samples were electrotransferred to 0.45-μm nitrocellulose filters in standard Towbin buffer (20Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44643) Google Scholar), containing 10% (v/v) methanol for 30 min at a current density of 2.5 mA/cm2 in a Millipore semi-dry blotting apparatus. After reversible staining of the transferred protein bands with Ponceau-S, the filters were processed for reaction with antibody (PABHK1) raised against synthetic peptide with the sequence HKAAVIGDTIGDPLK, corresponding to positions 720–734 of AVP (9Sarafian V. Kim Y. Poole R.J. Rea P.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1775-1779Crossref PubMed Scopus (170) Google Scholar). Immunoreactive bands were visualized by successive incubations of the membrane filters with horseradish peroxidase-conjugated sheep anti-rabbit immunoglobulin G and buffer containing 0.03% (w/v) H2O2, 0.5 mg/ml diaminobenzidine, and 0.03% (w/v) NiCl2 (21Harlowe E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1988Google Scholar). Data were fitted by nonlinear least squares analysis (22Marquardt D.W. J. Soc. Ind. Appl. Math. 1963; 11: 431-441Crossref Google Scholar) using the Ultrafit nonlinear curve-fitting package from BioSoft (Ferguson, MO). Two programs were employed to model the overall topology of the V-PPase: TopPred II and MEMSAT (membrane structureand topology). The TopPred II program, developed by Manuel G. Claros and Gunnar von Heijne (Karolinska Institute, Stockholm, Sweden) for Macintosh computers is a public domain software package for predicting the topology of both prokaryotic and eukaryotic membrane proteins by the concerted application of hydropathy analyses, the “positive-inside” rule and “charge-difference” rule (23Sipos L. von Heijne G. Eur. J. Biochem. 1993; 213: 1333-1340Crossref PubMed Scopus (252) Google Scholar). The MEMSAT program, developed by Jones et al. (24Jones D.T. Taylor W.R. Thornton J.M. Biochemistry. 1994; 33: 3038-3049Crossref PubMed Scopus (700) Google Scholar) for IBM PCs, is based on expectation maximization. From the distributions of amino acids compiled from membrane proteins, or portions thereof, of defined topology, the log-likelihood ratios (si) for domain classes are calculated for each of the 20 amino acids according to the expressionsi = ln (qi/pi) wherepi is the relative frequency of occurrence of amino acid i in all the sequences in the data set andqi is the relative frequency of occurrence of the same amino acid in a particular domain. These sivalues or propensities are then used to equate a given sequence with a given topology. The deduced amino acid sequences of the V-PPases encoded by the cDNAs isolated from A. thaliana (AVP, GenBank™ accession no. M81892) (9Sarafian V. Kim Y. Poole R.J. Rea P.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1775-1779Crossref PubMed Scopus (170) Google Scholar), Beta vulgaris(BVP1, L32792; BVP2, L32791) (25Kim Y. Kim E.J. Rea P.A. Plant Physiol. 1994; 106: 375-382Crossref PubMed Scopus (63) Google Scholar), andHordeum vulgare (HVP, D13472) (12Tanaka Y. Chiba K. Maeda M. Maeshima M. Biochem. Biophys. Res. Commun. 1993; 190: 962-967Google Scholar) were processed in parallel using both programs. A revised topological model of the V-PPase was derived from the deduced sequences of the polypeptides encoded by four cDNAs: AVP from A. thaliana(9Sarafian V. Kim Y. Poole R.J. Rea P.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1775-1779Crossref PubMed Scopus (170) Google Scholar), BVP1 and BVP2 from B. vulgaris(25Kim Y. Kim E.J. Rea P.A. Plant Physiol. 1994; 106: 375-382Crossref PubMed Scopus (63) Google Scholar), and HVP from H. vulgare (12Tanaka Y. Chiba K. Maeda M. Maeshima M. Biochem. Biophys. Res. Commun. 1993; 190: 962-967Google Scholar). The model shown in Fig. 2 was the only one of the three predicted by the TopPred II and MEMSAT programs of Claros and von Heijne and Jones et al. (24Jones D.T. Taylor W.R. Thornton J.M. Biochemistry. 1994; 33: 3038-3049Crossref PubMed Scopus (700) Google Scholar) capable of accommodating a cytosolic orientation for both the C terminus and the hydrophilic loop containing the N-ethylmaleimide (NEM)-reactive cysteine, Cys634, inferred from the characteristics of apoaequorin fusions (26Knight H. Trewavas A.J. Knight M.R. Plant Cell. 1996; 8: 489-503Crossref PubMed Scopus (708) Google Scholar) and the results of peptide mapping and Cys mutagenesis, respectively (8Kim E.J. Zhen R.-G. Rea P.A. J. Biol. Chem. 1995; 270: 2630-2635Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 10Zhen R.-G. Kim E.J. Rea P.A. J. Biol. Chem. 1994; 269: 23342-23350Abstract Full Text PDF PubMed Google Scholar). Examination of the structure of the Mr 66,000 subunit of the V-PPase by TopPred II consisted of three main stages. (i) The first stage was the construction of hydrophobicity profiles using a trapezoid sliding window (27von Heijne G. J. Mol. Biol. 1992; 225: 487-494Crossref PubMed Scopus (1395) Google Scholar). Depending on the height and width of the hydrophobicity maxima and the preset “upper cutoff” and “lower cutoff” values for the computed hydrophobicity indices, spans were categorized as either “certain” or “putative.” (ii) The second stage was enumeration of the difference in representation of positively charged amino acid residues between the two sides of the membrane and tests of the adherence of any given model to the positive-inside rule, with the bias in favor of Arg and Lys residues in hydrophilic loops with a cytosolic disposition in most polytopic membrane proteins (28von Heijne G. EMBO J. 1986; 5: 3021-3027Crossref PubMed Google Scholar). (iii) The third stage was application of the charge-difference rule (29Hartmann E. Rapoport T.A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 89: 5786-5790Crossref Scopus (485) Google Scholar), wherein the net charge difference between the 15 N-terminal and the 15 C-terminal residues flanking the most N-terminal transmembrane span is computed. Transmembrane orientation is correlated with the disposition of charged residues in the immediate vicinity of the first membrane span. The segment C-terminal to the first span is generally positively charged with respect to the N-terminal flanking regions in membrane proteins possessing a luminally oriented N terminus (29Hartmann E. Rapoport T.A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 89: 5786-5790Crossref Scopus (485) Google Scholar). Deployment of the MEMSAT program entailed analysis of segments of the sequence of the V-PPase in terms of their likelihood of being located within a particular topological element. Based on statistical analysis of the distribution of amino acids in membrane proteins, the MEMSAT program ranks amino acids according to their propensities for being associated with each of five types of topological element: two classes of hydrophilic loop, designated cytoplasmic (inside) loop (Li) and luminal (outside) loop (Lo), and three classes of transmembrane helix domain, designated helix inside (Hi), helix middle (Hm), and helix outside (Ho) (24Jones D.T. Taylor W.R. Thornton J.M. Biochemistry. 1994; 33: 3038-3049Crossref PubMed Scopus (700) Google Scholar). The consensus structure consistent with the predictions from both programs, the disposition of Cys634, and the C-terminal apoaequorin fusion data was a 15-span model containing a luminally localized N terminus and cytoplasmically localized C terminus (Fig. 2). While MEMSAT ranked a 16-span model highest, with the additional span encompassing residues 743–761, two models containing 14 and 15 spans ranked just below this model. In the 14-span model, the two lowest scoring transmembrane spans in the 16-span model (V and VI) were excluded, thus preserving the orientation of the N and C termini and the remaining C-terminal spans. In the 15-span model, the last transmembrane span in the 16-span model (XVI) was excluded, thus transferring the C terminus from the luminal to the cytosolic face of the membrane while preserving the orientation of a" @default.
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• W1990337490 title "Acidic Residues Necessary for Pyrophosphate-energized Pumping and Inhibition of the Vacuolar H+-pyrophosphatase byN,N′-Dicyclohexylcarbodiimide" @default.
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