Subunit composition and Ca2+-ATPase activity of the vacuolar ATPase from barley roots

ARCHIVES

Vol.

OF BIOCHEMISTRY

294, No. 2, May

AND

BIOPHYSICS

1, pp. 341-346,1992

Subunit Composition and Ca*+-ATPase Activity of the Vacuolar ATPase from Barley Roots Frances M. DuPont1 and Peter J. Morrissey U.S. Department of Agriculture, Agricultural Research Service, Western Regional Research Center, 800 Buchunun Street, Albany, California 94710

Received

July

26,1991,

and in revised

form

December

6, 1991

The vacuolar ATPase was purified from a tonoplastenriched membrane fraction from barley (Hordeurn vulgare cv CM72) roots. The membranes were solubilized with Triton X- 100 and the membrane proteins were separated by chromatography on Sephacryl S-400 followed by fast protein liquid chromatography on a Mono-Q column. The purified vacuolar ATPase was inhibited up to 90% by KN09 or 80% by dicyclohexylcarbodiimide (DCCI). The ATPase was resolved into polypeptides of 116,68,63,46,42,34,32, 17,13, and 12 kDa. An additional purification step of centrifugation on a glycerol gradient did not result in loss of any polypeptide bands or increased specific activity of the ATPase. Antibodies against the purifled holoenzyme inhibited proton transport by the native ATPase. Two peaks of solubilized Ca”ATPase were obtained from the Sephacryl S-400 column. A peak of Caa+-ATPase copurified with the vacuolar ATPase during all of the purification steps and was inhibited by NO; and DCCI. It is proposed that this Ca2’ATPase is a partial reaction of the plant vacuolar ATPase. The second Caa+-ATPase was greatly retarded on the Sephacryl S-400 column and eluted after the main protein peak. It was not inhibited significantly by NO; or DCCI. The second Ca2+-ATPase is a major component of ATP hydrolysis by the native membranes. o 1ee2~~~,demio

Plws,Inc.

The vacuolar ATPases, or V-type ATPases (l), are proton pumping ATPases that acidify endomembrane compartments of eukaryote cells. V-type ATPases have been purified from plants (l-3), fungi (4), and animals (5-9). The V-type ATPase provides the driving force for accumulation of ions, sugars, and amino acids in the plant vacuole. It may play a role in salt tolerance since it provides the energy for sequestering Na+ and Cl- in the vac1 To whom ooo3-9861/92

correspondence

should

be addressed.

$3.00

Copyright 0 1992 hy Academic Press, Inc. AU rights of reproduction in any form reserved.

uole (10-14). As part of an ongoing project to discover the mechanisms that underlie salt tolerance, we purified the V-type ATPase from a relatively salt-tolerant variety of barley. Purification was a necessary step in defining the characteristics of the V-type ATPase from barley roots because contamination by other ATPases interfered with measurement of the V-type ATPase in the native membranes. In particular, there was a high rate of Ca2+-stimulated ATPase. In this paper we describe the purification and subunit composition of the vacuolar ATPase from barley roots. The Ca2+-stimulated ATPase that was associated with the vacuolar membrane fraction was separated into two activities, one of which copurified with the vacuolar ATPase. MATERIALS

AND METHODS

Plant material. Barley (Ho&urn vulgare cv CM72) seeds were germinated on cheesecloth laid on stainless steel grids that were suspended over plastic trays. Each tray contained 7 liters of aerated nutrient medium (15). Trays were kept in the dark at 22°C. The pH of the nutrient medium was tested daily after the seeds germinated and adjusted to pH 5.6 with Ca(OH)2. Roots were harvested after 7 days. Typical yields were 70 to 100 g fresh weight of roots per tray, starting with 80 g fresh weight of dry seeds.

Membrcmepre~n. A tonoplast-enriched membrane fraction was prepared (16) with the following modifications. The homogenization buffer contained 0.2 or 5 mu PMSF? Roots (SO g) were ground in 80 ml of homogenization buffer, the homogenate was poured off and filtered through cheesecloth, fresh buffer was added to the roots, and the procedure was repeated four times. The volume of homogenate was adjusted to 400 ml. The tonoplast-enriched membranes were collected by differential centrifugation followed by discontinuous gradient centrifugation

’ Abbreviations used: BHT, hutylated hydroxyltoluene; DCCI, dicyclohexylcarbodiimide; DTT, dithiothreitol; ER, endoplasmic reticulum; FPLC, fast protein liquid chromatography; Mea, I-morpholineethanesulfonic acid; CI,EIO, polyoxyethylene 10 tridecyl ether; PAGE, polyacrylamide gel electrophoresis; Pipes, 1,4-piperazinediethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate, TCA, trichloroacetic acid; Tris, tris(hydroxymethyl)aminomethane. 341

342

DUPONT

AND

(16). They were added to 60 ml of 0.25 M sorbitol and 2 mM D’IT in 5 mM Pipes KOH, pH 7.2, and centrifuged in a Beckman 45Ti rotor3 for 35 min at 35,000 rpm. The pellet was resuspended in approximately 1 ml of the same buffer, or in 0.25 M sucrose, 2 mM DTT, and 5 mM Pipes KOH, pH 7.2, and frozen at -70°C until use. Solubtiization and purification of the V-ATPase. The vacuolar ATPase was purified by the method of Parry et al. (2) with the following modifications. Membranes were mixed with Triton X-100 solubilization buffer on ice to give a final concentration of 5 mM Tris-Mea, pH 8.0, 10% glycerol, 5 mM DTT, 1 mM Tris-EDTA, 4 mu MgClz, 0.4 mg/ml Type IV-S phosphatidylcholine from Sigma, and 12.5 mM BHT, and a ratio of 25 mg Triton X-100 to 1 mg membrane protein. Membranes were stirred for 5 min and then allowed to stand on ice for 25 min. The solubilized membrane proteins were separated by chromatography on a Sephacryl S-400 column at 4°C. The column buffer included 0.3% Triton X-100. The fractions were assayed for ATPase activity and the fractions from the peak of NOi-inhibited ATPaae were pooled, as were the fractions from a separate peak of Ca*+-stimulated ATPase. The NO;-inhibited ATPase was further purified by FPLC on an HR 5/5 Mono-Q column (Pharmacia). The column buffer was almost identical to that used by Parry et al. (2) and included the detergent C,sE,s at 0.1% and 0.1 mg/ml phosphatidylcholine. The sample was eluted with a salt gradient of O-l M KCl. Glycerolgradients. The peak of NOT-inhibited ATPase from FPLC was layered onto a gradient consisting of 12 ml of 10 to 40% or 20 to 40% (w/w) glycerol plus 5 mM DTI’, 1 mM Tris-EDTA, 2 mM MgClz, 0.4 mg/ml phosphatidylcholine, 0.1% C&iO, and 20 mM Tris-Mes, pH 8.0. The gradient was centrifuged at 24,000 rpm for 30 h in a Beckman SW28 rotor at 2°C.

MORRISSEY

RESULTS Purification

of the V-Type ATPase

The ATPase activity of the tonoplast-enriched fraction from barley roots was stimulated almost as much by Ca*’ as by Mg*+ and was inhibited less than 50% by NO;. In order to proceed with characterization of the ATPase, it was necessary to purify it and separate it from contamination by other ATP-hydrolyzing enzymes. The ATPase activity was resolved into four peaks by chromatography on a Sephacryl S-400 column after solubilization with Triton X-100 (Fig. 1). The ATPase in the void volume (peak 1) was partially inhibited by NO; and was stimulated slightly more by Ca*’ than by Mg*+. The void volume probably contained incompletely solubilized protein. The ATPase in fractions 28 to 32 (peak 2) was greatly inhibited by NO; and was stimulated somewhat more by Mg*+ than by Ca*+ (Fig. 1). The small peak of ATPase that was associated with the main protein peak (peak 3) was stimulated by NO; . . The ATPase which eluted after the main protein peak (peak 4) was stimulated much more by Ca*+ than by Mg*+ and was slightly inhibited by NO; (Fig. 1, peak 4). The pyrophosphatase activity of the tonoplast

ATPase assays. ATPase was assayed with a reaction mix consisting of 10 mg/ml Type IV-S phosphatidylcholine, 50 mM choline Cl, 1 mM MgClz, 1 mM Tris-ATP, and 30 mM Tris-Mes, pH 8.0. CaCl, was substituted for MgCls to assay for Ca’+-stimulated ATPase. ATPase was assayed for 20 min at 37’C, and Pi was determined by a modification of the method of LeBel et al. (17). Proton transport was assayed as previously described (18). Protein assays. Proteins were assayed using a naphthol blue black dye binding assay (19). Protein samples were dissolved in SDS, precipitated with TCA, collected and washed on 0.45-wrn Millipore filters, and stained with naphthol blue black. After destaining, the spots were cut from the filter paper and extracted with a NaOH and ethanol mix, and the Am was determined. Bovine y globulin was used as a protein standard. Protein analysis. Protein analysis was carried out by SDS-polyacrylamide gel electrophoresis. Samples were delipidated (2) and proteins were solubilized in 2% SDS, 25 mM mercaptoethanol, 10% glycerol, and 50 mM Tris-Cl, pH 6.8. The samples were heated for 5 min at 80°C. Proteins were separated on 1.5-mm-thick 12% SDS-polyacrylamide gels, with 4.5% stacking gels, prepared by the method of Laemmli (20). Gels were silver-stained by the method of Morrissey (21). Antibodies. Two batches of polyclonal antibodies against the purified ATPase (FPLC) were prepared from mouse Ascites fluid. The antibodies were precipitated with ammonium sulfate, dialyzed against phosphatebuffered saline, desalted by washing with DEAR-cellulose, concentrated by centrifugation in Centricon microconcentration tubes, and resuspended in phosphate-buffered saline. For immunoblots, the membrane samples were separated by electrophoresis on a 10% gel, blotted to Immobilon, reacted with antibody, and detected with alkaline phosphatase (22).

’ Mention of a specific product name by the U.S. Department of Agriculture does not constitute an endorsement and does not imply a recommendation over other suitable products.

0

10

20

30

40

50

0

FIG. 1. Separation of ATPase activity by Sephacryl S-400 chromatography of the tonoplast membrane fraction after solubilization with Triton X-100. Fractions were collected at 20-min intervals, with a flow rate of 3.5 to 4 ml per hour. (A) Assayed for ATPase with 30-~1 aliquots per fraction. Mg2+-ATPase was assayed with (0) or without (0) 150 mM KNOs . Ca”-ATPase (0) was assayed with 1 mM CaClz and 50 mM choline Cl. (B) Protein (A). This experiment is typical of approximately 30 column runs for preparation of NO;-inhibited ATPase or Ca*+ATPase. For routine preparations, only the region of peak 2 and/or peak 4 was assayed for ATPase.

BARLEY

--

VACUOLAR

343

ATPase TABLE

-1’

Purification

of

II

the Vacuolar ATPase

Based on Data in Table

stage of purification

Yield of NO;-inhibited ATPase (“I,)

Protein (%I

TX-100~solubilized membranes Sephacryl peak 2 FPLC peak Glycerol gradient

100 22 3 0.8”

I

Purification NO;-inhibited ATPase (-fold)

100 39 19 5

1 1.8 6.2 5.1”

of Inhibition by NO; (%)

32 70 92 91

a Approximate. -0

5

10

15 20 25 Fraction Number

30

35

40

FIG. 2. Purification of the vacuolar ATPase by FPLC on Mono Q. Sephacryl S-400 fractions enriched in NOT-inhibited ATPase (Sephacryl peak 2) were pooled and approximately 5 ml of sample, containing Triton X-100, was applied to the column with a Superloop. The column was run at 0.5 ml/min and l-ml fractions were collected. The program parameters were O-15 min, 0.00 M KC1; 15-23 min, 0.00-0.05 M KC1; 2367 min, 0.05-0.20 M KC1; 67-73 min, 0.20-1.00 M KCl. Mg’+-ATPase was assayed with 50 mM choline Cl with (A) or without (0) 150 mM KNOB. Am (-). KC1 gradient (- - -). Experiments were performed over 25 times for routine preparation of the NO,-inhibited ATPase with similar results.

fraction from barley roots was low compared to tonoplast fractions from other tissues, and only a small amount of pyrophosphatase was detected in the Sephacryl fractions. This coincided with the main protein peak (not shown). In order to further purify the vacuolar ATPase, the fractions containing the peak of NO;-inhibited ATPase from the Sephacryl column (peak 2) were pooled and

loaded onto a Mono-Q column for separation by FPLC (Fig. 2). The column was eluted without KC1 until the Triton X-100 was removed and then subjected to a gradient of increasing KCl. A peak of NOB-inhibited ATPase eluted at approximately 80 mM KCI. This was a lower concentration of KC1 than was required to elute the vacuolar ATPase from red beet, and the KC1 gradient used was more gradual than that used for red beet (2). The FPLC-purified ATPase was applied to a glycerol gradient and centrifuged for 18 or 30 h and a single peak of ATPase was collected. To evaluate the enrichment of vacuolar ATPase during the purification steps, the ATPase activity of the Triton X-100 supernatant, the two Sephacryl peaks, the FPLC peak, and the glycerol-gradient-purified ATPase were compared (Tables I and II). NOB-inhibited ATPase was calculated in order to estimate the amount of V-type ATPase in each fraction. Also, Ca’+- and Mg2+-ATPase

TABLE

Purification Total

Stage of purification TXlOO-solubilized membranes Sephacryl peak 2 Mono-Q peak Glycerol gradient peak Sephacryl peak 4’

Protein recovered bid 3.6 0.78 0.11 0.03* 0.07

ATPase

+Ca’+

+Mgx

7.5 1.0 0.2

5.7 1.1 0.4 0.1 0.7

0.1

1.2

I

of the Vacuolar recovered

(pmol

+M% f KN03

ATPase” PJmin) NO; inhibited

ATPase

+Ca’+

3.9 0.3 0.03

1.8 0.8 0.4

2.1 1.2

0.01

0.1

0.5

0.2

2.1 18.2

1.9

specific

+M$+ 1.6 1.4 3.5 3.2 11.4

activity

(pmol +Mg” + KNOB 1.1 0.4 0.3 0.3 8.2

Pi/mg/min) NO, inhibited 0.5 0.9 3.2 2.9 3.2

a Tonoplast-enriched membranes were solubilized with a ratio of 25 mg Triton X-100 to 1 mg protein and the membrane proteins were separated by Sephacryl S-400 chromatography. Peak 2 was applied to a Mono-Q column and eluted with KCl. The peak of NO;-inhibited ATPase from the Mono-Q column was layered onto a 20 to 40% (w/w) glycerol gradient, centrifuged for 30 h at 24,000 rpm, and assayed for ATPase. ATPase was assayed with 10 mg/ml phosphatidyl choline and 50 mM choline chloride, plus 1 mM Ca‘+, 1 mM Me, or 1 mM M%+, and 150 mM KNO,. NO;-inhibited ATPase is calculated as the difference in activity in the presence or absence of KNOB. The results of a representative experiment are shown. The purification by Sephacryl column chromatography followed by FPLC was repeated approximately 30 times. The glycerol gradient procedure was repeated 4 times. b Exact amount of protein was difficult to determine. ’ From a separate experiment.

344

DUPONT TABLE

Effect

Additions MgZ M%+ Mg= Ca2+ Ca2+ Ca*+

III

of Inhibitors on the Ca2+ and from the Sephacryl S400 and ATPase

+ DCCI + KNOB t DCCI + KNO 3

Sephacryl peak (Ca’+-ATPase) 1OOb 92+ 5 a7 -t 27 140 * 18 124 + 22 140 + 49

4

AND

Mg2+-ATPase Activities Mono-Q Columns

specific

activity”

Sephacryl peak (V-ATPase)

27 30 88 36 36

100’ f 19 f 14 f 22 + 35 + 28

(%) 2 FPLC of peak (V-ATPase)

2

1OOd 67 f 22 22* 7 94rt 6 68 + 24 26+ 7

’ ATPase was assayed with 10 mg/ml phosphatidylcholine and 50 mM choline chloride, plus 1 mM CaCl, or 1 mM MgClx, and with 100 nmol DCCI/mg protein or 150 mM KNOs present where indicated. Assayed at 37°C. b Data for each of three experiments were normalized and averaged, f SD. Activity in the presence of M%+ was taken to he 100%. ‘Data for each of four experiments were normalized and averaged. d Data for each of three experiments were normalized and averaged.

were compared for each fraction. There was an initial 30% increase in ATPase activity upon solubilization of the membranes with Triton X-100 (data not shown). Sephacryl S-400 chromatography followed by FPLC of Sephacryl peak 2 gave a 2-fold increase in the specific activity of Mg2+-ATPase, a 6.2-fold increase in NOB-inhibited ATPase, and no enrichment of Ca2+-ATPase (Tables I and II). In contrast, the specific activity of the Ca2’ATPase in the Sephacryl peak 4 was enriched g-fold over that in the Triton X-100 supernatant (Table I). The effects of Ca2+, Mg2+, NO;, and DCCI on the ATPase activity of the various fractions were compared in more detail (Table III). Percentage inhibition by NO, increased during the purification of the V-type ATPase, ranging from 30 to 60% in the original membranes (data not shown), 60 to 80% in the Sephacryl peak 2, and 70 to 90% in the purified vacuolar ATPase obtained by FPLC. The percentage inhibition by DCCI also increased from 30 to 60% in the original membranes (data not shown) to 60 to 80% in the Sephacryl peak 2. In individual experiments the percentages of inhibition by NO, and DCCI were nearly identical. For the FPLC-purified ATPase, however, the percentage inhibition by DCCI varied considerably between experiments, ranging from 67% to only 13%. The Ca2+-ATPase activity of the FPLC-purified ATPase was somewhat lower than the Mg2+-ATPase activity, but in each experiment the Ca2+ATPase and Mg 2f-ATPase were inhibited to a similar extent by NO, and to a similar extent by DCCI. It seemed likely that the Ca2+- and Mg2+-ATPase activities of the FPLC peak were both the activity of the vacuolar ATPase, and also that during purification some degree of sensitivity to DCCI was lost. The Ca2+-ATPase from the Sephacryl

MORRISSEY

column peak 4 was stimulated up to 60% more by Ca2+ than by Mg2+. It was not inhibited significantly by DCCI (7%), and the effect of NO& varied from slight inhibition to slight stimulation. There was no effect of azide on any of the fractions (data not shown). SDS-PAGE was used to compare the polypeptide compositions of the original membranes, peak 2 from the Sephacryl S-400 column, the FPLC-purified vacuolar ATPase, and the ATPase obtained from the glycerol gradient (Fig. 3). The FPLC-purified ATPase resolved into 10 polypeptides, of mol wt 115, 68, 53, 45, 42, 34, 32, 17, 13, and 12 kDa. The 115kDa protein was not always present and varied in relative intensity. The 53-kDa polypeptide frequently was resolved into two or three polypeptide bands. SDS-PAGE was also used to examine the polypeptide composition of peak 4 from the Sephacryl S-400 column but the polypeptide composition varied between experiments (data not shown). A range of mol wts from 14 to 115 kDa was observed, which suggested that the proteins

FIG. 3. SDS-polyacrylamide gel electrophoresis of tonoplast membranes (Lane l), the vacuolar ATPase peak 2 from the Sephacryl S-400 column (Lane 2), the vacuolar ATPase peak from the FPLC column (Lane a), and the vacuolar ATPase peak from the glycerol gradient (Lane 4). The gel was silver-stained. Molecular weights were determined by comparison with the following standards: myosin (200 kDa), j3-galactosidase (116.2 kDa), phosphorylase B (97 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa). The experiment, including the glycerol gradient, was performed three times with identical results. The same distribution of subunits for the FPLC-purified ATPase was observed in many additional gels.

BARLEY

VACUOLAR

in peak 4 were retarded by binding to the column, rather than because of low mol wt. Vacuolar ATPase subunits did not appear to be present. Purification of the Ca2’ATPase will require additional procedures. Two separate preparations of polyclonal antibodies were prepared against the purified V-type ATPase that was collected from the FPLC column. The antibodies were made against the holoenzyme. One preparation reacted strongly with the 66kDa band and faintly with the 34kDa band on an immunoblot of the purified enzyme. The other reacted strongly with the 53-kDa band and faintly with the 68-kDa band. The effect of the antibodies on proton transport by the ATPase in the native membranes was tested by incubating the membranes with antibody for 10 min prior to adding ATP. ATP-dependent proton transport was inhibited up to 80% by either antibody. DISCUSSION The vacuolar ATPase accounted for less than half of the ATP hydrolysis in the tonoplast-enriched fraction as defined by NOB-inhibited and DCCI-inhibited ATPase. Proton transport was supported only by Mg2+ and not by Ca2+ (data not shown), but ATP hydrolysis was stimulated by Ca2+ as much as or more than by Mg2+. It was necessary to purify the V-type ATPase in order to clearly define and characterize the V-type ATPase activity, and to separate it from any other ATPases in the preparation. One source of contaminating ATP hydrolysis in membrane preparations from wheat and barley, but not oats, is a highly active Ca 2+-ATPase (23,24). Much of the Ca2’stimulated ATPase was washed from barley membranes with 150 mM KCl, and purified plasma membrane fractions from barley or wheat had little Ca2+-ATPase (23, 24). When the 100,OOOg supernatant and pellet were compared, the greatest amount of Ca2+-ATPase activity was in the 100,OOOg supernatant, and when the membranes were separated by centrifugation on continuous sucrose gradients, the majority of the Ca2+-ATPase did not enter the gradient (23). It was suggested that there is a large amount of soluble Ca2+-ATPase in a barley root homogenate which sticks to the membranes but is easily removed (23). In addition to the soluble Ca2+-ATPase, a peak of Ca2+ATPase coincided with the peak of vacuolar ATPase activity on a continuous sucrose gradient and it was not possible to remove this Ca2+-ATPase with various washing procedures (23 and unpublished data). The data in this paper suggest that there were two Ca’+-stimulated ATPases in the tonoplast fraction, which were separated by Sephacryl S-400 chromatography of the Triton X-100 solubilized membranes. One Ca2+-stimulated ATPase was separated from the vacuolar ATPase by chromatography on the Sephacryl column. It was not inhibited by DCCI and it was only slightly inhibited by NO;. Purification by Sephacryl col-

ATPase

345

umn chromatography increased the specific activity of the Ca2+-stimulated ATPase at least ninefold. Neither the origin nor the function of this Ca2+-ATPase is known. It may be associated with the vacuolar membranes or with contaminating membranes, or may represent trapped vesicular contents. It is unlikely to be a Ca2+-translocating ATPase, since it does not require Mg2+. Hall et al. (26) demonstrated a Ca2+-ATPase activity in wheat hypocotyl cells using a lead precipitation technique and electron microscopy. After incubation at pH 5.5 the lead phosphate deposit was principally associated with ER and dictyosomes. Work is underway to purify the Ca2+-ATPase, as a first step in determining its origin and function. The second Ca2+-stimulated ATPase appeared to be the V-type ATPase itself, Ca’+-ATPase copurified with Mg’+-ATPase during Sephacryl column chromatography and FPLC. The Ca2+- and Mg2+-stimulated ATPase were inhibited to a similar extent by NO, and by DCCI. There are no other reports of Ca2+-stimulated ATPase being associated with a V-type ATPase from plants, but there are reports for other systems that both FIFo and V-type ATPases can be stimulated by Ca2+ as well as by Mg”+ (8,27). Although the proton-pumping activities of the Vtype ATPase are supported only by Mnzf or Mg2+ (6-g), the Ca2+-stimulated ATPase may be a partial reaction of the ATPase that is useful for studies of the mechanism of action of the ATPase (8). The observation of a high Ca2+-ATPase activity and the decreased sensitivity to DCCI for the FPLC-purified ATPase from barley may indicate a degree of uncoupling of the DCCI binding region from the region of ATP hydrolysis. The V-type ATPase has a complicated subunit composition. An external, hydrophilic knob is loosely attached to a hydrophobic transmembrane segment (29-32). The external knob is released by incubation in the cold with Mg2+ and ATP and is composed of at least five subunits of approximately 66, 57, 41, 34, and 33 kDa (2, 7, 28-30, 32,33). The transmembrane proton channel is composed of several subunits of approximately 16, 17, 20, and 39 kDa (28,32). The subunit composition of the purified Vtype ATPase from barley roots was similar to the subunit composition of V-type ATPases that were purified from red beet storage roots (a), mung bean hypocotyls (l), and oat roots (3). The reported mol wt for the subunits of the three plant ATPases are listed in Table IV and compared with the subunit composition and nomenclature for the lysosomal ATPase from rat liver (28). The overall subunit compositions are very similar for the ATPases from distantly related organisms and from a variety of endomembrane compartments, although there is not complete agreement on mol wts and number of subunits. For example, a 115-kDa subunit has been purified along with the ATPase in some instances, but it is missing in others (8). Immunological cross-reactivity has been demonstrated only for the 66- and 57-kDa subunits from plants, yeast, and animals (34). The presence of

346

DUPONT TABLE

Comparison from Four

IV

of the Subunit Composition of ViVc-ATPases Plant Species and the Lysosomal ViVo-ATPase Subunit

Subunit letter” Acl A B B’ C Ac2 D E CT -

AND

Lysosome” 115’J 72h 57h 55 41 3YJ 34 33 2oef 17'"

Barley roots 115 ssh 53h 45 42 34 32,31 17+9 13,12

(kDa)

Mung bean hypocotylb 8 6Sh 57 s 44 38,37 32 16ef 13,12

Red beet storage tissue’

MORRISSEY 3. Ward, stract]

J. M., and Sze, H. (1991)

4. Kane, Chem.

P. M., Yamashiro, 264,19,236-19,244.

5. Cidon, 6. Gluck,

S., and Nelson, S., and Caldwell,

7. Moriyama, 241-247. Oat rootad

100 67h 55h 52 44

Plant

Physiol.

96(suppl.),

C. T., and Stevens,

12. [Ab-

T. H. (1989)

J. Biol.

N. (1986) J. Biol. Chem. 261,9222-9227. J. (1987) J. Biol. Chem. 262, 15,780-15,789.

Y., and Nelson,

N. (1989)

Biochim.

Biophys.

Acta 980,

8. Xie, X.-S., and Stone, D. K. (1988) J. Bzbl. Chem. 263,9859-9867. 9. Wang, Z. Q., and Gluck, S. (1990) J. Biol. Chem. 265,21,957-21,965.

70 60

10. Binzel, M. A., Hess, F. D., Bressan, R. A., and Hasegawa, P. M. (1988) Plant Physiol. 66, 607-614. 11. Blumwald, E., and Poole, R. J. (1987) Plant Physiol. 83,884-887.

44 36

12. Fan, T. W.-M., Higashi, R. M., Norlyn, Proc. Natl. Acad. Sci. USA 86,9856-9860.

33

32,29

16=J

16er 13,12

a Following the nomenclature of Moriyama and Nelson (7). b Ref. (1). c Ref. (2). d Ref. (3). e Hydrophobic subunit. ’ DCCI-binding was demonstrated. B Visible in gel in the paper cited. h Identified by antibody cross-reactivity or sequence data.

subunits between 12 and 15 kDa is mentioned in a few reports (1,3,6,9). The 12- and 13-kDa subunits now have been reported for mung bean (l), a dicotyledonous plant, and for the monocots oat (3) and barley. Observation of the 12- and 13-kDa subunits may depend on the gel system used, since many published gels do not resolve proteins of this mol wt, or they may be unique to plants. If regulation and function of the V-type ATPases differs from one tissue, organism, or endomembrane compartment to another, one might observe differences in subunit composition. Purification of the V-type ATPase from barley roots confirms the general similarity of these ATPases in animals, plants, and fungi. Detailed sequences for individual subunits will be needed to clarify the relationships of the minor polypeptides described for the ATPase from various species. REFERENCES 1. Matsuura-Endo, C., Maeshima, M., and Yoshida, S. (1990) Eur. J. B&hem. 187,745-751. 2. Parry, R. V., Turner, J. C., and Rea, P. A. (1989) J. Biol. Chem. 264, 20,025-20,032.

13. Garbarino, 14. Kaestner,

J., and Epstein,

J., and DuPont, F. M. (1989) Plant Physiol. 89, l-4. K. H., and Sze, H. (1987) Plant Physiol. 83,483-489.

15. Epstein,

E., and Norlyn,

J. D. (1977)

16. DuPont, Physiol.

F. M., Tanaka, 86, 717-724.

C. K., and Hurkman,

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G. G., and Beaudoin,

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A. R. (1977)

C. (1973)

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U. K. (1970) Nature 227, 680-685. J. H. (1981) Anal. B&hem. 117.307-310.

22. Blake, M. S., Johnston, K. H., Russell-Jones, E. C. (1984) Anal. Biochem. 136, 175-179. 23. DuPont, F. M., and Hurkman, W. J. (1985) 862. 24. Kahr, M., and Moller, I. M. (1976) Physiol. C., Widell,

Y., and Nelson,

29. Apps, D. K., Percy, B&hem. J. 263,81-88.

J. M.,

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S., and Moller,

Z., and Weiss,

S. (1989)

N. (1989) and

56,502-

Physiol.

77,857-

38,153-158.

I. M. (1989)

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R. L. (1990)

G. J., and Gotschlich,

26. Hall, J. L., Kinney, A. J., Dymott, A., Thorpe, D. A. (1982) Histochem. J. 14,323-331. 27. Gromet-Elhanan, 3650.

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249-251.

W. J. (1988)

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