Functional Comparisons between Plant Plasma Membrane H+-ATPase Isoforms Expressed in Yeast*

To examine the functional properties of the three major isoforms of plasma membrane H+-ATPase expressed in Arabidopsis thaliana (AHA1, AHA2, and AHA3), we employed a system for the heterologous expression of functional plant plasma membrane H+-ATPase in yeast (Villalba, J. M., Palmgren, M. G., Berberian, G. E., Fergu-son, C., and Serrano, B (1992) J. Biol. Chen. 267,12341-12349). Each isoform was expressed efficiently but ap- peared to be retained in the endoplasmic reticulum of yeast. All isoforma displayed qualitatively similar enzy- matic properties, but quantitative differences were found. When compared with AHA3 , AHAl and AHA2 had an apparent higher turnover rate for ATP hydrolysis, eshibited a 10-fold higher apparent affinity for ATP, and a 3-fold higher sensitivity toward vanadate. In addition, AHA2 had a slightly lower apparent affinity for H+ and seemed to be more susceptible to activation by lysophos-phatidylcholine than did AHA1 and AHA3. This study represents the first comparison of the functional properties of isoforms of the plant plasma membrane €I+- ATPase. The plasma membrane H+-ATPase When determining the pH dependence of ATPase activity, varying volumes of two reaction mixtures as but with 50 m~ MES-TYis, pH 5.5, and 50 m MOPS-Tris, pH 7.5, respectively, were mixed, and pH was measured in the final reaction mixture. When determining the ATP dependence ofATPase activity, MgCl2 was kept constant at 10 m, and 5 m~ phosphoenolpyruvate, 1 m NADH, 25 pg of pyruvate kinase, and 25 pg of lactate dehydrogenase (both enzymes in glycerol) were supplied to the reaction mixture (1 ml). ATP hydrolysis was determined by fol-lowing the reduction of NADH at 340 nm. electrophoresis blotting performed as (Villalba monoclonal against membrane H*-ATPase was utilized.

of A. thaliana is expressed predominantly in the phloem of roots, leaves, and stems, and in pollen (DeWitt et al., 19911, whereas expression ofAHA2 is mainly in root epidermis (Sussman, 1992).l Reporter gene analysis with AHAl has not yet been published.
The role of multiple isoforms for the plant plasma membrane H+-ATPase is essentially unknown. If functional differences exist among the plasma membrane H+-ATPase isoforms, combined with tissue-or cell-specific expression, it seems likely that they might impart unique properties to certain cells. It is possible that the plasma membrane H+-ATPase exists as multiple isoforms to allow for differential regulation of H+-ATPase at various levels (i.e. transcriptional, post-transcriptional, translational, or post-translational).
For example, plasma membrane H+-ATPase is known to be regulated transcriptionally by auxin (Hager et al., 1991).
As a consequence of their conserved primary structure, all of the known plant plasma membrane H+-ATPase isoforms are predicted to have essentially the same tertiary structure. Despite this similarity, it seemed possible that functional differences might exist among the isoforms. Tu date, functional characterization of plant plasma membrane H+-ATPase isoforms has proven difficult due to the presence and the similarity of multiple isoforms in many tissues. Concomitant expression of isoforms in the same cells or tissue is a deterrent in their characterization. Tu determine whether inherent functional differences exist among these isoforms, we have employed a recently developed system for heterologous expression of plant plasma membrane H+-ATPase which permits the convenient analysis of enzymatic activity (Vlllalba et al., 1992). This system involves expression of functional plant H+-ATPase in the endoplasmic reticulum (ER)' of yeast cells. By employing this system, three A. thaliana H+-ATPase isoforms have been produced individually in yeast in the same cellular environment. This has enabled us to analyze various biochemical parameters of the isoforms.

MATERIALS AND METHODS
Construction of Plasmids-Plasmids pRS-891, containing the A H A l gene, and pMP-142, containing A H A 2 , have been described previously (Villalba et al., 1992; Palmgren and Christensen, 1993). pRS-541 was produced by cloning the AHA3 cDNA (Pardo and Serrano, 1991a) into pBS (Stratagene). TheAHA3 cDNAwas a kind gift from Dr. J. M. Pardo and Professor R. Serrano (Universidad Politecnica, Valencia, Spain). A 1081-base pair FokI fragment of pRS-541, containing the ATG start codon and an internal BglII site, was made blunt by Klenow treatment and digested by BglII. The 227-base pair digestion product which contained the initiation ATG was subcloned simultaneously with the 3.0-kb BglII-KpnI fragment of pRS-541, containing the rest of the AHA3 gene, into EcoRV-KpnI-digested pBluescript SK-(Stratagene) to produce pMP-137. These steps removed eight spurious out-of-frame ATG codons 5' to the correct ATG. The complete AHA3 gene was excised as a 3.2-kb EcoRI fragment, subcloned into pBluescript SK-, digested partially with EcoRI, treated with Klenow, and excised as a 3.2-kb (EcoRI)-SpeI fragment by SpeI. This fragment was ligated to the 7.6-kb (XhoI)-SpeI fragment of pRS-136, in which the XhoI sticky end had been blunted by Klenow treatment, in order to produce pMP-169.
Yeast Strains and Culture Conditions-The yeast strains used in this study were: RS-72 (Cid et al., 1987), RS-933 ( R S -7 m p 3 5 1 (Hill et al., 1986)), RS-934 (RS-72/pRS-891 (Villalba et al., 1992)), MP-142 (RS-72/ pMP-136 (Palmgren and Christensen, 1993)). and . Yeast cells were made competent for plasmid uptake by treatment with lithium acetate and polyethyleneglycol according to Ito et al. (1983). The synthetic growth media were as described (Villalba et al., 1992). In order to express only plasmid-borne enzyme, yeast cells were cultured in galactose-containing medium until growth reached the stationary phase after which they were stored at 4 "C without shaking. After incubation in the cold for 24 h, the cells were pelleted, resuspended in the same volume of glucose medium (30 "C), and cells were harvested after shaking a t 30 "C for 1 h. Upon prolonged growth in glucose medium, the level of AHA3 expressed diminished significantly (to below 25% ofmaximum; Fig. l), while expression ofAHAl andAHA2 remained the same (data not shown). The reason for this instability of AHA3 is unknown, and no proteolytic degradation of the polypeptide could be observed in Western blots immunodecorated with a monoclonal plant H*-ATPase antibody (Villalba et al., 1991;Fig. 1) or with polyclonal antisera directed against the N terminus, the central part, and the C terminus of AHA3 data not shown).
Biochemical Methods-For analysis of subcellular distribution of ATPase, a microsomal membrane fraction prepared by differential centrifugation according to Villalba et al. (1992) was layered on top of a continuous gradient composed of 2653% (w/w) sucrose in 10 m Tris-HCl, pH 7.6, 1 m M EDTA, and 1 m dithiothreitol. After overnight centrifugation at 30,000 revolutiondmin (Beckman rotor Ti-SW40). fractions of 1 ml were collected from the top of the gradient.
For purification of the ER fraction containing plant ATPase, a microsomal membrane fraction from 250 ml of glucose culture (see above) was applied to a discontinuous sucrose gradient made of 5 ml of 29% (w/w) sucrose and 5 ml of 34% (w/w) sucrose. After overnight centrifugation at 30,000 revolutions/min (Beckman rotor Ti-SW40), membranes enriched in plant plasma membrane H+-ATPase were recovered at the 29/34 interface.
ATP hydrolysis was measured at 30 "C in 0.3 ml of reaction mixture containing 50 m MOPS adjusted to pH 6.5 with Tris, 3 m Na2ATP, 10 m M MgCI,, 50 m~ KN03 (to inhibit vacuolar ATPase), 5 m~ sodium azide (to inhibit mitochondrial ATPase), 0.2 m ammonium heptamolybdate (to inhibit acid phosphatase). Stocks of vanadate were boiled for 3 min prior to use to break down polymeric species. Assays were initiated by the addition of purified ER (5-10 pg of membrane protein) and stopped aRer 10-30 min as described (Baginski et al., 1967).
When determining the pH dependence of ATPase activity, varying volumes of two reaction mixtures as above but with 50 m~ MES-TYis, pH 5.5, and 50 m MOPS-Tris, pH 7.5, respectively, were mixed, and pH was measured in the final reaction mixture. When determining the ATP dependence ofATPase activity, MgCl2 was kept constant at 10 m, and 5 m~ phosphoenolpyruvate, 1 m NADH, 25 pg of pyruvate kinase, and 25 pg of lactate dehydrogenase (both enzymes in glycerol) were supplied to the reaction mixture (1 ml). ATP hydrolysis was determined by following the reduction of NADH at 340 nm.
SDS-polyacrylamide gel electrophoresis and Western blotting were performed as described (Villalba et al., 1992). A monoclonal antibody (Villalba et al., 1991) raised against the plant plasma membrane H*-ATPase was utilized.
Protein was measured according to Bradford (1976).

Expression of Plant H+-ATPase Isoforms under Different Conditions
Our objective in this study was to determine the enzymatic properties of three isoforms of the plasma membrane H+-ATPase expressed in the plant A. thaliana. Expression of plant plasma membrane H+-ATPase in yeast cells has proven to be a very powerful system for addressing the relationship between structure and function for this enzyme (Villalba et al., 1992;Palmgren and Christensen, 1993). This system allows the char- Strains were cultured in galactose medium until growth reached stationary phase (0 h). Yeast cells expressingAHA3 were transferred to glucose medium and growth was for an additional 1, 16, or 64 h before harvesting the cells. The antibody employed was a monoclonal antibody against plant plasma membrane H+-ATPase.
acterization of heterologously expressed plant H+-ATPase in membranes devoid of endogenous yeast ATPases (Villalba et al., 1992;Palmgren and Christensen 1993). The A. thaliana isoform genes were cloned in multicopy expression plasmids under control of the promoter of the yeast plasma membrane H+-ATPase gene (€"A1 ). This promoter confers high levels of constitutive expression and is, in addition, positively regulated by glucose (Eraso et al., Capieaux et al., 1989). The expression plasmids are introduced into Saccharomyces cerevisiae strain RS-72 (Cid et al., 1987). In this strain the constitutive promoter of the chromosomal PMAl gene has been placed under control of a galacose-dependent promoter by a gene disruption strategy (Cid et al., 1987). The resulting transformed strains (strain RS-934 expressing A H A 1 (Villalba et al., 1992), strain MP-142 expressing AHA2 (Palmgren and Christensen, 1993), and strain MP-170 expressingAHA3 (this paper)) would express yeast ATPase on galactose medium but not on glucose medium. The plant ATPases would be expressed on both media, b u t at somewhat higher levels on glucose medium. In addition, yeast cells had been transformed with the plasmid lacking the plant cDNA (control strain  or with plasmid containing the yeast H+-ATPase gene (control strain MP-213).
In order to investigate the expression of plant H+-ATPase isoforms, yeast cells putatively expressingAHAl,AHA2,AHA3, as well as the control strain RS-933 were cultured in galactose medium until growth reached the stationary phase. Total membranes from all four strains were analyzed by Western blot (Fig   1). All three plant plasma membrane H+-ATPase isoforms were expressed at high levels in the yeast cells. The AHA2 isoform migrated with slightly greater mobility than isoformsAHA1 and AHA3 on 7% acrylamide-SDS gels (Fig. 1). This is surprising since the predicted molecular masses of the ATPase isoforms from A. thaliana (Pardo and Serrano, 1989b) all are about 104 kDa (AHA1 = 104,182;AHA2 = 104,27O;AHA3 = 104,318). Other features than size that could influence mobility in SDS-polyacrylamide gel electrophoresis could be amount of SDS binding, structural features retained in SDS, or post-translational modifications. In order to rule out trivial explanations, perhaps caused by cloning artifacts, we sequenced the coding region of theAHA2 clone from both ends and the terminal sequences corresponded to the published sequences (Pardo and Serrano, 1989b;Harper et al., 1990). At the protein level, attempts to sequence the purified AHA2 protein by Edman degradation were unsuccessful since the N terminus appeared to be blocked. to glucose medium. It is expected that expression of functional plant H+-ATPase in the yeast plasma membrane should support growth of cells devoid of yeast ATPase. However, among the three A. thaliana H+-ATPase isoforms only one, AHA2, was able to complement endogenous yeast plasma membrane H+-ATPase (PMAl), albeit to a very low degree (Fig. 2), as reported previously (Palmgren and Christensen, 1993). The lack of complementation exhibited by AHA3 suggested that this particular isoform is retained in the ER of transformed yeast cells as is the case for the AHAl (Villalba et al., 1992) and

Different Distribution of Yeast H+-ATPase and AHA3
Total membranes from yeast cells grown on glucose for 16 h were fractionated by sucrose gradient centrifugation under isopycnic conditions. Membranes from strain MP-170 expressing AHA3 contained a large peak of plasma membrane H+-ATPase activity at 30% sucrose with a shoulder at 4 0 4 5 % sucrose (Fig.  3). This peak correspond to a novel fraction of enlarged ER (Villalba et al., 1992). On the contrary, a small peak at 4 0 4 6 % sucrose was present in membranes from strain MP-213 expressing plasmid-borne yeast ATPase (Fig. 3). These dense membranes correspond to the major domain of the yeast plasma membrane . The low level of the yeast ATPase is due to the low activity state of yeast ATPase after homogenization of water-washed, nonglucolyzing cells (Serrano, 1983). The middle of the gradient (around 40% sucrose) is enriched in mitochondria and rough ER (Villalba et al., 19921, and membranes a t this density isolated from strain MP-213 and "933 exhibited some ATPase activity (Fig. 3). Low density membranes equilibrating at 30% sucrose from strain MP-213 (expressing yeast ATPase) and control strain RS-933 (expressing no ATPases on glucose medium) displayed only negligible ATPase activity.
These results indicate that most of the AHA3 polypeptide expressed in transformed yeast cells was located in the novel fraction of ER in which AHAl and AHA2 also seem to be retained (Villalba et al., 1992;Palmgren and Christensen, 1993). This implied, that all three A. thaliana plasma membrane H+-ATPase isoforms could be expressed in yeast and purified in a membrane fraction devoid of contaminating ATPases.

Expression of Plant Plasma Membrane H+-ATPase
Isoforms in the ER of Yeast Analysis of the polypeptide composition of membrane vesicles derived from the endoplasmic reticulum of yeast cells transformed with each of the different A. thaliana plasma membrane H+-ATPase constructs used in this paper revealed that each isoform was expressed at high levels in this particular membrane fraction (Fig. 4A). Although the expression level varied somehow from membrane preparation to membrane preparation, AHA2 was always expressed to the highest degree followed by AHA3 and AHA1. Densitometric analysis indicated that the 95 kDa AHA2 band amounted to about 70% of total ER protein, while AHA3 and AHAl were expressed to somewhat lower degrees (about 55 and 40% of total ER protein, respectively). Interestingly, as for total membranes (see above), in our gel system AHA2 migrated with an apparent size smaller than predicted from its sequence compared with the other isoforms.
The high expression of AHA2 in the ER as compared with AHAl and AHA3 might explain why only this particular isoform complements yeast cells devoid of yeast ATPase (Fig. 2). Assuming that the ER of yeast has a maximal capacity for retaining polypeptides, the ER harboring this specific isoform may be saturated with plant H+-ATPase thus allowing for a certain nonspecific "spill-over" of enzyme to the plasma membrane (Palmgren and Christensen, 1993).

Characterization of the Plant Plasma Membrane
H+-ATPase Isoforms Synthesized in Yeast Specific Activity-Each of the AHA species expressed in the ER of transformed yeast cells were active ATPases as illustrated in Table I. The specific activity of each isoform in ER vesicles purified from yeast cells transferred to glucose medium for 1 h only and assayed at pH 6.5 and 3 m~ ATP varied significantly, and the specific activity ofAHA2 was about 2-fold higher than that of A H A l and AHA3 (Table I).
Differences in specific ATPase activity between AHA isoforms could either reflect differences in the amount of the various enzymes expressed in the ER or, alternatively, it could reflect fundamentally different rates of turnover of the catalytic cycle of the different enzymes. To address this issue equal amounts ofATP hydrolytic activity (0.2 pmol of Pi releasedmid ml; 10 p1 of ER membrane preparation from yeast cells transformed with each of the different isoforms) were run on an SDS-polyacrylamide gel, and the gel was stained with COOmassie Brilliant Blue (Fig. 4B). Densitometric analysis revealed that the bands representing AHAl and AHA2 were stained to the same degree while the intensity of the AHA3 band exceeded that of the AHAl and AHA2 bands about 2-fold.
This suggests that differences in ATPase activity between membranes expressing AHAl and AHA2 could be explained largely by different levels of active enzyme expressed in the membranes, whereas AHA3 seemed to have a somewhat lower turnover rate for ATP hydrolysis compared with AHAl and AHA2 or, alternatively, was not assayed under optimal assay conditions. It should be noted, however, that in these kinds of experiments there is no way to check for the amount of active versus inactive, or misfolded enzyme.
Effect of Mg2' a d Ca2+-All three plant H+-ATPase isoforms showed an absolute requirement for Mg2' pointing to MgATP as the true substrate (Table 11). M e could not be substituted at all by Ca2+ (Table 11). Ca2+ inactivates the ATPase activity of plasma membranes isolated from plant sources (Vara and Serrano, 1982). In the presence of M 2 + , Ca2+ in the millimolar range caused only a minor inhibition of the various AHA species. This inhibition is probably due to competition with Mg2' in binding to ATP.
pH Optimum-A critical parameter for the biological functioning of H+ pumps is their affinity for intracellular H+. Therefore, pH is an important parameter of enzyme function, and its influence on enzyme activity was examined, as shown in Fig. 5. ER vesicles expressingAHA1, A H A 2 , orAHA3 displayed essentially superimposable curves, which matched the pH dependence typically observed with plasma membrane H+-ATPase isolated from plant sources (reviewed by Sze, 1985;Briskin, 1990). The pH optima of the three isoforms were in all cases pH 6.5 (Fig. 6), although the pH optimum of AHA2 tended to approach pH 6.4 (Fig. 6), and activity fell off rapidly at higher values. A minor change was seen in the case of the AHA3 enzyme, which appeared more sensitive to inactivation at lower pH values. Essentially all enzymes have a characteristic pH profile which reflects folding of the polypeptide. Assuming, however, that the change in activity between pH 7.5 and 6.5 reflects binding of the transported proton at a specific site, AHA2 exhibited a slightly lower affinity for H+ compared with AHAl and AHA3 (apparent for H+: AHAl = 100 m; AHA2 = 150 m; AHA3 = 100 m). These alterations in pH sensitivity were small, however, and could not account for the low specific activity of AHA3 (see above).
K, for ATP-The most significant difference between the three isoforms was seen when the dependence on the enzyme reaction upon ATP was determined. The apparent affinity for ATP for the various isoforms expressed in yeast ER was measured employing a n ATP regenerating system. With 10 m Mg2' and variable ATP from 50 to 3000 p~, the kinetics for all three isoforms obey Michaelis-Menten behavior (Fig. 6)

. A H A l and
AHA2 exhibited a relatively low K, (0.15 m~) whereas AHA3 exhibited a high K, (1.5 mM) similar to fungal P-type H+ pumps (Goffeau and Slayman, 1981;Bowman and Bowman, 1986). Thus, the low affinity for ATP exhibited by AHA3 partly seemed to explain the low specific activity displayed by this enzyme a t 3 m~ ATP.
Ki for Vanadate-Vanadate is a potent inhibitor of enzymes which form phosphorylated intermediates during their reaction cycle, probably because it resembles the transition state analog of phosphate, and thus binds at the active site (Cantley et al., 1978). All three plant H+-ATPase isoforms were strongly inhibited by vanadate (Fig. 5). AHAl and AHA2 were characterized by having a 3-fold higher sensitivity toward vanadate as compared to AHA3 (apparent Ki for vanadate: AHAl = 3 p~; AHA2 = 3 p~; AHA3 = 10 p~; see Fig. 7).
Effect of Lysophosphatidylcholine-Lysophosphatidylcholine (lyso-PC) is an effector molecule of plant plasma membrane H+-ATPase (Palmgren, 1991). When the ATPase activity of ER vesicles expressing either isoform was titrated with this lipid, typical sigmoidal activation curves indicating cooperativity between lyso-PC molecules were seen in all cases (Fig. 8). ER vesicles purified from yeast are all of the same orientation (the ATP-binding site of heterologously expressed plant ATPase faces the extravesicular medium) and do not harbor latent ATPase activity (Villalba et al., 1992). Therefore, the increase in ATPase activity is likely to be due to true activation. AHA2 was activated to the highest degree by lyso-PC (maximal stimulation by lyso-PC: AHAl = 60%; AHA2 = 160%; AHA3 = 50%).
At 15 pg of membrane proteidml, half-maximal activation by lyso-PC was 20 pg/ml for all three AHA species (Fig. 8).

DISCUSSION
The cloning of the cDNAs for the three major A. thaliana plasma membrane H+-ATPase isoforms (Harper et al., , 1990Serrano, 1989a, 1989b) has allowed for their individual expression in the ER of yeast, thereby producing a means in which to characterize the kinetic properties of each isoform. Studying the properties of the three plant ATPase isoforms expressed in yeast is advantageous in that they are all present in the same environment. That is, lipid composition of the surrounding membrane is similar. Also, if cell type-specific ATPase activities were assayed at 30 "C in 300 111 of reaction mixture containing 50 m~ MOPS-BTP, pH 6.5,3 IIIM Na, ATP, 10 m~ MgCl,, 50 m~ KNO,, 5 m~ sodium azide, 0.2 IIIM ammoniumheptamolybdate, and 0.1 m~ EDTA. ER membrane vesicles were isolated from yeast cells grown for 1 h on glucose medium in order to boost expression of plant ATPase. The data shown are average values for seven independent ER membrane isolations. Activities were assayed at 30 "C in the absence or presence of the indicated concentrations of MgCl, and CaCl, in 300 pl of reaction mixture containing 50 m~ MOPS-BTP, pH 6.5, 3 m~ Na2ATP, 50 m~ KN03, 5 IIIM sodium azide, 0.2 IIIM ammonium heptamolybdate, and 0.1 m~ EDTA.

Strain or
The data shown are the average of three determinations obtained from one ER membrane isolation. variable splicing) or at the protein level (i.e. regulatory phosphorylation), this may be minimized when all three isoforms are expressed in a heterologous species such as yeast.

A H A l
Electrophoresis of ER isolated from transformed yeast cells clearly reveal that the known Arabidopsis plasma membrane H+-ATPase isoforms migrate with slightly different rates through the acrylamide-SDS gel system (Figs. 1 and 4). Biochemical heterogeneity in plant plasma membrane H+-ATPase preparations was first reported for native corn root plasma membrane H+-ATPase (Gallagher and Leonard, 1987). Two closely associated ATPase bands, both reacting with antibodies to Neurospora or plant plasma membrane H+-ATPase, have also been demonstrated in the plasma membrane isolated from barley roots (Dupont et al., 1988), corn roots (Grouzis et al., 1990), oat roots (Palmgren et al., 1990), sugar beet leaves, and A. thaliana . Anomalous migration in SDS-polyacrylamide gel electrophoresis is a feature of other P-type ATPases. The al-subunit of the Na+iK+-ATPase (Sweadner 1989(Sweadner , 1990 and the SERCAl isoform of sarcoendoplasmic reticulum Ca2+-ATPase (Lytton et al., 1992) both migrate with an apparent size larger than predicted from their sequence compared with isoforms of the same enzymes having only slightly different calculated molecular weights. Na+iK+-ATPase a-subunit mobility seems to reflect complex detergent-protein interactions that can be affected by experimental conditions and the existence of more than one band on gels may reflect different conformations of each isoform in detergent (Sweadner, 1990). Therefore, in light of the results presented here, what is seen in plant plasma membrane preparations as H+-ATPase doublets seems to be a direct visualization of the isoform heterogeneity of the preparation.
Why isoforms of the plasma membrane H+-ATPase? At least AHA2 (Sussman, 1992) and AHA3 (DeWitt et al., 1991) are expressed in a tissue-and developmental specific manner. Lingrel (1992) has pointed to two models of Na+/K+-ATPase a isoform diversity. The first model, the functional model, predicts that the catalytic or regulatory properties of each of the isoforms differ, and isoforms with a particular functional property would be expressed in a tissue or cell type where this particular property is required. The genetic model, on the contrary, assumes that functional differences among isoforms do not exist or that, if they do, these differences imply no significant physiological role. According to this scheme, only the overall level of enzyme in a particular cell or at a specific developmental stage is important. Utilizing the expression of plant plasma membrane H+-ATPase in yeast, we have characterized the substrate dependence properties of each of three isoforms expressed in the plant A. thaliana. The results of the present study indicate that AHAl and AHA2 have very similar apparent affinities for ATP (Fig. 6) and vanadate (Fig. 71, whereas AHA3 exhibit severalfold lower affinities for ATP and vanadate. In addition, it is possible that AHA3 turns over at a somewhat lower rate than do A H A l and AHA2. From analysis of the nucleotide sequence ofAh!Al, Al€42, andAHA3, it is not surprising that AHAl and AHA2 behave as very similar enzymes, whereas AHA3 is distinct. AHAl and AHA2 show 94.3% identity at the amino acid level, whereas AHA3 is only 87.7% and 88.5% identical to A H A l and-, respectively. The data presented in this study thus tend to support at the functional level the view that there are at least two AHA subfamilies present in A. thaliana.
The observed biochemical characteristics of the AHA subspecies are very similar to those described for the plasma membrane H+-ATPase of various plant materials, that is a pH op-timum for ATP hydrolysis around pH 6.5, K,,, for ATP between 0.1-1.0 m, and Ki for vanadate between 1 and 10 m (Briskin, 1990). The plasma membrane H+-ATPase activity of a highly purified plasma membrane fraction from etiolated A. thaliana seedlings had a pH optimum of pH 6.6, and the concentration of vanadate required to inhibit the ATPase activity by 50% was 1-2 1.1~ (Olivari et al., 1993). Quantitatively, however, it is difficult to compare the results of the present study with previous reports, since earlier H+-ATPase preparations derived from plant material may have contained multiple isoforms. A separate question concerns the molecular mechanism by which the plasma membrane H+-ATPase is regulated posttranslationally. Lyso-PC is a lipid found in small amounts in plant plasma membranes which activates plant ATPase in vitro. The mechanism of activation by lyso-PC involves displacement of an autoinhibitory domain located at the C-terminal region of the enzyme (Palmgren, 1991;Palmgren et al., 1991;Palmgren and Christensen, 1993). All three A. thaliana AHA species synthesized in yeast were activated by lyso-PC ( Fig. 8), suggesting that all isoforms share a common mechanism of regulation. Interestingly, lyso-PC seemed to have a more potent effect on AHA2 compared with the other AHA species (Fig. 81, suggesting that this particular isoform is more tightly regulated than other isoforms. The plant plasma membrane H+-ATPase is a phosphoprotein in vivo, and it has been hypothesized that kinase-mediated phosphorylation of the ATPase is the means by which the catalytic activity is altered post-translationally (Schaller and Sussman 1988). In the presence of M$+, the H+-ATPase activity of oat root plasma membrane vesicles is inhibited by Ca2+ (80% inhibition at 1 m; Vara and Serrano, 1982). When purified oat root plasma membrane vesicles were incubated with [y-32PlATP, radioactivity was incorporated into the ATPase, at serine and threonine residues. The majority of this kinasemediated ATPase phosphorylation was found to be strictly dependent on the presence of calcium (Schaller and Sussman 1988). Taken together, these data suggest that the negative effect of Ca2+ on the H+-ATPase activity, if any, is indirect and sustained by a phosphorylation mechanism. Accordingly, none of the A. thulium plasma membrane H+-ATPase isoforms produced in yeast were sensitive to Ca2+ (Table 11).
The plant plasma membrane H+-ATPase belongs to a super family of cation-translocating ATPases, the P-type ATPases (Pedersen and Carafoli, 1987). The properties of isoforms of other P-type ATPases have been compared by means of heterologous expression systems. The isoforms that have been characterized are the d -, a2-, and a3-isoforms of Na+/K+-ATPase (Jewel1 and Lingrel, 1991;Blanco et ul., 1993) and the SERCA1, SERCA2a, SERCMb, and SERCA3 isoforms of sarcoplasmic reticulum Ca2+-ATPase (Lytton et al., 1992;Verboomen et al., 1992). The isoforms of each ATPase display qualitatively similar enzymatic properties but the quantitative properties of Na+/ K+-ATPase and Ca2+-ATPase isoforms are not identical in all respects. Isoforms of Na+/K+-ATPase and Ca2+-ATPase, respectively, exhibits significant differences in affinities for their various ligands, thus supporting the hypothesis that the purpose of multiple isoforms is to provide ATPases with unique catalytic or regulatory properties in the various differentiated cells in which each is specifically expressed.
This study represents the first comparison of the functional properties of plant plasma membrane H+-ATPase isoforms. If the isoforms in Arabidopsis possess the substrate dependence properties we have observed in yeast, it is tempting to speculate about their physiological significance. AHA3 is expressed in phloem cells and have an unusually low affinity for ATP, suggesting that these cells maintain an elevated ATP which would allow this particular isoform to operate at a significant rate.