©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Site-directed Mutagenesis of Vacuolar H-pyrophosphatase
NECESSITY OF Cys FOR INHIBITION BY MALEIMIDES BUT NOT CATALYSIS (*)

(Received for publication, October 24, 1994)

Eugene J. Kim (§) Rui-Guang Zhen (§) Philip A. Rea (¶)

From the Department of Biology, Plant Science Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A characteristic feature of the vacuolar H-translocating inorganic pyrophosphatase (V-PPase) of plant cells is its high sensitivity to irreversible inhibition by N-ethylmaleimide (NEM) and other sulfhydryl reagents. Previous investigations in this laboratory have demonstrated that the primary site for substrate-protectable covalent modification of the V-PPase by ^14C-labeled NEM maps to a single M(r) 14,000 V8 protease fragment (V8) (Zhen, R.-G., Kim, E. J., and Rea, P. A.(1994) J. Biol. Chem. 269, 23342-23350). Here, we describe site-directed mutagenesis of the cDNA encoding the V-PPase from Arabidopsis thaliana, its heterologous expression in Saccharomyces cerevisiae and single substitution of all 9 conserved Cys residues to either Ser or Ala. In all cases, except one, Cys mutagenesis exerts little or no effect on either the catalytic activity or susceptibility of the enzyme to inhibition by NEM. By contrast, and in complete agreement with the results of peptide mapping experiments, substitution of Cys, the sole conserved cysteine residue encompassed by V8, with Ser or Ala generates enzyme that is insensitive to NEM but active in both PP(i) hydrolysis and PP(i)-dependent H translocation. The specific requirement for Cys for inhibition by NEM and the dispensability of all of the conserved Cys residues, including Cys, for V-PPase function indicate that the inhibitory action of maleimides reflects steric constraints imposed by the addition of a substituted alkyl group to the side chain of Cys rather than direct participation of this amino acid residue in catalysis.


INTRODUCTION

Two primary electrogenic H pumps reside on the vacuolar membrane of plant cells: a V-type H-ATPase (V-ATPase) (^1)(Sze et al., 1992) and a H-translocating inorganic pyrophosphatase (V-PPase) (Rea and Poole, 1993). The V-ATPase is common to the membranes bounding the acidic intracellular compartments of all eukaryotic cells, but the V-PPase appears to be restricted to the vacuolysosomal membranes of plants and possibly the chromatophores of a few species of photosynthetic bacteria (Rea and Poole, 1993). Elucidation of the role played by pyrophosphate (PP(i)) as an alternate energy source for a number of critical metabolic reactions, specifically sucrose mobilization via sucrose synthase (Huber and Azakawa 1986; Black et al., 1987) and glycolysis via PP(i):fructose-6-phosphate 1-phosphotransferase (Black et al., 1987; Dennis and Greyson, 1987), has given rise to the notion that, rather than simply dissipatively hydrolyzing PP(i), plants have retained or developed the capacity to scavenge a significant fraction of the energy contained in this phosphoanhydride. By extension, recognition of the capacity of the V-PPase for vacuolar energization (Rea and Sanders, 1987) and the results of enzyme localization studies indicating that the V-PPase may be the sole enzyme responsible for the disposal of cytosolic PP(i) (Quick et al., 1989) have implicated this enzyme not only as a scavenger of the free energy of PP(i) but also as an important element in the regulation of steady state cytosolic PP(i) concentrations (Rea and Poole, 1993).

To understand the relationship between its cellular function and molecular organization, it is necessary to define the structure-function relations of the V-PPase. To date, however, two inherent characteristics of the enzyme have impeded such analyses: its novelty and pronounced sequence conservation. There are no known homologs of the V-PPase; it bears no systematic resemblance to soluble PPases at the polypeptide level (Cooperman et al., 1992; Rea et al., 1992b) and is considered to belong to a fourth category of primary ion translocase, distinct from the previously defined F-, P- and V-ATPases (Pedersen and Carafoli, 1987; Rea et al., 1992b). Those V-PPase sequences that are known show unusually high levels of sequence similarity; the four published deduced amino acid sequences for the major M(r) 66,000 substrate-binding subunit (^2)of the V-PPase are more than 85% identical (Kim et al., 1994a). The identification of amino acid residues that might be critical for catalysis by the application of sequence alignment procedures has consequently been largely unproductive. As a result, there has had to be a near exclusive reliance on protein chemical methods for the initial delineation of catalytically important amino acid residues. One such approach has been the use of maleimides to modify and localize residues that might be involved in V-PPase function.

A well characterized property of the V-PPase is its sensitivity to inhibition by the sulfhydryl reagent, N-ethylmaleimide (NEM). NEM irreversibly inhibits the enzyme in a substrate (``Mg + PP(i)'') protectable manner with single site kinetics. On the basis of this finding and the fact that NEM and the membrane-impermeant cysteine reagent, 3-(N-maleimidylpropionyl) biocytin, inhibit the V-PPase with similar kinetics and compete for a common binding site on the M(r) 66,000 substrate-binding subunit, a single residue located in a cytosolically disposed extramembranous domain is inferred to undergo covalent modification in both cases (Zhen et al., 1994). Peptide mapping of this residue through selective labeling of the V-PPase with ^14C-labeled NEM, purification of the M(r) 66,000 subunit and its digestion with V8 protease generates a single ^14C-labeled band (V8) migrating at M(r) 14,000 on Tris-tricine gels that aligns with the carboxyl-terminal segment of the substrate-binding subunit. Because V8 encompasses only 1 cysteine residue at position 634 that is conserved between the V-PPases from Arabidopsis thaliana (Sarafian et al., 1992), Beta vulgaris (Kim et al., 1994a), and Hordeum vulgare (Tanaka et al., 1993), this residue, located in putative hydrophilic loop X, is concluded to contain the cytosolically oriented sulfhydryl group whose alkylation by maleimides is responsible for inactivation of the enzyme (Zhen et al., 1994).

Although instructive, the results of these protein chemical investigations are subject to two limitations. (i) They provide insight into the topology of the V-PPase but are incapable of resolving the direct participation of Cys in catalysis and/or substrate binding. The fact that reaction of an enzyme with a group-specific reagent causes irreversible inhibition does not necessarily imply that the functional group is in the active site. Covalent modification of a nonessential residue could, for instance, result in a conformational change that inactivates the enzyme. By the same token, protection against inhibition, by substrate, does not mean that the susceptible group is in the active site. It is equally likely that conformational changes accompanying substrate binding result in the occlusion of reactive residues that are otherwise remote from the active site. (ii) Technical constraints have prohibited direct identification of the modified residue (Zhen et al., 1994). There is a possibility, albeit small, that the ^14C-labeled residue is not a cysteine residue or that nonsequenceable amino-terminally blocked peptides, for example, other than V8 are responsible for the signal seen on fluorograms. Thus, whenever practicable, it is important to test the necessity of specific amino acid residues for catalysis and/or substrate binding by approaches other than through the use of group-specific reagents so as to circumvent some of the interpretative difficulties associated with these compounds.

Recent experiments have demonstrated that when constructs of the yeast-Escherichia coli shuttle vector pYES2 containing the entire open reading frame of the cDNA (AVP) encoding the substrate-binding subunit of the V-PPase from Arabidopsis are employed to transform Saccharomyces cerevisiae, vacuolarly localized functional enzyme active in PP(i)dependent H translocation is generated (Kim et al., 1994b). 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 in Saccharomyces, 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 this pump. In the specific context of the significance of Cys, site-directed mutagenesis has the potential of not only providing independent criteria of identity for the Cys residue, or residues, required for inactivation of the enzyme by maleimides but also a means of determining whether one or more of these residues is essential for catalysis.

In this communication, we report the single substitution of all of the conserved Cys residues of the V-PPase from Arabidopsis. Analyses of the patterns of inhibition of heterologously expressed wild type and mutant enzyme by NEM demonstrate the necessity of Cys for inhibition of the V-PPase by maleimides but dispensability of this, and all other conserved Cys residues, for catalytic activity.


MATERIALS AND METHODS

Microorganisms

S. cerevisiae, haploid strain BJ5459 (MATa, ura3-52, trp1, lys2-801, leu2Delta1, his3-Delta200, pep4::HIS3, prb1Delta1.6R, can1, GAL) (Jones, 1991) was used for heterologous expression of the V-PPase. This strain, which is vacuolar protease-deficient, yielded higher and more consistent V-PPase activities after heterologous expression of AVP than the strain (AACY1) used before (Kim et al., 1994b). Transformation with the 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; Kim et al., 1994b), was by the LiOAc/PEG method (Schiestl and Gietz, 1989). The resulting Ura transformant colonies were picked and grown in AHC medium (0.17% (w/v) yeast nitrogen base without amino acids, 0.5% (w/v) ammonium sulfate, 1% (w/v) acid-hydrolyzed casein, 0.002% (w/v) adenine hemisulfate, 50 mM potassium phosphate, pH 5.5) containing 2% (w/v) galactose for subsequent membrane isolation. E. coli DH5alpha 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.

Site-directed Mutagenesis

All mutagenesis was performed directly on pYES2-AVP vector. E. coli CJ236 (dut ung) cells were transformed with pYES2-AVP, grown in liquid medium containing helper phage, VCS-M13 (Stratagene), and single-stranded uracilated template DNA was isolated after the addition of kanamycin. Site-specific mutations were generated using a Muta-Gene M13 in vitro mutagenesis kit (Bio-Rad) by second strand synthesis from the uracilated template (Kunkel, 1985; Kunkel et al., 1987) using mutant oligonucleotide primers. The mutagenic oligonucleotides were designed to singly substitute each conserved Cys codon with a Ser or Ala codon on the basis of the cDNA sequence of AVP (Sarafian et al., 1992). The sequences of the nine oligonucleotides (positions of conserved Cys codons shown in bold typeface and positions of degeneracy shown in brackets) were as follows: Cys Ser/Ala, CCGATT[T/G]CTGCGGTGATT; Cys Ser/Ala, GCTAAG[T/G]CCGCTGAGATT; Cys Ser/Ala, CAAGCCT[T/G]CTACTTACGAC; Cys Ser/Ala, CCAGAACC[T/G]CCAAGCCTG; Cys Ser/Ala, GCATCA[T/G]CCGCTGCTCTTG; Cys Ser/Ala, TCTTGGTT[T/G]CTTTGATCAC; Cys Ser/Ala, TTCCTT[T/G]CTGTTTGTGTTGG; Cys Ser/Ala, CAGATTCA[T/G]CCAGAACTGG; Cys Ser/Ala, GCCACA[T/G]CTGTCAAGATC. The annealing, synthesis, and ligation reactions were performed according to the manufacturer's recommendations (Bio-Rad manual 170-3571). In all cases, mutagenesis was confirmed by sequencing the mutated region before yeast transformation, and, in some cases, the sequence of the targeted region of the AVP insert of pYES2-AVP was further verified after extraction of the vector from the yeast transformants. Sequence analysis was by the dideoxynucleotide chain termination method (Sanger et al., 1977).

Preparation of Microsomes

Yeast microsomes enriched for the V-PPase were prepared by a modification of the procedure described by Kim et al. (1994b). Cell cultures were grown by diluting 200 ml of stationary phase cells into 2 liters of AHC medium supplemented with 2% (w/v) galactose. The cells were grown for 20 h at 30 °C to an A of approximately 1.0 and collected by centrifugation. After resuspension in 10 mM dithiothreitol, 100 mM Tris-HCl (pH 9.4), the cells were incubated for 20 min at 37 °C with gentle shaking. The cells were pelleted, resuspended in 100 ml of YP medium (1% (w/v) yeast extract, 2% (w/v) Bacto-Peptone) containing 0.7 M sorbitol, 1% (w/v) galactose, 5 mM dithiothreitol, and 100 mM Tris-Mes (pH 7.5), and converted to spheroplasts by the addition of 75 mg of zymolyase 20T (ICN). The suspension was incubated for 60-90 min at 30 °C with gentle shaking, pelleted by centrifugation at 4,000 times g for 10 min, and resuspended in 50 ml of ice-cold homogenization medium (10% (w/v) glycerol, 1.5% (w/v) polyvinylpyrollidone, 5 mM Tris-EGTA, 2 mg/ml bovine serum albumin, 50 mM Tris-ascorbate, pH 7.6) containing 1 mM phenylmethylsulfonyl fluoride and 1 µg/ml leupeptin. After lysis by homogenization in a 50-ml glass Dounce homogenizer, the crude lysate was cleared of cell debris and unbroken cells by centrifugation at 4,000 times g for 10 min. The pellet was resuspended in another 50 ml of homogenization medium, homogenized again, and recentrifuged. The supernatants from both low speed centrifugations were pooled and centrifuged at 100,000 times g for 35 min. The pellet was resuspended in suspension medium (1.1 M glycerol, 2 mM dithiothreitol, 1 mM Tris-EGTA, 2 mg/ml bovine serum albumin, 5 mM Tris-Mes, pH 7.6) containing 1 mM phenylmethylsulfonyl fluoride and 1 µg/ml leupeptin, layered onto a discontinuous sucrose density gradient consisting of 10% (w/w) and 28% sucrose, dissolved in suspension medium, and centrifuged at 100,000 times g for 2 h. Partially purified V-PPase-containing microsomes were withdrawn from the 10-28% interface, diluted more than 10-fold with suspension medium, and pelleted by centrifugation at 100,000 times g for 35 min. The resulting membrane pellet was washed free of dithiothreitol by three rounds of resuspension in NEM suspension medium (10% (v/v) glycerol, 2 mg/ml bovine serum albumin, 5 mM Tris-Mes, pH 8.0) and centrifugation at 100,000 times g for 35 min. The final microsome preparation was used immediately or stored at -85 °C.

Reaction of V-PPase with N-Ethylmaleimide

The standard mixture for reaction with NEM contained 30 mM Tris-Mes (pH 8.0), NEM (0-20 µM), 200 µM potassium fluoride, and the indicated concentrations of ligands (Tris-PP(i)plus or minus MgSO(4)). Membrane protein was added, and the mixture was incubated for 5 min at 0 °C. Reaction was terminated by the addition of 0.5 mM dithiothreitol, and aliquots of the mixture were assayed for PPase activity.

Measurement of PPase Activity

PPase activity was measured as the rate of liberation of P(i) from PP(i) at 37 °C by the method of Ames(1966) in reaction medium containing 300 µM Tris-PP(i), 1.3 mM MgSO(4), 50 mM KCl, 500 µM potassium fluoride, 5 µM gramicidin-D, and 30 mM Tris-Mes (pH 8.0).

Potassium fluoride was included in the NEM reaction and PPase assay media to diminish PP(i) hydrolysis by contaminating yeast-soluble PPase. Yeast-soluble PPase is exquisitely sensitive to inhibition by potassium fluoride (K = 20 µM) whereas the V-PPase is relatively insensitive (K = 3.4 mM) (Baykov et al., 1993b; Kim et al., 1994b). Inclusion of 100-500 µM potassium fluoride thereby largely abolishes soluble PPase-catalyzed consumption of protective substrate during incubation with NEM and minimizes the contribution of soluble PPase to total PP(i) hydrolysis during the activity assays.

Measurement of PP-dependent HTranslocation

PP(i)-dependent H translocation was assayed fluorimetrically using acridine orange (2 µM) as transmembrane pH difference indicator in assay media containing microsomes (50 µg), 1 mM Tris-PP(i), 100 mM KCl, 0.4 M glycerol, 1 mM Tris-EGTA, and 5 mM Tris-HCl (pH 8.0). Reaction was initiated by the addition of 1.3 mM MgSO(4), and the decrease in fluorescence was measured at 540 nm (Rea and Turner, 1990).

Protein Estimations

Protein was estimated by a modification of the method of Peterson(1977).

Chemicals

N-Ethylmaleimide and potassium fluoride were purchased from Sigma, zymolyase 20T was from ICN, helper phage VCS-M13 was from Stratagene, and the Muta-Gene in vitro mutagenesis kit was from Bio-Rad. All of the general reagents were of the highest quality and obtained from Fisher, Research Organics (Cleveland, OH), or Sigma.


RESULTS

NEM Sensitivity of Heterologously Expressed Wild Type V-PPase

The cDNA encoding the substrate-binding subunit of the V-PPase from A. thaliana is expressed in a functional state in S. cerevisiae. Moreover, like the endogenous enzyme of vacuolar membrane vesicles prepared from B. vulgaris and Vigna radiata (Britten et al., 1989; Rea et al., 1992a; Zhen et al., 1994), the heterologously expressed wild type enzyme associated with partially purified microsomes isolated from S. cerevisiae BJ5459, after transformation with pYES2-AVP, is susceptible to (Mg + PP(i))-protectable, free PP(i)-potentiated inhibition by NEM (Fig. 1A). Quantitative protection from inhibition is conferred by Mg + PP(i)versus membranes incubated with NEM in the presence of PP(i) alone, indicating that Mg and PP(i), in combination, decrease the reactivity of the NEM-reactive site, or sites, on the enzyme.


Figure 1: Effects of Mg + PP(i) (MgPP) and Tris-PP(i) alone (free PP) on inhibition of heterologously expressed wild type (A) and Cys Ser- or Cys Ala-substituted A. thaliana V-PPase (B) by NEM. Membrane vesicles purified from pYES2-AVP-transformed S. cerevisiae BJ5459 expressing wild type or C634S or C634A mutant AVP were treated with NEM for 5 min at 0 °C in reaction medium containing MgPP(i) (0.3 mM Tris-PP(i) + 1.3 mM MgSO(4)) or free PP(i) (0.3 mM Tris-PP(i)). Reaction with NEM was terminated by the addition of 0.5 mM dithiothreitol, and aliquots of the mixture were assayed for PP(i) hydrolysis as described under ``Materials and Methods.''



These findings are in complete agreement with the results of previous investigations of plant vacuolar membrane vesicles showing that Mg alone confers negligible protection from inhibition by NEM by comparison with Mg + PP(i), in combination, while free PP(i) has a potentiating action versus membranes treated with NEM in the absence of both Mg and PP(i) (Britten et al., 1989). A specific requirement for the simultaneous presence of Mg and PP(i) for quantitative protection from NEM is consistent with the results of steady state reaction kinetic modeling analyses, which demonstrate that magnesium, probably dimagnesium (Mg(2)PP(i)), pyrophosphate is the active substrate species (Baykov et al., 1993a; Leigh et al., 1992).

Effect of Cys Ser or Cys Ala Substitutions on NEM Sensitivity and Catalytic Activity

Alignment of the amino acid sequences of the V-PPases from Arabidopsis (Sarafian et al, 1992) and H. vulgare (Tanaka et al., 1993) and the two isoforms from Beta (Kim et al., 1994a), deduced from the nucleotide sequences of their respective cDNAs, discloses a total of 9 conserved Cys residues corresponding to positions 19, 78, 128, 136, 308, 343, 411, 444, and 634 in the sequence from Arabidopsis (Fig. 2). To determine which, if any, of these residues is the site of action for inhibition of the enzyme by NEM, each conserved Cys was singly mutated to a Ser residue at positions 19, 78, 128, 136, 308, 343, 411, and 444 and a Ser or Ala residue at position 634. Cys Ser substitutions were employed to conserve the electronegativity of the side chain. The Cys Ala substitution at position 634 was introduced to assess the effects of eliminating this electronegative center while minimizing the structural deformation that might otherwise arise from the introduction of a bulkier substituent. By testing the susceptibility of the mutated forms of the V-PPase to (Mg + PP(i))-protectable, free PP(i)-potentiated inhibition by NEM, it was possible to examine the effects of mutagenesis on catalytic activity, NEM inhibitability, and substrate binding in parallel.


Figure 2: Tentative topological model of V-PPase from A. thaliana showing positions of conserved Cys residues. The putative transmembrane spans (boxedsequences) were predicted from the HELIXMEM program of PC/GENE. The conserved Cys residues, common to the deduced sequences from A. thaliana (Sarafian et al., 1992), B. vulgaris (isoforms 1 and 2) (Kim et al., 1994a), and H. vulgare (Tanaka et al., 1993), are shown in white against a blackbackground.



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 mutagenic oligonucleotides designed to substitute each conserved Cys codon with a Ser or Ala codon. After amplification of the mutated plasmid in E. coli DHalpha and confirmation of the mutations by DNA sequencing, S. cerevisiae BJ5459 (Ura) was transformed with the mutated plasmid, and the resulting Ura transformants were grown in liquid culture under inducing conditions. Partially purified microsomes were prepared from the transformants and V-PPase activity was characterized. The results are summarized in Fig. 1, Fig. 3, and Fig. 4.


Figure 3: NEM sensitivity of V-PPase singly mutated from Cys to Ser at positions 19, 78, 128, 136, 308, 343, 411, and 444. Membranes purified from S. cerevisiae BJ5459 expressing mutated V-PPase were tested for inhibition by NEM (0-20 µM) in media containing MgPP(i) (circle) or free PP(i) (bullet) as described in Fig. 1.




Figure 4: PP(i)-dependent H translocation by membrane vesicles prepared from pYES2-AVP-transformed S. cerevisiae BJ5459 expressing either wild type or mutant (C634S or C634A) V-PPase. The membranes were incubated with 50 µM NEM in reaction medium containing MgPP(i) (0.3 mM Tris-PP(i) + 1.3 mM MgSO(4)) or free PP(i) (0.3 mM Tris-PP(i)) for 5 min at 0 °C. Reaction with NEM was terminated by the addition of 0.5 mM dithiothreitol, and the membranes were washed free of NEM and ligands. Aliquots (50 µg) of the washed membranes were assayed for H translocation (intravesicular acidification) with the fluorescent transmembrane pH difference indicator acridine orange in a total reaction volume of 1 ml after the addition of 1.3 mM MgSO(4) to medium containing 1 mM Tris-PP(i).



All of the substitutions, except for C634S and C634A, exert little or no effect on the activity of the V-PPase or its sensitivity to inhibition by NEM (Fig. 3). Cys Ser substitutions at positions 19, 78, 308, 343, and 411 yield enzyme exhibiting a pattern of (Mg + PP(i))-protectable, free PP(i)-potentiated inhibition by NEM similar to wild type V-PPase ( Fig. 1and Fig. 3). Likewise, although C128S, C136S, and C444S mutants are slightly less sensitive to inhibition by NEM in the presence of free PP(i) than wild type enzyme, they are nevertheless quantitatively protected by Mg + PP(i) (Fig. 3). Furthermore, the specific activity of the V-PPase approximates wild type in all cases. Hence, wild type enzyme and the C19S, C78S, C128S, C136S, C308S, C343S, C411S, and C444S mutants exhibit activities of 30, 36, 19, 16, 22, 27, 25, 37, and 20 µmol/mg/h, respectively. In striking contrast, substitution of Cys generates enzyme that is insensitive to NEM irrespective of whether Mg + PP(i) or free PP(i) are included in the NEM reaction medium (Fig. 1). Replacement of Cys with either Ser or Ala yields heterologously expressed enzyme that is active in both PP(i) hydrolysis (Fig. 1B) and PP(i)-dependent H translocation (Fig. 4) but insensitive to NEM concentrations in excess of 50 µM. The PP(i) hydrolytic activities of C634S and C634A, 28 and 26 µmol/mg/h, respectively, approximate wild type enzyme as do both the rates and extents of PP(i)-dependent intravesicular acidification. It therefore appears that although Cys is required for inhibition of the V-PPase by NEM, it is not required for PP(i) hydrolysis or PP(i)-dependent H translocation. The finding that the C634S and C634A mutants are indistinguishable with respect to both NEM insensitivity and catalytic activity (Fig. 1B) implies that both functional attributes are independent of whether the amino acid at position 634 possesses a polar side chain containing an electronegative center (-CH(2)-SH or -CH(2)-OH) or nonpolar (-CH(3)) substituent.


DISCUSSION

The mutational analyses described demonstrate that only one conserved Cys residue, Cys, is required for inhibition of the V-PPase by NEM. Together with the results from previous protein chemical investigations of the mode and site of action of maleimides (Zhen et al, 1994), these studies identify the sulfhydryl group of Cys as the reactive moiety. Two inferences therefore follow. (i) A cytosolic orientation must be assigned to the putative hydrophilic loop (loop X) encompassing Cys (residues 594-646, Fig. 2). The studies showing that NEM, a permeant maleimide, and 3-(N-maleimidylpropionyl) biocytin, an impermeant maleimide, inhibit the enzyme with similar kinetics in a substrate-protectable manner and compete for a common binding site (Zhen et al., 1994) in conjunction with the demonstrated necessity for a Cys residue at position 634 by mutagenesis, unequivocally establish a cytosolic disposition for this residue and those immediately flanking it. (ii) Earlier speculations concerning the location of the NEM-reactive site, or sites, are refuted. It has been noted that Cys and Cys, located in putative hydrophilic loop II, are flanked by sequences possessing a spacing and alternation of acidic and basic amino acid residues equivalent to those regions of the soluble PPase from Saccharomyces known, from site-directed mutagenesis and x-ray crystallography, to be critical for catalysis (Rea et al., 1992b; Cooperman et al., 1992). Hence, while the V-PPase and soluble PPases appear to be remote evolutionarily, it has been proposed that they may share convergent motifs related to the need for both classes of enzyme to interact with the same substrates, inhibitors, and activators (MgPP(i), Mg(2)PP(i), Mg, Ca). Further, in view of the proximity of Cys and Cys to these motifs, the sensitivity of the V-PPase to covalent modification and inhibition by maleimides has been attributed to alkylation of one or both of these Cys residues (Rea et al., 1992b). Although the direct participation of loop II residues other than Cys in substrate binding and/or turnover is not addressed here, the finding that C128S and C136S mutants are not only active in catalysis but also retain sensitivity to (Mg + PP(i))-protectable, free PP(i)-potentiated inhibition by NEM clearly contradicts their proposed involvement in inhibition by maleimides.

An unexpected but simplifying finding and an insight that could not have been gained other than through mutagenesis is that C634S and C634A mutants retain catalytic activity. The situation with the V-PPase is therefore reminiscent of the A subunit of yeast V-ATPase (Taiz et al., 1994) and E. colilac permease (Kaback, 1992). In the case of the V-ATPase, C261S mutants acquire insensitivity to NEM while retaining wild type hydrolytic activity (Taiz et al., 1994). In the case of lac permease, only Cys appears to be important for transport, but even this residue is not essential (Kaback, 1992). When Cys is replaced with Val and each of the other Cys residues is replaced with Ser, about 30% of the initial rate of transport and about 60% of the steady state level of accumulation of lactose is achieved by the ``C-less'' permease versus wild type, although the former is rendered insensitive to NEM (van Iwaarden et al., 1991). By analogy with these two transporters and the dispensability of all of the conserved Cys residues of the V-PPase, the inhibitory action of maleimides on wild type enzyme is specifically attributed to structural deformation imposed through the introduction of a bulky substituted alkyl group on Cys. Thus, while the substrate protectability of enzyme inhibition and covalent modification of Cys by maleimides indicates that this residue is close to or conformationally coupled with the substrate-binding site, direct participation of this or any other Cys residue in the catalytic cycle of the V-PPase is improbable.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Contributed equally to this work.

To whom correspondence should be addressed. parea{at}mail.sas.upenn.edu.

(^1)
The abbreviations used are: V-ATPase, vacuolar H-adenosinetriphosphatase (EC 3.6.1.3); AVP, cDNA encoding A. thaliana vacuolar H-pyrophosphatase; Mes, 2-(N-morpholino)ethanesulfonic acid; NEM, N-ethylmaleimide; V-PPase, vacuolar H-pyrophosphatase (EC 3.6.1.1).

(^2)
The mass of the substrate-binding subunit of the V-PPase, computed from its deduced amino acid sequence, is 80-81 kDa. This polypeptide, however, migrates at M(r) 66,000 on SDS gels.


ACKNOWLEDGEMENTS

We thank members of the Daldal Laboratory (Plant Science Institute) for advice on site-directed mutagenesis, Professor Beth Jones (Carnegie Mellon University, Pittsburgh, PA) for the kind gift of S. cerevisiae strain BJ5459, and colleague Dr. Konrad Howitz for stimulating discussions.


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