©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Nucleotide Labeling and Reconstitution of the Recombinant 58-kDa Subunit of the Vacuolar Proton-translocating ATPase (*)

Sheng-Bin Peng

From the (1)Division of Molecular Transport, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9121

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Evidence suggests that ATP hydrolysis catalyzed by the clathrin-coated vesicle proton-translocating ATPase requires at least four polypeptides of molecular masses of 70, 58, 40, and 33 kDa (Xie, X.-S., and Stone, D. K.(1988) J. Biol. Chem. 263, 9859-9867). To further investigate the subunit requirements for ATP hydrolysis, histidine-tagged, 58-kDa polypeptide was expressed in insect Sf9 (Spodoptera frugiperda) cells. After purification by Ni-nitrolotriacetic acid chromatography, the 58-kDa protein was found to lack significant ATPase activity. However, the subunit was photoaffinity labeled with [-P]ATP, [C]ADP, or S-labeled ADP and UV irradiation in a divalent cation-dependent manner. The labeling was saturable with an apparent K of 4 µM for both ATP and ADP. ATP and ADP competition labeling experiments indicate that the two nucleotides share the same binding site. When reconstituted with recombinant 70-kDa subunit and a biochemically prepared catalytic sector (V) depleted of the 70- and 58-kDa subunits, the 58-kDa component restores Ca-activated ATP hydrolysis to a specific activity of 0.19 µmol P mg protein min, thus demonstrating that ATP hydrolysis in vacuolar type proton pumps is dependent upon the 58-kDa subunit as well as multi-subunit interactions.


INTRODUCTION

Over the past decade, the importance of the vacuolar H ATPases (V-type ATPase) has become increasingly apparent. These ATP-driven proton pumps acidify a variety of intracellular compartments in eukaryotic cells(1, 2, 3) , and the acidification of lysosomes, secretory vesicles, and storage vacuoles plays a critical role in many basic cellular processes. Vacuolar acidification is necessary for protein degradation in lysosomes, ligand-receptor processing in endosomes, peptide maturation in secretory vesicles, neurotransmitter uptake in synaptic vesicles, and the concentration of nutrients in storage vacuoles. From a biomedical standpoint, V-type ATPases are of interest because of their role in the pathogenesis of osteoporosis (4, 5, 6) and the systemic acidosis of uremia (7). More recent evidence suggests that V-type ATPase may be involved in resistance to chemotherapeutic agents in cultured tumor cells(8) .

V-type ATPases structurally resemble F-F ATP synthetases and are complex hetero-oligomers with molecular masses of greater than 500 kDa. At least 10 polypeptides are considered candidate subunits; these have apparent molecular masses of 116, 70, 57, 58, 50, 40, 38, 34, 33, and 17 kDa(9, 10) . Attempts to determine subunit composition by means of the resolution and reconstitution approach has led to the definition of several functional domains of the clathrin-coated vesicle H-ATPase. These are a peripheral ATP hydrolytic sector (V)(11) , a bafilomycin sensitive proton channel (V)(12, 13) , and a soluble regulatory element(14) . Dissociation of the peripheral components of purified pump leads to the generation of an ATP hydrolytic sector, which functions in the presence of Ca-ATP but not MgATP(11) . Although the components of this active catalytic sector, V, are under active investigation, the relevance of Ca-activated ATPase activity to native enzyme function was established with the demonstration that dissociated pump components could be reassembled and transformed from a Ca-activated to Mg-activated mode by a novel, 50-57-kDa polypeptide heterodimer(10) .

At the present time, it is not known whether this Ca-activated ATPase activity is physiologically important. Myosin and kinesin switch from a Mg-activated ATP-hydrolytic state to a Ca-activated mode with removal of actin and microtubules, respectively(15, 16) . This transition serves to prevent futile ATP hydrolysis, and such a phenomenon may occur during the biogenesis of V-type H-ATPases. Alternatively the Ca-ATPase activity of V may resemble that of chloroplast F (CF), wherein Ca-ATPase activity is manifested through several nonphysiological manipulations, such as protease and detergent treatment, or heating(17, 18) . Irrespective of this issue, however, analysis of the polypeptide requirements for Ca-activated ATP hydrolysis has provided a useful assay for subunit identification in V-type proton pumps. Moreover, the subunit-dependent reversibility of the transition (10) further supports its potential cellular importance.

Full definition of the molecular structure of the enzyme and the function of individual subunits by a purely biochemical resolution and reconstitution approach has proven difficult because of limited starting materials for such experiments. As an alternative, we are currently reconstituting partial reactions of the V-type ATPase with recombinant polypeptides. By this strategy, we have recently reconstituted recombinant 40- (19) and 33-kDa (20) subunits to biochemically prepared fractions that were deficient in these components and have demonstrated that both are subunits essential for ATP hydrolysis. We have also cloned and expressed the 70-kDa polypeptide in Sf9 cells. Although this purified component could not support ATP hydrolysis, it was labeled by [-P]ATP and UV irradiation. When reconstituted to a 70-kDa-depleted fraction, it restored Ca-activated ATP hydrolytic activity. This evidence is consistent with the proposal that the 70-kDa subunit is the actual ATP hydrolytic center of the V-type ATPase(21) .

With respect to the 58-kDa component, the subject of this study, cDNAs encoding this subunit have been cloned from many different sources (22-30). In addition, there is evidence that isoforms of the 58-kDa subunit are present in eukaryotic tissues(22, 23, 26, 28) . Amino acid sequence analysis reveals that the primary structure of the 58-kDa subunit is highly conserved and contains an ATP binding motif. In this respect, the 58-kDa subunit resembles the subunit of F-ATPase. Although several laboratories have conducted labeling experiments with ATP, nucleotide analogues, and/or inhibitors (31-35), the 58-kDa component was labeled in only one study, where it was found that the photoactivable ATP analogue -P-labeled 3-O-(4-benzoyl)benzoyl-ATP bound to this subunit(31) . However, in this instance, the 70-kDa polypeptide, which was consistently labeled by nucleotide in all other studies(32, 33, 34) , was not labeled. Therefore, to date, the view that the 58-kDa subunit of the V-type ATPase is consistently a nucleotide binding polypeptide is equivocal.

We have previously cloned cDNAs coding for the 58-kDa subunit from human kidney (22) and bovine brain.()In the current study, we have expressed the 58-kDa protein in insect Sf9 cells with a recombinant baculovirus. When purified, the recombinant 58-kDa subunit can be photoaffinity labeled with [-P]ATP, [C]ADP, and S-labeled ADP and UV irradiation in a divalent-cation dependent manner, indicating that the 58-kDa subunit contains a nucleotide binding domain. When reconstituted with biochemically prepared 40- and 33-kDa subunits and a recombinant 70-kDa polypeptide, the 58-kDa subunit restores Ca-activated ATP hydrolysis to a specific activity of 0.19 µmol of P mg of protein min, thus demonstrating that ATP hydrolysis in vacuolar-type proton pumps is dependent upon the 58-kDa subunit as well as multi-subunit interactions.


EXPERIMENTAL PROCEDURES

Plasmid Construction

The coding region for the 58-kDa subunit of the V-type H-ATPase was amplified by polymerase chain reaction using cloned cDNA (22) as a template and two primers (5`-CTGCCATATGGCCATGGAGATAGACAGCAGG-3` and 5`-GCGTGATCATCTAGAGCGCAGTGTCAGGCGCGAGG-3`), which were designed to contain NdeI and BclI restriction sites and initiator and stop codons at their 5`-ends, respectively, to enable cloning into the expression vector p(36) . The amplification reaction was performed in a Gene Machine thermal cycler using the following conditions: 1 min at 94 °C and 5 min at 68 °C, for a total of 30 cycles. The polymerase chain reaction product was size fractionated by agarose gel electrophoresis, from which a single 1.6-kilobase fragment was identified and purified. The fragment was identical to that previously reported(22) , as determined by restriction mapping and direct DNA sequencing by the dideoxy termination method(37) . Expression vector p was constructed by replacing the NdeI and BamHI fragment of p with the polymerase chain reaction-amplified fragment, which had been digested with NdeI and BclI. Another expression vector, p, which expresses a fusion protein containing 10 histidine residues and a factor Xa recognition sequence at the amino terminus of the 58-kDa protein, was constructed by replacing the NdeI and HindIII fragment of p with NdeI-HindIII fragment of p.

Expression of the 58-kDa Subunit in Insect Sf9 Cells Using a Baculovirus Vector

The bacterial expression vector p was digested with XbaI, and the resultant 1.6-kilobase fragment, which contained the entire coding region for the 58-kDa subunit, was cloned into XbaI sites of the baculovirus expression vector p. The resultant plasmid, p, was characterized by restriction mapping and partial DNA sequencing. Plasmid DNA was prepared with alkaline lysis and purified by two rounds of cesium chloride gradient centrifugation(38) .

Spodoptera frugiperda (Sf9) cells were grown in monolayer or in suspension culture at 27 °C in either Grace's or IPL-41 medium with 10% heat-inactivated fetal bovine serum plus 0.1% pluronic polyol F68. Cells were split 1:5 for propagation every 3-4 days. Recombinant baculovirus was generated by cotransfection of Sf9 cells by the lipofection method with purified vector p DNA and linearized AcRP23-LacZ viral DNA(21, 39) . Positive recombinant viral clones were isolated by plaque assay and identified by their ability to direct the expression of the 58-kDa protein, as determined by SDS-PAGE()and Western blot analysis.

For expression and production of the 58-kDa protein, Sf9 cells (2 10 cells/ml medium) were grown in suspension culture at 27 °C, infected with pure recombinant virus, and maintained in culture for designated time periods to optimize expression.

Purification of the Recombinant 58-kDa Subunit from Sf9 Cells

500 ml of Sf9 cells were infected with pure recombinant baculovirus, incubated in suspension culture for 48 h at 27 °C, and harvested by centrifugation at 4,000 g for 15 min. The cells were then resuspended in 30 ml of sonication buffer consisting of 50 mM NaHPO (pH 8.0), 500 mM NaCl, 0.1 mM phenymethysulfonyl fluoride, and sonicated for 1 min. The preparation was examined under the microscope to achieve efficient cell lysis with minimal sonication. The lysate was then centrifuged at 40,000 rpm for 1 h at 4 °C with a Beckman 70 Ti rotor. The resultant supernatant was used to purify the recombinant 58-kDa protein by the following steps.

Step 1: Ni-NTA Resin Chromatography

A 3-ml Ni-NTA resin column was prepared and equilibrated with sonication buffer as described(40) . Supernatant (30 ml) was loaded onto the column, which was then washed with 30 ml of sonication buffer, 30 ml of wash buffer (20 mM Tris/HCl (pH 8.0), 0.01% CE, and 20% glycerol), and 200 ml of wash buffer containing 20 mM imidazole (pH 8.0). The protein was eluted with 20 ml of wash buffer plus 200 mM imidazole, and 1-ml fractions were collected and analyzed by SDS-PAGE.

Step 2: CM-Sepharose (CL-6B) Chromatography

Selected fractions (5 ml) from Ni-NTA chromatography were combined and diluted with 3 volumes of buffer A (10 mM Tris/MES, pH 7.0, 1 mM EDTA, 0.01% CE, 1 mM dithiothreitol, 20% glycerol) and loaded onto a 2-ml CM-Sepharose column, which had been pre-equilibrated with buffer A. The pass-through fraction was saved for the next step.

Step 3: DEAE-Sepharose (CL-6B) Chromatography

The fraction from step 2 (20 ml) was loaded onto a pre-equilibrated 2-ml DEAE-Sepharose column, which was sequentially washed with 10 ml of buffer A and 20 ml of buffer A plus 100 mM NaCl. The 58-kDa subunit was eluted with 10 ml of buffer A containing 400 mM NaCl, and 1 ml fractions were collected and analyzed by SDS-PAGE. The peak fractions were dialyzed against 300 volumes of buffer A without dithiothreitol and used for reconstitution of ATP hydrolysis and nucleotide labeling experiments.

Antibody Preparation and Western Blot Analysis

Polyclonal antibody against SDS-denatured, recombinant 58-kDa subunit, expressed in Escherichia coli BL21(DE3) with p, was raised in a New Zealand White female rabbit with the same method to prepare polyclonal antibodies against 40- and 33-kDa proteins(19, 20) . For Western blot analysis, cell lysates and proteins were separated on a 11% SDS-polyacrylamide gel and transferred electrophoretically to nitrocellulose paper. Immunodetection was performed using Amersham ECL Western blotting system.

Purification of Clathrin-coated Vesicle H-ATPase and Generation of a Fraction Depleted of the 70- and 58-kDa Subunits

The clathrin-coated vesicle H-ATPase complex was solubilized with CE and purified from clathrin-coated vesicles of bovine brain as described(9) . Generation of the 70- and 58-kDa-depleted fraction was performed generally as follows. Briefly, proton pump complex (750 µg of purified protein) from the final glycerol gradient was treated with 3 M urea on ice for 1 h. The mixture was adjusted to pH 7.1 with MES and loaded on a 15-ml CM-Sepharose column, which was pre-equilibrated with 3 M urea in a buffer containing 30 mM Tricine-Na, pH 7.5, 0.02% CE, 0.5 mM EDTA, 3 mM dithiothreitol, and 10% glycerol. The proteins were eluted with 0-500 mM NaCl in the same buffer without urea. Fractions depleted of 70- and 58-kDa polypeptides were combined, saturated (NH)SO was added to a final concentration of 65%, and the mixture was centrifuged at 80,000 g for 25 min. The pellet was dissolved in the above buffer and was layered over a 10-30% linear glycerol gradient prepared in the same buffer (as above, without urea, and without glycerol). After centrifugation at 180,000 g for 22 h at 4 °C, fractions were harvested from the bottom of the tube, subjected to SDS-PAGE, analyzed for Ca-activated ATPase activity, and used for reconstitution experiments described under ``Results.'' Purified proton-translocating ATPase and the 70- and 58-kDa subunit-depleted fraction utilized for reconstitution experiments are illustrated in Fig. 2. The protein bands illustrated in Fig. 2were quantitatively evaluated on an LKB 2202 Ultrascan Laser Densitometer to determine the degree of subunit depletion.


Figure 2: SDS-PAGE of purified recombinant 58- and 70-kDa proteins and biochemically prepared proton pump and polypeptides. SDS-PAGE was performed as described under ``Experimental Procedures.'' Lane1, purified clathrin-coated vesicle proton pump from bovine brain; lane2, purified ATP catalytic sector (V); lane3, biochemically prepared V depleted of 70- and 58-kDa polypeptides; lane4, purified recombinant 58-kDa protein; lane5, purified recombinant 70-kDa subunit.



Measurement of ATPase Activity

ATPase activity was measured by the liberation of P from [-P]ATP. The assay solution consisted of 30 mM KCl, 50 mM Tris/MES (pH 7.0), 2.5 mM CaCl, and 2 mM [-P]ATP (200-400 cpm/nmol). ATPase samples were preincubated with 2.5 µg of phosphatidylserine for 10 min at room temperature. Reactions were initiated by the addition of 100 µl of assay solution. After incubation for 30 min at 37 °C, the reactions were terminated by the addition of 1 ml of 1.25 N perchloric acid, and liberated P was extracted and counted as described(41) .

Nucleotide Labeling of the Recombinant 58-kDa Subunit

Photoaffinity labeling of the recombinant 58-kDa subunit was performed as described (21, 42) with modification. A reaction mixture (20 µl) containing 0.2 µg of purified 58-kDa protein, 10 mM Tris/MES (pH 7.0), 10% glycerol, radioactive ATP or ADP, and other components as indicated in the legends to figures was incubated at room temperature for 5 min and then irradiated for 8 min on ice at a distance of 35 mm from the UV source (model R-51, Spectronics Corp., Westbury, NY). The reactions were terminated by the addition of 2.2 µl of 10 SDS-PAGE sample buffer. After SDS-PAGE, the gel was dried and autoradiographed on Kodak X-Omat film at -80 °C for 24-48 h. Incorporation of [-P]ATP was determined by scanning autoradiograms with an Image Quan densitometer (Molecular Dynamics 300A computing densitometer). Relative radioactive nucleotide incorporation was quantified as absorbance area on the basis of whole band analysis. Exposure time of the film was adjusted to ensure that the signal would fall well within the linear range of detection.

Miscellaneous

Protein determination (43) and SDS-PAGE (44) were performed as reported. The sources of all enzymes, vectors, and chemical reagents used in this study have been previously described(9, 19, 20, 21) .


RESULTS

Expression of the 58-kDa Subunit of V-type ATPase

Two bacterial expression vectors, p and p, each containing the entire encoding region for the 58-kDa subunit, were used to express the 58-kDa protein in E. coli. However, the resultant recombinant 58-kDa protein in E. coli was aggregated and functionally inactive. Repeated attempts to solubilize, denature, and refold the protein were unsuccessful (data not shown).

As an alternative, we constructed and purified a recombinant baculovirus containing the cDNA sequence encoding the 58-kDa protein that was used to infect Sf9 cells. The recombinant baculovirus directed the expression of a fusion protein containing a histidine tag at the amino terminus. As shown in Fig. 1, the 58-kDa protein was expressed in Sf9 cells infected with recombinant virus. The time course of expression of the 58-kDa protein in Sf9 cells after infection with the virus was studied to optimize conditions for protein production. At designated time points over a 96-h course of infection, aliquots of cells were removed and analyzed by SDS-PAGE and Western blot analysis. Recombinant 58-kDa protein was detected as early as 36 h postinfection by polyclonal antiserum against recombinant 58-kDa polypeptide (Fig. 1B, lane3) and was readily visualized by Coomassie Blue-stained, SDS-PAGE 48-h postinfection (Fig. 1A, lane4). The production of recombinant 58-kDa protein reached a maximal level by 60-72 h (Fig. 1A, lanes5 and 6), constituting approximately 3-5% of the total cellular proteins. The 58-kDa protein was not detected by SDS-PAGE and Western blot analysis in the cells that were not transfected with recombinant virus (Fig. 1, A and B, lanes1).


Figure 1: SDS-PAGE and Western blot analysis of the 58-kDa polypeptide expressed in Sf9 cells transfected with a recombinant baculovirus. Total lysates of infected Sf9 cells were subjected to SDS-PAGE analysis (panelA). PanelB illustrates the Western blot analysis of the soluble portion of the cell lysates, defined as the supernatant obtained after centrifuging the total cellular lysates at 150,000 g for 1 h. Lane1, non-transfected Sf9 cells, lanes2-7, Sf9 cells infected with recombinant baculovirus for 24, 36, 48, 60, 72, or 96 h, respectively.



Purification of the Recombinant 58-kDa Protein from Sf9 Cells

Although significant expression of the 58-kDa protein was obtained in Sf9 cells 48-72 h postinfection (Fig. 1), recombinant protein was largely aggregated. Western blot analysis of soluble fractions of Sf9 cell lysates demonstrated that infected Sf9 cells produced detectable soluble recombinant protein (Fig. 1B) 36-72 h after infection and that optimal production of soluble 58-kDa protein (Fig. 1B, lane4) occurred at 48 h. The 58-kDa protein was partially purified from soluble extract by Ni-NTA column chromatography(20, 40) . Further purification was achieved by CM-Sepharose and DEAE-Sepharose column chromatography, as illustrated in Fig. 2, lane4. As expected, the recombinant protein is of a slightly greater molecular mass than that obtained from native bovine tissue because of the additional histidine tag and factor Xa site residues at the NH terminus.

Generation of the 58- and 70-kDa Subunit-depleted Catalytic Core of Vacuolar H-ATPase from Bovine Brain and Reconstitution of ATP Hydrolysis

Proton-translocating ATPase, purified from clathrin-coated vesicles of bovine brain (Fig. 2, lane1), was partially dissociated with 3 M urea. After (NH)SO precipitation and glycerol gradient centrifugation, a fraction consisting of at least four polypeptides (70-, 58-, 40-, and 33-kDa components) was obtained (Fig. 2, lane2). By CM-Sepharose chromatography and glycerol gradient centrifugation, a fraction depleted of 58- and 70-kDa subunits, which consisted largely of the 40- and 33-kDa polypeptides, was generated (Fig. 2, lane3). Densitometric analysis of this fraction, compared with that of whole enzyme, revealed that the fraction was depleted of 70- and 58-kDa components by approximately 96%. To date, we have been unable to achieve selective depletion of only the 58-kDa subunit from V. This problem, however, was overcome by the experiments shown in . In this study, recombinant 70-kDa subunit (Fig. 2, lane5)(21) , the 58-kDa subunit, and a biologically prepared V depleted of 70- and 58-kDa subunits ( Fig. 2lane3) were tested singly and in combination for their ability to support Ca-activated ATP hydrolysis. As shown in , the recombinant 58-kDa, 70-kDa, or biochemically prepared fraction alone cannot support significant ATP hydrolysis. The mixture of the 70- and 58-kDa components or the addition of either the 58- or 70-kDa subunit to the biochemically prepared fraction restored only trivial ATP-hydrolytic activity. However, when both the 70- and 58-kDa polypeptides were added to the biochemically prepared fraction, significant Ca-activated, N-ethylmaleimide-sensitive ATP-hydrolytic activity (0.45 nmol P min assay; 0.19 µmol P mg of protein min) was observed. There was no significant Mg-activated ATPase activity when all these components were reconstituted under these conditions.

Fig. 3illustrates the influence of various concentrations of recombinant 58-kDa polypeptide on the reconstitution activity of ATP hydrolysis. The stimulation of Ca-activated ATPase activity by the 58-kDa component was saturable, and maximum activity was reached when 4 µg of 58-kDa polypeptide was added to a mixture of recombinant 70 kDa (1 µg) and biochemically prepared V depleted of 70- and 58-kDa subunits (0.4 µg).


Figure 3: Effect of various concentrations of 58-kDa polypeptide on reconstitution of Ca-activated ATP hydrolysis. ATPase activities were measured by the liberation of P from [-P]ATP as described under ``Experimental Procedures,'' using 0.4 µg of biochemically prepared V depleted of 70- and 58-kDa polypeptides, 1 µg of 70-kDa protein, and different concentrations of 58-kDa polypeptide as indicated.



Nucleotide Labeling of the Recombinant 58-kDa Protein

Nucleotide labeling of recombinant 58-kDa protein was attempted using radioactive ATP or ADP and ultraviolet irradiation. As shown in Fig. 4A, the 58-kDa protein was photoaffinity labeled by [-P]ATP when the reaction mixture contained divalent cations (Mg or Ca). Although the 58-kDa protein is labeled when no additional divalent was added to the reaction mixture (Fig. 4A, lane3), this labeling was eliminated by the addition of 5 mM EDTA (Fig. 4A, lane4), indicating that the ATP labeling is truly divalent dependent. In contrast, N-ethylmaleimide, an inhibitor of V-type H-ATPase, did not inhibit ATP-labeling, even at 10 mM (data not shown). Bafilomycin, a specific inhibitor of V-type H-ATPase, also did not inhibit ATP labeling either even at 10 µM, a concentration of about 1,000-fold greater than that required to inhibit V-type H pumps (data not shown).


Figure 4: Photoaffinity labeling of the recombinant 58-kDa polypeptide. Reaction mixtures (20 µl) containing 0.2 µg of the 58-kDa protein, 10 mM Tris/MES, pH 7.0, 10% glycerol, radioactive nucleotide, and other components as indicated were UV irradiated for 8 min on ice, followed by SDS-PAGE and autoradiography as described under ``Experimental Procedures.'' PanelA, [-P]ATP labeling. All lanes contained 1 µl of [-P]ATP (10 µCi/µl, 3000 Ci/mmol). Specific reaction conditions were: lane1, 2 mM MgCl; lane2, 2 mM CaCl; lane3, no additional divalent cation; lane4, 5 mM EDTA. PanelB, radioactive ADP labeling. Lane1, S-labeled ADP (1 µl, 5 µCi/µl, 1095 Ci/mmol), 2 mM CaCl; lane2, [C]ADP (10 µl, 1 µCi/50 µl, 57.1 mCi/mmol), 2 mM CaCl.



In addition, the 58-kDa polypeptide was also labeled by [C]ADP and S-labeled ADP, as demonstrated in Fig. 4B. Photoaffinity labeling of the 58-kDa polypeptide by [-P]ATP and S-labeled ADP exhibited saturation kinetics. 20 µM of either ATP or ADP was sufficient to saturate photolabeling as demonstrated in Fig. 5. The apparent K for both ATP and ADP, determined from double reciprocal plots of response curve shown in Fig. 5, was 4 µM, indicating that the ATP and ADP had similar binding affinity to the 58-kDa polypeptide.


Figure 5: Kinetics of photoincorporation of radioactive nucleotides into recombinant 58-kDa polypeptide. The 58-kDa polypeptide was UV irradiated in the presence of 2 mM MgCl and indicated concentrations of [-P]ATP (0.25 µCi/µm ATP, -) or S-labeled ADP (0.2 µCi/µM ADP, -). Incorporation of radioactive nucleotides into the 58-kDa polypeptide was determined from the densitometric scans of the autoradiograms as described under ``Experimental Procedures.''



The competition of cold ATP and cold ADP on photoaffinity labeling of the 58-kDa polypeptide by [-P]ATP is shown in Fig. 6. Half-maximal protection against photo-incorporation of [-P]ATP into 58-kDa polypeptide occurred with cold ATP and ADP at approximately 280 and 300 µM, respectively, and complete protection was afforded by 1 mM of either ATP or ADP. This result implies that ATP and ADP share the same binding site.


Figure 6: Competition by cold ATP or ADP for the [-P]ATP binding to the recombinant 58-kDa polypeptide. The 58-kDa protein was UV irradiated in the presence of 2 mM of MgCl, 1 µl of [-P]ATP (10 µCi/µl, 3000 Ci/mmol), and indicated concentrations of cold ATP () or cold ADP (). Photoincorporation of [-P] ATP into the 58-kDa polypeptide was determined from the densitometric scans of the autoradiograms as described under ``Experimental Procedures.''




DISCUSSION

A major issue in the field of V-type proton pumps centers upon the subunit composition of these enzymes and the role of the individual components in pump function. Reconstitution of function with biochemically prepared pump fractions suggested that at least four polypeptides of 70, 58, 40, and 33 kDa are necessary for ATP hydrolysis (11). As an alternative, we have taken another route for further resolution of subunit composition and function, in which we clone, express, and purify the candidate subunits and then attempt to reconstitute ATPase activity. In this way, we have shown that 40- (19) and 33-kDa (20) polypeptides are essential for ATP hydrolysis and that they are genuine subunits of the V-type ATPase. We also demonstrated that recombinant 70-kDa polypeptide alone can be labeled by [-P]ATP and is necessary for ATP hydrolysis(21) .

In the current investigations, we have turned our attention to the 58-kDa subunit. Previously, we have successfully purified and renatured the 40- and 33-kDa subunits, which were expressed in E. coli. Initially, we pursued a similar approach with the 58-kDa subunit but were unable to renature the purified protein to yield a reconstitutively active form because of the aggregation of overexpressed 58-kDa protein in E. coli. Consequently, we adopted an expression and purification protocol using baculovirus and Sf9 cells, which we previously utilized for the 33- (20) and 70-kDa subunits(21) .

The 58-kDa component of V-type ATPase has been considered to be a potential nucleotide binding protein because of its similarity in amino acid sequence to the (and ) subunit of F-ATPase and specifically because of the presence of a nucleotide binding motif. To date, several laboratories have attempted to label V-ATPase with radioactive nucleotides or analogues. In fact, in only one instance has the 58-kDa subunit been labeled by a nucleotide analogue(31) . The current study demonstrates that the 58-kDa subunit can be labeled by radioactive nucleotides and that labeling requires a divalent cation (Mg or Ca) (Fig. 4). The divalent cation dependence of the labeling is similar to that of the subunit of F-ATPase from E. coli(45) . Earlier investigations indicated that the subunit binding by nucleotide was divalent cation independent(46) . Based on results of current and previous experiments(21) , the 58-kDa polypeptide demonstrates a higher ATP affinity (K of 4 µM ATP) than the 70-kDa subunit (K of 35 µM ATP).

The labeling of the recombinant 58-kDa subunit with nucleotides is striking in consideration of the observations that labeling of holoenzyme occurs only on the 70-kDa component. Several possible explanations for this phenomenon exist. First, the fact that this polypeptide is recombinant is potentially worrisome in that improper folding or disordered tertiary structure might arise during expression itself or during the isolation procedure. By developing a protocol for harvesting a soluble 58-kDa polypeptide, this possibility has been minimized. More convincing is the fact that the recombinant 58-kDa polypeptide used for the labeling experiments is reconstitutively active, as shown in .

A second potential problem of this nature might arise from the presence of 10 histidine residues at the amino terminus. Attempts at removing these residues with factor Xa was not successful because of additional cleavages of the polypeptide. Again, the fact that the histidine-tagged 58-kDa polypeptide is reconstitutively active indicates this is not likely to cause wholesale distortions in structure. Analysis of the structure of the subunit of F, which is presumably analogous to the 58-kDa subunit of V-type pumps, reveals that the amino terminus of the subunit protrudes from the top of F and that there is evidentially a high degree of freedom in rotation in this terminal domain(47) . Thus, it is possible that the amino terminus of the 58-kDa subunit of the V-type proton pump is structurally noncontiguous (and noncommunicative) with the nucleotide binding domain and that the presence of additional amino terminus residues is inconsequential.

A third possibility, and the mostly likely, is that under most circumstances, the ATP binding site of the 58-kDa subunit is inaccessible to nucleotide exchange in the native enzyme and thus is functionally and structurally analogous to the subunit of F, which contains a ``nonexchangeable'' ATP (and divalent metal) binding site. Thus, in isolated V-type pumps, this site may already be occupied by nucleotide, and only by preparing nascent, recombinant protein can the site become accessible to labeling. At present, there is no proven role for nucleotides occupying nonexchangeable sites in F, although it has been proposed that these sites become occupied during the biogeneses of the catalytic sector and may play a stabilizing, structural assembly role. If so, it will be of interest to determine if this is the case in V-type pumps by analyzing the nucleotide requirements, if any, for the assembly or association of the 70- and 58-kDa components.

The most important achievement of this investigation lies in the reconstitution of purified and recombinant 58-kDa polypeptide. As demonstrated in , recombinant 58-kDa polypeptide alone or other fractions used for reconstitution in this experiment have trivial Ca-activated ATPase activity. When reconstituted to a biochemically prepared V depleted of 70- and 58-kDa subunits and recombinant 70-kDa fraction, the 58-kDa polypeptide resulted in a significant Ca-activated ATP hydrolysis to a specific activity of 0.19 µmol P min mg of protein. Without the 58-kDa subunit, the reconstitution with other polypeptides demonstrated only minimal ATPase activity. These data indicate that the 58-kDa component is necessary for maximal ATP hydrolysis. Like the na-tive catalytic sector from bovine brain-coated vesicles, the reconstituted ATPase activity was N-ethylmaleimide sensitive ().

In addition to demonstrating, for the first time, the essential role of the 58-kDa polypeptide in V-type ATPase, this work further underscores our point that maximal ATP hydrolysis requires multiple components and their interactions. An accepted view is that ATP hydrolysis actually occurs on the 70-kDa or 70- and 58-kDa components and that the 40- and 33-kDa subunits render the catalytic site active. As shown in , recombinant 70- and 58-kDa components together did not support significant ATP hydrolysis, and any three out of four components (70, 58, 40, and 33 kDa) demonstrated only minimal ATPase activity. Therefore, at least these four subunits are essential for full function of catalytic sector. With this observation, we are now prepared to undertake the reassembly of the catalytic sector from wholly recombinant components.

  
Table: Reconstitution of Ca-activated ATPase with recombinant 58-kDa subunit and other polypeptides of the V-type ATPase

ATPase activities were measured by the liberation of P from [-P]ATP as described under ``Experimental Procedures,'' using 1 µg of purified recombinant 58-kDa protein, 1 µg of recombinant 70-kDa protein (21), 0.4 µg of biochemically purified Vc depleted of 70- and 58-kDa polypeptides, and 5 mMN-ethylmaleimide as indicated. Denaturation of the 58-kDa subunit was achieved by boiling for 10 min. All of the fractions utilized in this experiment are as shown in Fig. 2.



FOOTNOTES

*
This work was supported by Grant DK-33627 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

S.-B. Peng, X.-S. Xie, and D. K. Stone, unpublished observations.

The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; CE, polyoxyethylene 9-lauryl ether; MES, 2-(N-morpholino)ethanesulfonic acid; NTA, nitrolotriacetic acid.


ACKNOWLEDGEMENTS

I thank Drs. Dennis K. Stone, Xiao-Song Xie, and Bill P. Crider for helpful discussions and suggestions and Ying Zhang for construction of the bacterial expression vector p. Superb technical and administrative assistance were provided by Shung-Ching Tsai and Kay Martin.


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