Planta

Planta (1988) 173:509-518

9 Springer-Verlag 1988

Phosphorylation of the plasma-membrane H +-ATPase of oat roots by a calcium-stimulated protein kinase G. Eric Schaller and Michael R. Sussman* Department of Horticulture and Cell/Molecular Biology Program, University of Wisconsin, Madison, WI 53706, USA

Abstract. When plasma-membrane vesicles isolated from oat (Arena sativa L.) root cells were incubated with [7-32P]ATP, the H+-ATPase was found to be phosphorylated at serine and threonine residues. Phosphotyrosine was not detected. Endogenous ATPase kinase activity was also observed in plasma-membrane vesicles isolated from potato (Solanum tuberosum L.) root cells as well as from yeast (Saccharomyces cerevisiae). Identity of the phosphorylated oat root M r = 100000 polypeptide as the ATPase was confirmed using conventional glycerol density-gradient centrifugation to purify the native enzyme and by a new procedure for purifying the denatured polypeptide using reversephase high-performance liquid chromatography. Kinase-mediated phosphorylation of the oat root plasma-membrane H +-ATPase was stimulated by the addition of low concentrations of Ca 2+ and by a decrease in pH, from 7.2 to 6.2. These results demonstrate that kinase-mediated phosphorylation of the H+-ATPase is a plausible mechanism for regulating activity. They further indicate that changes in the cytoplasmic [Ca 2+] and pH are potentially important elements in modulating the kinase-mediated phosphorylation. Key words: Arena (ATPase) - Calcium and ATPase - H +-ATPase - Plasma membrane - Protein phosphorylation - Solanum (ATPase).

Introduction

The plasma membrane of higher plants and fungi contains an electrogenic, proton-pumping ATPase * To whom correspondence should be sent EDTA = ethylenediaminetetraacetic acid; EGTA=ethylene glycol-bis-(7-aminoethyl ether)-N,N,N',N'tetraacetic acid; M~- relative molecular mass; RP-HPLC = reverse-phase high-performance liquid chromatography; SDSPAGE=sodium dodecyl sulfate polyacrylamide gel electrophoresis Abbreviations:

(H +-ATPase) which has been investigated in electrophysiological and biochemical studies (Goffeau and Slayman 1981; Serrano 1985), and more recently, with recombinant-DNA techniques (Hager et al. 1986; Serrano et al. 1986). The H+-ATPase contains a single Mr = 100 000 polypeptide and uses the chemical energy of ATP to drive the extrusion of protons into the external medium. This ATPfueled proton electrochemical gradient, generated across the plasma membrane, is used to drive solute-uptake systems, and maintains the turgot, pH, and ionic composition of the cell required for growth. The activity of the higher-plant H+-ATPase is affected within seconds after treatment with stimuli such as fusicoccin and blue light. The fungal toxin fusicoccin induces growth in plant cells apparently by stimulating the ATPase, which leads to acidification and loosening of the cell wall, a prerequisite for plant cell growth (Rayle and Cleland 1977). Fusicoccin has been reported to stimulate the ATPase in isolated plasma membranes as well as in whole cells (see Marr6 1979; Bertl and Felle 1985; Rasi-Caldogno et al. 1986). Irradiation with blue light leads to stomatal opening, preceded by activation of the H +-ATPase in guard cells. Stimulation of the ATPase causes proton extrusion from these cells, hyperpolarization of the membrane potential and increased K + uptake. This in turn causes the guard cells to swell osmotically, and the stomata open (Assmann et al. 1985). In addition, recent studies indicate that turgor pressure and the phytotoxin, syringomycin, also exert rapid effects on plant tissue, possibly through an alteration in the activity of the plasma-membrane H+-ATPase (Bidwai et al. 1987; Bidwai and Takemoto 1987; Reinhold et al. 1984). The reaction mechanism of the plasma-membrane H +-ATpase has been studied in some detail (Briskin 1986) but little is known about how the enzyme is physiologically regulated. The stimula-

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G.E. Schaller and M.R. Sussman: Phospharylation of plasma-membrane H +-ATPase

tion of H+-ATPase by fusicoccin and blue light occurs rapidly enough to indicate allosteric regulation by some metabolite or by a covalent modification such as phosphorylation/dephosphorylation. In this paper we present results demonstrating that the plasma-membrane proton pump of higher plants is a substrate for an endogenous, membrane-associated, calcium-stimulated protein kinase, establishing a possible means of regulation. In addition, we compare kinase-mediated phosphorylation of the higher-plant plasma-membrane H +-ATPase to that of the yeast plasma-membrane H+-ATPase. A preliminary account of these results has been presented in Schaller and Sussman (1987). Material and methods Isolation of plasma membranes. Plasma-membrane vesicles were isolated from roots of 7-d-old etiolated oat (Arena sativa L., cv. Stout; Olds Seed Co., Madison, Wis., USA) seedlings grown in vermiculite, or from roots of potato (Solanum tuberosum L., cv. Norland; a gift from Dr. B. McCown, Madison, Wis. USA) plants, as described by Surowy and Sussman (1985). Plasma-membrane vesicles were isolated from Saccharomyces eerevisiae using the cell-wall digestion and sonication procedure described by Sussman and Slayman (1983). The ATPase assays were also as described in Surowy and Sussman (1985), and specific activities varied between 1 and 3 lamol, rain- 1. (rag protein)- 1. Greater than 95% of this ATPase activity was insensitive to 5raM KN3, 100mM KNO3 and 0.1raM (NH4)6MOTOzg'H20 (ammonium molybdate), inhibitors, in the order given, of the mitochondrial, vacuolar and a nonspecific cytoplasmic phosphatase. The activity was sensitive to appropriate concentrations of vanadate, dicyclohexylcarbodiimide and diethylstilbesterol, inhibitors of the plasma-membrane proton pump.

ethylenediaminetetraacetic acid (Na~-EDTA) (Figs. 1~4), or by 400 gl of 10% trichloroacetic acid (Figs. 5-8), followed by chilling on ice. Reaction blanks, in which membranes were treated with excess Na2-EDTA or trichloroacetic acid prior to ATP addition, showed an absence of phosphorylated polypeptides. Samples were then centrifuged for 30 rain in a microfuge, and supernatants discarded. For reactions terminated with trichloroacetic acid, pellets were rinsed once with chilled 250 mM sucrose prior to dissolution in electrophoresis sample buffer. This sucrose rinse was found to be necessary to prevent aggregation of the Mr = 100000 ATPase polypeptide on subsequent sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Pellets were dissolved in 20 lal of electrophoresis sample buffer and phosphorylated membrane proteins separated by SDS-PAGE according to Laemmli (1970). Following electrophoresis, gels were stained with Coomassie blue overnight, then destained and dried. Autoradiographs were made by exposing Kodak XOmat film (Eastman-Kodak, Rochester, N.Y., USA) to the dried gels at - 80 ~ C. Autoradiograph bands were scanned using a Zeineh Soft Laser Scanning Densitometer, Model SL-504-XL (Biomed Instruments, Fullerton, Cal., USA), and the peak areas quantitated by cutting out and weighing the peak tracings.

In-vivo phosphorylation. Oat roots, 10 g, were radioactively labeled prior to plasma-membrane extraction by incubation with 3.7-10 v Bq of carrier-free [32p]inorganic phosphate in 25 ml of uptake buffer (1 mM Mes-KOH, pH 6.0, 1 mM CaC12, 50 m M KC1) for 6 h at 20 ~ C, with gentle agitation. The roots were then rinsed with distilled water, homogenized, and fractionated to purify the plasma membrane as performed with nonradioactive tissue. Measurement of the radioactivity present in the external solution demonstrated that 20% of the phosphate supplied was taken up during the 6-h uptake period.

In-vitro kinase assay. For the experiments shown in Figs. 1-4,

Purification of the plasma-membrane H+-A TPase. The ATPase was solubilized with lysolecithin from Triton X-100-washed vesicles according to Serrano (1984). Solubilized enzyme was applied to a glycerol density gradient (25-55% w/v glycerol) and centrifugation carried out for 33 h at 49000 rpm in a Beckman SW 55Ti rotor. Fractions were analyzed for ATPase activity and by SDS-PAGE on 8% (w/v) acrylamide gels, as in Surowy and Sussman (1985).

plasma-membrane vesicles (50-100 gg protein) were added to kinase buffer (50 mM 2-(N-morpholino)ethanesulfonic acid (Mes), 2 m M ATP, 6 m M MgC12, adjusted to pH 6.5 with NaOH) containing 3.7-105 Bq of [~-32p]ATP ( > 1.85.101'~ Bq" mmol-1; Catalogue No. NEG-002Z, New England Nuclear, Boston, Mass., USA), and incubated at 20~ for indicated periods of time. For the experiments shown in Figs. 5-8, where the effect of calcium upon the kinase was examined, plasma membranes were first washed in 50 mM Mes, 10 mM MgC12, 0.2 mM ethylene glycol-bis(7-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), pH 6, and centrifuged at 150000.g for 45 min in a Beckman SW 55Ti ultracentrifuge rotor (Beckman Instruments, Palo Alto, Cal., USA). The pellet was then resuspended and preincubated for 15 rain in a solution containing 50 mM Mes, 6 mM MgCI2, 1.5 mM EGTA, and an appropriate amount of C a C l z ' 6 H 2 0 to give the desired concentration of free calcium. The pH of the solution was adjusted with N a O H or HC1, and the exact pH of the reaction determined for the final reaction mixture. The free-calcium concentration was calculated from the association constant for Ca" E G T A as described by Pershadsingh and McDonald (1980). The reaction was started by the addition of ATP; incubation was at 20 ~ C. The reaction volume was typically 200 ~tl and the reaction was terminated either by the addition of 50 gl of 0.1 M disodium

Molecular size of the native, undenatured H +-A TPase. Sedimentation properties of the solubilized oat H +-ATPase were determined in a manner previously used by Dufour and Goffeau (1980) to characterize the H+-ATPase of the yeast Schizosaceharomyces pombe. To determine the sedimentation coefficient of the native, undenatured H +-ATPase lipoprotein particle, solubilized plasma-membranes were analyzed by sucrose gradient centrifugation. Triton-washed plasma membranes were solubilized with lysolecithin in the usual manner but in a buffer containing 8% sucrose (w/w) instead of glycerol. The solubilized extract was supplemented with 30 ~g catalase per 0.5 ml extract, and 0.5-ml portions of the extract were applied to 10-27% (w/w) sucrose gradients. Centrifugation was carried out at 5~ C in an SW 55Ti swinging-bucket rotor using tubes, 51 mm long, 13 mm diameter, at 35000 rpm for variable time periods. Fractions of 0.5 ml were collected from the bottom of the gradient, and sucrose concentrations measured with a refractometer. Gradient fractions were analyzed for ATPase activity as previously described, and for catalase activity according to instructions provided by the supplier (Sigma Chemical Company, St. Louis, Mo., USA). Sedimentation coefficients were estimated as in Dufour and Goffeau (1980), using the computation model of MeEwen (1967) and the relative molecular mass (Mr) esti-

G.E. Schaller and M.R. Sussman: Phospharylation of plasma-membrane H §

511

mated using the equations of Tanford et al. (1974). For these calculations the partial specific volume of lipid in the oat H +ATPase particle was assumed to be similar to that observed with the yeast H +-ATpase.

Reverse-phase high-performance liquid chromatography (RPHPLC). A Varian Model 5000 High Pressure Liquid Chromatograph (Varian Instrument Group, Sugar Land, Tex., USA) equipped with a Hewlett Packard Model 1040A Diode Array Spectrophotometric Detector (Hewlett-Packard, Palo Alto, Cal., USA) was used to separate and analyze membrane components. In a typical experiment, 1 mg of plasma-membrane vesicles was collected by centrifugation, and the pellet solubilized in 100 lal of anhydrous trifluoroacetic acid at room temperature immediately prior to injection. A 30-rain linear gradient, from 60 to 80% (v/v) ethanol containing 0.1% trifluoroacetic acid, was run at a constant flow rate of 1 ml-min-1. A Vydac C-4 5-gin reverse-phase stainless-steel column (250 mm long, 46 mm diameter; The Separations Group, Hesperia, Cal., USA) was used for the separation. Fractions (2 ml) were collected and analyzed for protein by the method of Lowry (1951) and by SDS-PAGE. Maximum recovery of hydrophobic proteins was achieved when stop solution containing SDS, bromphenol blue and glycerol was added to fractions before evaporation of ethanol, water and trifluoroacetic acid. Prior to electrophoresis, 5 gl of 2 M Tris was added to raise the pH of samples. Phosphoamino-acid analysis. Gels (0.75 mm thick) stained with Coomassie blue (Sigma) were rinsed overnight at room temperature in a large volume of distilled water. Visible bands were cut from the gels and homogenized in a small volume of 50 mM ammonium bicarbonate (pH 8.0) using a glass-teflon motordriven homogenizer. Peptides labeled with 3zp were released from gel particles by overnight incubation at 37 ~ C with trypsin (I0 gg.m1-1) in the presence of 0.1 mM vanadate to inhibit phosphatases that may be present in the trypsin preparation, and of 0.1 mM calcium chloride to inhibit trypsin self-digestion. This procedure eluted more than 90% of the radioactivity from gel slices, as judged by liquid scintillation counting. The eluted samples were then evaporated to dryness and resuspended in 500 gl of 6 N HCI. Following 2 h at 110~ C in vacuo, the solution was again evaporated to dryness and resuspended in 5 2 5 gl of high-voltage electrophoresis buffer (945 ml H20, 50 ml glacial acetic acid, 5 ml pyridine) containing 5 gg of each phosphoamino-acid standard (phosphotyrosine, phosphothreonine and phosphoserine). Samples were then subjected to thin-layer electrophoresis on 20-cm-long plastic-backed cellulose sheets (Eastman Kodak) for 2 h at 500 V, followed by ninhydrin (0.5% in acetone) visualization of standards, and autoradiography (McDonough and Mahler 1982). Calcium measurements. The amount of calcium bound to the membranes and present in the reaction solutions was determined with a Varian SpectrAA-20 Atomic Absorption Spectrophotometer (Varian Instrument Group). Sample preparation procedures were as described in a technical bulletin provided with the instrument (" Analytical Methods for Tissue Spectroscopy", Publication No. 85-10009-00, Varian Techtron Printing, Springvale, Australia). Lipid analysis. Phosphorylated plasma-membrane lipids were extracted and analyzed by thin-layer chromotography according to Varsanyi et al. (1983). Chemicals. ATP was purchased from Boehringer-Mannheim Biochemicals, Indianapolis, Ind., USA; RP-HPLC solvent from Burdick and Jackson Laboratories, Muskegon, Mich., USA;

Fig. 1. Phosphorylated polypeptides from oat, potato and yeast plasma-membrane vesicles analyzed by SDS-PAGE. Lanes 1 3 , 7, 9 and 11 are autoradiograms while lanes 6, 8 and 10 are Coomassie-stained profiles. Oat vesicles were incubated with [y-32P]ATP for 5 rain in the absence (lanes 1, 2, 3, 5) or presence (lane 4) of 0.02 M non-radioactive ATP. In lane 3, 0.05 M hydroxylamine was added after the reactions was terminated. In lane 5, 0.02 M non-radioactive ATP was added before the reaction was terminated. Kinase reactions were terminated with 20 mM N a 2 E D T A . Lane 2 is a blank in which EDTA was added prior to commencement of the reaction. Lane 6 is a typical Coomassie-stained polypeptide profile of the oat plasma-membrane vesicles used in this study. Lanes 7 and 9 show a similar 5-min in-vitro kinase reaction performed with potato (lane 7) and yeast (lane 9) vesicles. Lanes 8 and 10 show a typical Coomassie-stained polypeptide profile of the potato and yeast vesicles, respectively. Lane 11 is an autoradiogram of oat plasma-membrane vesicles labeled in-vivo with 32p-inorganic phosphate. Arrows denote positions of Mr=100000 (100K); 50000 (50K); 18000 (18K); 15000 (15K); 10000 (10K); and 3 000 (3K) phosphoproteins, as determined by comparison with M r standards and reagents used in SDS-PAGE from Bio-Rad Laboratories, Richmond, Cal., USA. All other routine chemicals and catalase (Catalogue No. C-10) were purchased from Sigma.

Results

Kinase activities associated with plant and fungal plasma membranes. When plasma-membrane vesicles were isolated from root cells of oat, incubated at 20 ~ C for 30 rain with [~-32p]ATP and analyzed by autoradiography following SDS-PAGE, radioactivity was found associated with several polypeptides. The most heavily labeled proteins had the following molecular masses : 100000; 50000 (broad); 18 000; 15 000; 10 000; and 3 000 (Fig. 1, lane 1). A reaction blank, in which membranes were treated with excess E D T A (Fig. 1, lane 2) prior to ATP addition, showed n o 32p incorporation. Incorporation was also prevented by the addition of a large excess of nonradioactive ATP prior to starting the reaction (Fig. i, lane 4). There was no change in incorporation, however, when excess nonradioactive ATP (Fig. 1, lane 3) was

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G.E. Schallerand M.R. Sussman:Phospharylationof plasma-membraneH+-ATPase

added just before the reaction was terminated. Lack of a decrease by a nonradioactive ATP chase and by hydroxylamine rule out contributions from phosphorylated intermediates and indicate instead that the observed incorporation is mediated by protein-kinase activity associated with the plasma membrane. With oat root vesicles, the extent of phosphate incorporation into polypeptides was found to be independent of their relative abundance in the membrane, as judged by general Coomassie stain (Fig. 1, lane 6). Similar kinase activity was also observed in plasma-membrane vesicles isolated from potato (Fig. 1, lane 7, autoradiogram, and lane 8, Coomassie stain) and from the yeast Saccharomyces cerevisiae (Fig. 1, lane 9, autoradiogram, and lane 10, Coomassie stain). Although there were differences in the number and Mr of phosphorylated peptides, all three species displayed a prominent phosphorylated polypeptide band at Mr = 100000. Results qualitatively similar to those obtained with these in-vitro experiments were obtained when plasma-membrane vesicles were labeled in vivo. When oat roots were excised and labeled with [32p]phosphate in distilled water for 3 h prior to homogenization, phosphorylated polypeptides at Mr=100000; 50000; 18000; 15000; 10000 and 3000 were observed (Fig. 1, lane 11). The relative levels of incorporation in the in-vitro and in-vivo labelilng experiments were not quantitatively identical. In particular, the intensity of the M r = 100 000 polypeptide was reduced in-vivo compared to the other phosphoproteins.

Purification of the phosphorylated A TPase. At present, a proton pump (H +-ATPase) is the only enzyme from plant plasma membranes which has been purified to homogeneity. The procedure is similar to that utilized in the purification of the cation-translocating ATPases of animal plasma membranes and relies on a final density-gradient centrifugation to separate the native, nondenatured ATPase lipoprotein complex. The purified enzyme contains a single Mr = 100000 polypeptide, as judged by Coomassie-stained SDS-PAGE (Serrano 1984; Anthon and Spanswick 1986). In order to determine rigorously whether the Mr = 100 000 phosphoprotein observed in oat-root vesicle preparations is this same enzyme, we purified the ATPase from in-vitro-labeled membranes by two different procedures. In the first procedure, the ATPase was purified by detergent solubilization followed by conventional glycerol density-gradient centrifugation (Serrano 1984). In this proce-

dure the ATPase emerges as a "solubilized" enzyme in a unique particle of high Mr, containing both protein and lipid, and possibly representing the functional unit of the ATPase. The success of purification relies on the fact that the ATPase aggregate migrates at a rate different from that of other solubilized proteins. Oat plasma-membrane vesicles were first washed with a low concentration of Triton X-100 to remove loosely bound protein without releasing the ATPase. The ATPase was then solubilized with a high conenctration of lysolecithin and resolved from other proteins by centrifugation through a 25-55% (w/v) glycerol gradient. Using this procedure with in-vitro 32p-labeled vesicles, we observed that the 32p remained associated with the Coomassie-stained Mr= 100 000 ATPase catalytic subunit as it co-purified with ATPase activity (Fig. 2, fraction 6). In contrast, radioactive Mr = 15 000 and Mr = 18 000 phosphoproteins migrated slower than the ATPase during centrifugation (Fig. 2, fractions 4, 5). Radioactivity associated with the Mr=50000 phosphoprotein remained present as a weak diffuse band in fractions 4 through 6, while the prominent Mr = 10 000 radioactive band pelleted through the gradient and was present mostly in fraction 13. As mentioned, the success of this purification technique relies on the observation that the native, solubilized enzyme consists of a high-Mr aggregate of protein and lipid. Several results confirm that the separation of plasma-membrane proteins by density-gradient centrifugation is different from size-fractionation such as that observed with SDSPAGE. Examination of the gradient fractions by SDS-PAGE shows that mobility on the gradient is independent of the molecular weight of the denatured polypeptide. For example, in Fig. 2A it can be seen that polypeptides with Mrs of 80000; 65 000; and 25 000 all have greater mobility on the glycerol density gradient than does the M r = 100000 polypeptide. We have also determined the sedimentation coefficient for the solubilized ATPase and from this calculated a molecular weight for the active particle. The rate of sedimentation of the solubilized ATPase through a sucrose density gradient was estimated from the computation model of McEwen (1967), in a manner previously applied to the solubilized yeast tt+-ATPase (Dufour and Goffeau 1980). Assuming that the oat H +-ATpase has a similar lipid composition to the yeast H+-ATPase, yielding a density of 1.28 g. cm-3 for the ATPase-lipid complex, we calculate S2o,w= 10.9 S for the solubilized ATPase. The ATPase migrated very similarly to catalase, a soluble globular protein with a known density of 1.37 g.

G.E. Schaller and M.R. Sussman: Phospharylation of plasma-membrane H +-ATPase

Fig. 2A-C. Separation of lysolecithin-solubilized oat plasmamembrane phosphoproteins by glycerol density-gradient centrifugation. Following a 15-min in-vitro kinase reaction with [y_32P]ATP, oat vesicles (i rag) were washed with Triton X-100, solubilized with lysolecithin, and applied to a 5-ml 25% (w/v) to 55% (w/v) glycerol density gradient (Serrano 1984). Following centrifugation, 400-gl fractions were collected. A shows a Coomassie-stained SDS-PAGE analysis using 200 gl of the fractions obtained from the centrifuged gradient (fraction 1 is top of the gradient, while fraction 13 is the gradient pellet); B shows an autoradiogram of the same gel as in A; C shows results of ATPase assays performed with aliquots (100 I~1)from the same gradient displayed in the upper figure. 100% ATPase activity represents 0.21 gmol Pi released.rain-1.fraction 1

cm -3 and sedimentation coefficient of 11.4 S (Martin and Ames 1961). Although catalase has an Mr of 250000, the M~ of the ATPase particle is expected to be substantially more than that of catalase. This is because the ATPase particle contains lipid, lowering the particle density, and because membrane proteins have a higher frictional coefficient in aqueous solutions than soluble globular proteins such as catalase. Using a density of

513

1.28 g" cm-3 and a frictional ratio fifo = 1.5, we calculate an Mr for the ATPase-lipid complex of 447000, and an Mr for the ATPase protein of 309 000. These results indicate that the solubilized ATPase is an aggregate of three Mr= 100000 polypeptides. Supporting this value, cross-linking experiments indicate that the solublized ATPase exists as a trimer of catalytic subunits (Anthon and Spanswick 1986) and radiation inactivation experiments indicate that the minimum functional unit of the membrane-associated enzyme is at least a dimer (Briskin et al. 1985). In a second procedure, we purified the H +ATPase from in-vitro-labeled membranes by RPHPLC. Plasma-membrane vesicles were treated with anhydrous trifluoroacetic acid immediately prior to injection, and protein and lipid components were resolved using a linear 60-80% (v/v) ethanol gradient containing 0.1% trifluoroacetic acid throughout. This system is based on that employed by Gerber et al. (1979) for resolving peptide fragments of bacteriorhodopsin, another very hydrophobic integral membrane protein. By using a brief treatment in anhydrous trifluoroacetic to solubilize completely the lipid and protein components, we were able to inject crude membrane vesicles directly onto the reverse-phase column. A high starting concentration of ethanol (60% v/v) was required to prevent aggregation and ensure high yields of the eluted ATPase. We observed that greater than 90% of the total plasma-membrane protein eluted rapidly, before the ethanol concentration reached 63% (v/v) (Fig. 3). In contrast, the Mr=100000 polypeptide did not elute until the ethanol concentration reached 69% (v/v). The Mr= 100000 polypeptide was present as a phosphoprotein and was identified as the ATPase by its extreme hydrophobicity and by comparison with the chromatographic mobility of the purified enzyme. The M~= 100000 polypeptide of the oat plasma membrane had a mobility identical to that of the detergent-purified enzyme of oat, and similar to that of the purified H +-ATPase of N e u r o s p o ra crassa and the purified Na+,K+-ATPase of pig kidney (results not shown). It is interesting to observe that the major phosphorylated product resulting from incubation of [y-32p]ATP with oat plasma-membrane vesicles elutes as a single peak at 65% (v/v) ethanol (Fig. 3). This material migrates on SDS-PAGE at Mr = 3 000 and is identical to the low-molecularweight phosphorylated species observed earlier in both in-vitro and in-vivo labeling experiments with oat vesicles. Chromatographic studies with authentic standard lipids using RP-HPLC as described

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G.E. Schaller and M.R. Sussman: Phospharylation of plasma-membrane H §

Attempts to use two-dimensional gel electrophoresis to determine whether the phosphorylated polypeptide and the ATPase are identical have not been successful. We were unable to obtain conditions in which the very hydrophobic ATPase would focus as a sharp band, rather than a smear. Other investigators have experienced similar difficulties (Vai et al. 1986). However, since the phosphorylated polypeptide co-chromatographs with the ATPase polypeptide on glycerol gradient centrifugation and RP-HPLC, it is reasonable to conclude that the ATPase is a phosphoprotein.

Fig. 3A-C. Separation of plasma-membrane protein and lipids by reverse-phase high-pressure liquid chromatography. A 1-mg sample of oat plasma-membrane vesicles was phosphorylated in vitro for 30 min with [7-32p]ATP using standard conditions (see Material and methods), centrifuged in a microfuge, and washed to discard unincorporated radioactivity. Membrane pellets were then dissolved in 100 gl of anhydrous trifluoroacetic acid and immediately injected onto a Vydac C-4 RP-HPLC column. The flow rate was 1 ml-min -~ and a 30-rain linear gradient of ethanol containing 0.1% trifluoroacetic acid was used to resolve polypeptides. A is a Coomassie-stained SDSPAGE analysis using 1.5 of the 2.0 ml fractions obtained from the HPLC; B is an autoradiogram obtained from a separate SDS-PAGE analysis of an HPLC run performed under conditions similar to A; and C is the protein content (bar graph), measured according to Lowry (1951), of the 0.5 ml remaining from fractions obtained in B. C also depicts the ethanol gradient used for protein elution

above, and thin-layer chromatography according to Varsanyi et al. (1983), indicate that this lowmolecular-weight phosphorylated species is mainly phosphatidic acid, the product of [y-g2P]ATP and diglyceride, as catalyzed by an endogenous diglyceride kinase.

Phosphoamino-acid analysis. In order to identify the amino acids phosphorylated in the ATPase, a large-scale in-vitro labeling experiment for 30 min at 20~ was performed. Following SDSPAGE, the gel was Coomassie-stained and subjected to autoradiography to locate the radioactive polypeptides. Gel slices containing the prominent Mr=100000 and Mr=18000 phosphoproteins were then homogenized and treated overnight with trypsin, a procedure that eluted more than 95% of the 32p from gel particles. Following a 2-h digestion at 110 ~ C in 6 N HC1, the eluted radioactivity was subjected to thin-layer electrophoresis according to McDonough and Mahler (I982). As shown in Fig. 4, we observed only phosphoserine in the Mr=18000 phosphoprotein, but both phosphothreonine and phosphoserine, in approximately equal amounts, in the Mr= 100000 phosphoprotein. In addition to the radioactive amino acids, a radioactive spot co-chromatographing with inorganic phosphate was observed. Radioactive phosphotyrosine was not detected in either of the two samples. Effect of calcium and p H on kinase activities associated with plasma-membrane vesicles of A. sativa and S. cerevisiae. Calcium ions stimulated the protein-kinase activity associated with oat-root plasma membranes (Figs. 5, 6) but had no effect on that associated with yeast (Fig. 7). Endogenous calcium found in the buffer and associated with membranes was chelated with EGTA (1.5 raM). Varying quantities of CaCI2" 6 HzO were then added to give different concentrations of "free" calcium, calculated according to Pershadsingh and McDonald (1980). At 1.5 mM EGTA, maximal calcium stimulation was observed with 0.79 mM added calcium. Direct measurements of calcium in the buffers and bound to membranes, by atomic absorption spectrophotometry, demonstrated that endogenous calcium contributed 12.8 I~M calcium. Taking this value into account, and using the equi-

G.E. Schaller and M.R. Sussman: Phospharylation of plasma-membrane H +-ATPase

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FREE [Co 2+] (#M) Fig. 5. Effect of free-Ca2+ concentration on 32p incorporation from [y-32p]ATP into the Mr = 100000 polypeptide of oat plasma-membrane vesicles. Oat plasma membranes were incubated at pH 6.2 for 2 rain at 20~ C, in the presence of 1.5 mM EGTANa2 and varying amounts of CaCI2 to give the free-calcium concentrations shown. Incorporation of 32p into the Mr= 100000 polypeptide was quantitated by scanning an autoradiogram of an SDS polyacrylamide gel with a laser densitometer. Incorporation is expressed as percent of the maximum observed

Fig. 4. Phosphoamino-acid analysis of oat plasma-membrane

phosphoproteins using thin-layer electrophoresis. Lanes 1 and 2 represent autoradiograms of the hydrolysate products from Mr= 18000 (lane 1) and Mr=100000 (lane 2) phosphoprotein separated by SDS-PAGE. Arrows denote positions of inorganic phosphate (P0, phosphoserine (P-ser), phosphothreonine (Pthr), and phosphotyrosine (P-tyr), as determined by standards librium c o n s t a n t for E G T A - C a 2+ binding rep o r t e d by Pershadsingh a n d M c D o n a l d (1980), we calculated that in the presence o f 1.5 m M E G T A , maximal stimulation occurred at less than 7 p M free calcium (Fig. 5). (This calculation was m a d e using an a p p a r e n t association c o n s t a n t at p H 6.2 o f 10szs.) Stimulation was readily a p p a r e n t at less than 1 g M free calcium. The effect o f p H u p o n kinase-mediated phosp h o r y l a t i o n was also examined. The stimulatory effect o f calcium o n p h o s p h o r y l a t i o n o f the Mr = 100000 A T P a s e and on other polypeptides in oatr o o t p l a s m a m e m b r a n e s was observed at all p H values examined, a b o v e p H 4.7 (Fig. 6A, B). In contrast, i n c o r p o r a t i o n o f 32p into the Mr = 3000 lipid species was unaffected by the presence o f calcium. P h o s p h o r y l a t i o n o f the A T P a s e polypeptide showed a m a r k e d p H - d e p e n d e n c e : at p H below 5 or a b o v e 7.5, there was little 3Zp i n c o r p o r a t i o n . I n c o r p o r a t i o n o f 32p into other polypeptides was

Fig. 6A, B. Effect of pH on 32p incorporation from [y-32p]ATP into polypeptides of oat plasma-membrane vesicles. An autoradiogram of a 9% acrylamide SDS-PAGE is shown. Lanes 1 4 in A show incorporation in the absence of calcium at, in this order, pH 4.7, 5.8, 6.7 and 7.8. Lanes 1-8 in B show incorporation in the presence of 2.4 mM added calcium at pHs 4.7, 5.2, 5.8, 6.2, 6.7, 7.2, 7.8, 8.4. All reactions were for 2 rain at 20~ C in the presence of 1.5 mM EGTA-Naz

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15

MINUTES Fig. 8. Time course of 32p incorporation into the Mr= 100000 phosphoprotein of oat (circles) and yeast (triangles) plasmamembrane vesicles in the absence (open symbols) and presence (closed symbols) of 30 NM added free calcium. Incorporation is expressed as a percent of the maximum observed with each species, and was determined by measuring the area under peaks when autoradiograms were scanned with an optical densitometer

Fig. 7A, B. Effect o f p H on 32p incorporation from [7-32P]ATP into polypeptides of yeast plasma-membrane vesicles. An autoradiogram of a 9% acrylamide SDS-PAGE is shown. Lanes 1-7 in A show, in this order, incorporation in the absence of calcium at pH 4.7, 5.2, 5.8, 6.2, 6.7, 7.8, 8.4. All reactions were for 2 rain at 20~ in the presence of 1.5 mM EGTA. In B, an identical kinase reaction was performed at pH 6.2 in the absence (lane 1) or presence (lane 2) of 30 laM added free calcium

reduced below pH 5, but did not show such a decline at the higher pHs. These experiments were performed in the presence of a fixed amount of total calcium in excess of the EGTA concentration, since the apparent EGTA-Ca 2 + association constant is pH-dependent. A calcium concentration of 2.37mM and an E G T A concentration of 1.5 m M were used. Under these conditions the free-calcium concentration only varied from 0.93 mM to 0.87 m M between pH 5,8 and pH 8.4, the region where the ATPase exhibited a unique pH dependence. The strong pH dependence for phosphorylation of the ATPase was also observed when lower Ca 2 + concentrations were tested. At lower pH, 32p incorporation into yeast plasma-membrane polypeptides resembled that observed with oats. However, in contrast to the results with the higher plant, the reduction at alkaline pH was much less marked (Fig. 7 A). As shown in Fig. 8, the time course for incorporation of 32p into the M r = 100000 ATPase polypeptide of oat-root plasma membranes was rapid for the first 5 rain and then leveled off. Calcium

stimulated the steady-state incorporation approximately threefold. Quantitation of the calcium effect on phosphorylation of oat-root plasma-membrane polypeptides other than the M r = t 0 0 0 0 0 ATPase invariably showed a tenfold or greater stimulation (results not shown). The time course for incorporation of 32p into the Mr = 100000 ATPase polypeptide of yeast plasma membranes was unaffected by EGTA, and resembled that observed with oat-root plasma membranes in the presence of calcium (results not shown). Added calcium gave no stimulation (Fig. 7B), and at higher concentrations a slight decrease was observed. Discussion

Our results demonstrate that the plasma-membrane proton-pump of oat-root cells is phosphorylated in-vitro by a calcium-stimulated protein kinase. Previous experiments by others have demonstrated the existence of calcium-stimulated proteinkinase activity in the microsomal fraction of plant cells (Hetherington and Trewavas 1982; Salimath and Matin6 1983) but, to our knowledge, this is the first report to identify a specific plasma-membrane enzyme, the H+-ATPase, as a major substrate for the kinase. Other investigators have probably not noted a strongly phosphorylated Mr = 100 000 protein band on SDS-PAGE for three reasons. First, the ATPase is particularly susceptible to proteolysis and, unless great care is taken with the plasma-membrane isolation, is likely to be degraded; our plasma-membrane isolation procedure was designed to preserve ATPase activity.

G.E. Schaller and M.R. Sussman : Phospharylation of plasma-membrane H +-ATPase

Second, the ATPase will aggregate into a highmolecular-weight component that does not enter gels if the standard procedure of boiling samples prior to SDS-PAGE is utilized (Gallagher and Leonard 1987). Third, previous investigations of membrane-associated kinase activity were conducted at pH 7 or higher; we found that phosphorylation of the ATPase was highly pH-dependent, and at those pH values the ATPase would not be strongly phosphorylated. Calcium stimulated phosphorylation of the ATPase and other membrane proteins. Stimulation was readily apparent at less than I gM free calcium and reached a maximum by 7 gM free calcium. These values are consistant with stimulation based upon a calcium-calmodulin-regulated protein kinase (Marme and Dieter 1983) and are close to the level actually found in the cytoplasm of plant cells (Williamson and Ashley 1982). Phosphorylation of the ATPase was stimulated threefold by calcium, while other polypeptides showed at least a tenfold stimulation. Phosphorylation of the ATPase showed a unique pH dependence. Phosphorylation of the ATPase declined at pH 6.7 and above, a region where the phosphorylation of other plasma-membrane proteins did not decrease substantially. One interpretation of these results is that there exist multiple protein kinases, with differing sensitivities to pH and calcium concentration and differing affinities for the ATPase versus other membrane polypeptide substrates. An alternative explanation is that only one protein kinase exists, and pH or calcium are specifically altering the ATPase conformation, rendering the polypeptide less susceptible to kinase-mediated modificiation. Regardless of the interpretation, these data indicate that changes in the cytoplasmic pH and calcium concentration may operate in vivo to alter the phosphorylation of the ATPase. A preliminary study indicated that the M r = 100000 ATPase is also phosphorylated in vivo, strengthening the likelihood that our in-vitro investigations are physiologically relevant. A quantitative difference was noted, however, between the in-vivo and in-vitro phosphorylations. The ATPase was tess strongly labeled in vivo than under our standard in-vitro reaction conditions. This quantitative difference is probably a consequence of the previously discussed effect of pH on the phosphorylation of the ATPase. The cytoplasmic pH of plant cells is generally in the range of 7.1 to 7.3 (Brummer et aI. 1985; Roberts et al. J981), a region where we also found the ATPase to be poorly phosphorylated in relation to other membrane proteins when examined in-vitro (compare lane 11 in Fig. 1 with lanes 4 and 6 in Fig. 6).

517

We compared the phosphorylation of the oat plasma-membrane H § to that of the yeast plasma-membrane H+-ATPase. Like the higherplant ATPase, the yeast plasma-membrane ATPase is phosphorylated by a membrane-associated protein kinase (McDonough and Mahler 1982; Portillo and Mazon 1985; Yanagita etal. 1987). We observed two major differences in the kinasemediated phosphorylation of the respective ATPases: in higher plants the phosphorylation is stimulated by calcium and substantially inhibited at alkaline pH, while in exponentially growing yeast cells the phosphorylation is unaffected by calcium and not strongly inhibited at alkaline pH. Kinase-mediated phosphorylation of the oat plasma-membrane H+-ATPase is a means by which the activity of the ATPase could be regulated, and could mediate the effects of fusicoccin, blue light, turgor, and syringomycin upon ATPase activity. There are conflicting reports concerning how phosphorylation affects the ATPase. In experiments with an impure microsomal fraction isolated from corn roots, Zocchi (1985) reported that calcium-stimulated phosphorylation inhibited a H§ that presumably resides in the plasma membrane. In contrast, Bidwai et al. (1987) reported that syringomycin, a peptide toxin, stimulates the H § of the red-beet plasma membrane and this correlates with a syringomycin-induced stimulation of plasma-membrane protein-kinase activity (Bidwai and Takamoto 1987). Our phosphoamino-acid analysis indicates that there are multiple sites of phosphorylation of the ATPase in the in-vitro kinase reaction. Complex patterns of phosphorylation have been found with a number of proteins; glycogen synthase, for example, is regulated by five protein kinases which phosphorylate seven separate sites (Picton et al. 1982). In light of these observations, further characterization of the location of target sites in the ATPase and the effect that each site has on catalytic activity should provide approaches for better defining the role of proton-pump phosphorylation in plant growth.

We wish to acknowledge the assistance of Michael R. Culbertson for providing the rho - strain of Saccharomyces cerevisiae, Jon Behling for assistance in performing calcium measurements and Donald B. Katz and Terry K. Surowy for a critical reading of the manuscript. This study was supported by grants to M.R.S. from the Department of Energy (No. DE-AC0283ER13086), the U.S. Department of Agriculture (No. 87-CRCR-I-2357), the College of Agriculture and Life Sciences and the Graduate School of the University of Wisconsin, and by an N.I.H. Traineeship Fellowship awarded to G.E.S. and administered by the University of Wisconsin Cell and Molecular Biology Program.

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G.E. Schaller and M.R. Sussman: Phospharylation of plasma-membrane H +-ATPase

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