©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Disassembly and Reassembly of the Yeast Vacuolar H-ATPase in Vivo(*)

Patricia M. Kane

From the (1)Department of Biochemistry and Molecular Biology, State University of New York Health Science Center, Syracuse, New York 13210

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The vacuolar H-ATPase of the yeast Saccharomyces cerevisiae is composed of a complex of peripheral subunits (the V sector) attached to an integral membrane complex (the V sector). In the experiments described here, attachment of the V to the V sector was assessed in wild-type cells under a variety of growth conditions. Depriving the yeast cells of glucose, even for as little as 5 min, caused dissociation of approximately 70% of the assembled enzyme complexes into separate V and V subcomplexes. Restoration of glucose induced rapid and efficient reassembly of the enzyme from the previously synthesized subcomplexes. Indirect immunofluorescence microscopy and subcellular fractionation revealed detachment of the peripheral subunits from the vacuolar membrane in the absence of glucose, followed by reattachment in the presence of glucose. Rapid dissociation of vacuolar H-ATPases could also be triggered by shifting cells into a variety of other carbon sources, and reassembly could be generated by addition of glucose. Disassembly and reassembly of vacuolar H-ATPases in vivo may be a means of regulating organelle acidification in response to extracellular conditions, or a mechanism for assembling alternate complexes of vacuolar H-ATPases in different intracellular compartments.


INTRODUCTION

Vacuolar proton-translocating ATPases (H-ATPases)()are multisubunit complexes, found in all eukaryotic cells, that acidify one or more intracellular compartments(1) . Vacuolar H-ATPases from plants, animals, and fungi are very similar. All consist of a V sector, which is a complex of peripheral membrane proteins containing the ATP-binding sites, and a V sector, comprising a complex of integral membrane proteins containing the proton pore(2, 3, 4) . The vacuolar H-ATPases share sequence and structural similarities with the FF-ATPases of mitochondria, chloroplasts, and bacteria, and these similarities are believed to reflect an evolutionary relationship(5) . However, unlike the FF-ATPases, which can be separated into a soluble F complex that is capable of ATP hydrolysis(6) , and a membrane-bound F complex that appears to function as a proton channel(7) , a variety of biochemical studies indicate that the V sector of the vacuolar H-ATPases does not retain its ATPase activity when detached from the membrane(8, 9, 10, 11, 12) , and the V complex is not an open proton pore(12, 13) .

Studies of the assembly of the yeast vacuolar H-ATPase have shown that mutants lacking one subunit of the enzyme complex can still assemble the V complex in the cytoplasm under conditions where the V complex is not assembled, and the V complex can be assembled and transported to the vacuole in the absence of an assembled V complex(14, 15, 16, 17) . Independent V and V complexes were also seen in wild-type yeast cells, but it was not clear whether these complexes were assembly intermediates, products of breakdown of the vacuolar H-ATPase, or artifacts of the biochemical isolation(14) . This paper describes the reversible dissociation of the wild-type V complex from the V complex in vivo. Both the disassembly and subsequent reassembly of the complex occur rapidly and efficiently in response to changes in growth conditions, suggesting that these processes could play a role in regulation of vacuolar H-ATPase activity or in cellular distribution of the active enzyme.


EXPERIMENTAL PROCEDURES

Materials and Strains

Zymolyase 100T was purchased from Seikagaku America, Inc. TranS-label was purchased from ICN. Dithiobis(succinimidylpropionate) (DSP) was obtained from Pierce. C-Labeled molecular mass markers (high range) were obtained from Life Technologies, Inc. All secondary antibodies used for indirect immunofluorescence microscopy (fluorescein-conjugated and unconjugated) were obtained from Organon Teknika-Cappel. All other reagents were purchased from Sigma.

The wild-type yeast strain SF838-1D (genotype: MAT, ade6, leu2-3, leu2-112, ura3-52, pep4-3, gal2) was used in all experiments. Yeast media were prepared as described previously(18, 19) .

Immunoprecipitations

Immunoprecipitations were carried out under nondenaturing conditions as described(14) , with the following modifications. Cells were converted to spheroplasts in supplemented minimal medium lacking methionine that contained 2% glucose and 1.2 M sorbitol (SD-Met, 1.2 M sorbitol) by incubation for 20 min with 0.1 unit of zymolyase/10 cells. For each immunoprecipitation, 0.5 10 spheroplasts were suspended in 150 µl of SD-Met, 1.2 M sorbitol, shaken 20 min at 30 °C, and then incubated for the indicated labeling time with 50 µCi of TranS-label. At the end of the incubation, unlabeled methionine and cysteine were added to a final concentration of 0.33 mg/ml each, and the cells were pelleted by centrifugation. For samples with a subsequent chase, the pellets were resuspended in the appropriate medium to the same density as for labeling and shaken at 30 °C for varied times. After the labeling and chase times were completed, spheroplast pellets were solubilized in phosphate-buffered saline (PBS; 137 mM NaCl, 2.6 mM KCl, 12 mM sodium phosphate, pH 7.2) containing 1% (w/v) CE and a protease inhibitor mixture (final concentrations: 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 2 µg/ml chymostatin, 5 µg/ml aprotinin). DSP was added as 25 mM solution in dimethyl sulfoxide, to a final concentration of 0.67 mM. After 60 min, an equal volume of quenching buffer (50 mM Tris/HCl, pH 7.5, 10% glycerol, 1 mM EDTA) containing a 2-fold concentration of the protease inhibitor mixture was added to the samples to quench the cross-linker, and after another 15 min, samples were presorbed with Protein A-Sepharose CL-4B, as described. Antibody incubations were carried out as described(14) , except that 5 µl (12.5 µg) of purified 8B1 antibody (9) and 495 µl of PBS containing 5 mg/ml bovine serum albumin were added instead of a cultured supernatant containing this antibody. Immunoprecipitated proteins were washed, solubilized, and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography as described(14) . In some cases immunoprecipitation results were quantitated by exposing the gels overnight to the phosphor-containing screen and analyzing on a Fuji BAS100 phosphorimager.

In the experiment shown in , yeast cells were grown overnight, converted to spheroplasts, and labeled with TranS-label as described above except that 2% raffinose was used as the sole carbon source instead of 2% glucose. Unlabeled methionine and cysteine were added to initiate the chase period, but raffinose (to give a total concentration of 4%) and glucose (to give a final concentration of 2%) were then added directly to the labeling medium rather than spinning down the spheroplasts and resuspending them in fresh medium.

In the absence of the DSP cross-linker, a similar collection of subunits, with the exception of the 42-kDa subunit, could be immunoprecipitated from wild-type cells by the 8B1 antibody. The 42-kDa subunit appeared to be lost during immunoprecipitation without cross-linker even though it could be isolated with the complex by other means(14) . Addition of cross-linker allowed consistent immunoprecipitation of the 42-kDa subunit with the wild-type complex and allowed more reliable comparisons between different experiments. In the presence of DSP, several proteins in addition to the 100-, 36-, and 17-kDa subunits of the ATPase are co-precipitated consistently by the 10D7 antibody. These included a band at 75 kDa and a faint, diffuse band at 19 kDa that were co-precipitated previously in the absence of cross-linker(14) , as well as a band at 200 kDa and several at 40-60 kDa. Precipitation of these bands was characteristic of the 10D7 antibody, but the amount of protein in additional bands did not appear to change under conditions for disassembly and reassembly as the 100-, 36-, and 17-kDa proteins did.

Indirect Immunofluorescence Microscopy

Cells were grown to mid-log phase in YEPD (1% yeast extract, 2% peptone, 2% dextrose medium; Ref. 18), then diluted to approximately 1 10 cells/ml in YEPD or YEP (1% yeast extract, 2% peptone medium without dextrose) and incubated at 30 °C. After the indicated time of incubation, cells were fixed for 30 min in the same medium, then pelleted and fixed overnight(20) . Monoclonal antibodies 13D11 and 10D7 (15) were used at a 1:10 dilution and undiluted, respectively, and the signal was amplified as described(20) .

Fractionation of Spheroplast Lysates and Isolation of Vacuolar Vesicles

Cells grown overnight at 30 °C to early to mid-log phase in YEPD, YEP containing 2% raffinose, or YEP containing 3% glycerol, 2% ethanol were converted to spheroplasts and washed as described above, except that all incubations were carried out in the presence of the appropriate carbon source. Spheroplasts from approximately 2.5 10 cells were resuspended in 1 ml of the appropriate incubation medium containing 1.2 M sorbitol and incubated for 20 min at 30 °C. In some samples the incubation was continued for another 20 min in the presence of added 2% glucose to assess reassembly; otherwise, the cells were pelleted after the initial 20-min incubation then resuspended in 1 ml of PBS containing the same protease inhibitor mixture used for immunoprecipitations. DSP was added to a final concentration of 0.6 mM, and the mixture was incubated on ice for 60 min. The cross-linker was quenched by addition of 1 ml of quenching buffer, followed by incubation for 20 min on ice. After addition of another 3 ml of PBS containing the protease inhibitor mixture, the spheroplast lysate was homogenized in a Dounce homogenizer with 10 strokes of the A-pestle. The lysate was then centrifuged for 60 min at 35,000 rpm (147,000 g at r) in an SW50.1 rotor. Supernatant and pellet fractions were analyzed by Western blotting as described(14) .

Vacuolar vesicles were prepared as described(20) , except that spheroplasts were washed once with 1.2 M sorbitol, then incubated 15 min at 30 °C in either YEPD containing 1.2 M sorbitol or YEP containing 1.2 M sorbitol. After the incubation, the spheroplasts were pelleted by centrifugation, then lysed immediately. ATPase activities of isolated vesicles were measured at 37 °C using the coupled enzyme assay of Lotscher et al.(21) , and a Lowry (22) assay was used to quantitate protein. In order to assess the amount of ATPase activity specific for V-type ATPases, vacuolar vesicles were preincubated with 100 nM concanamycin A (Wako Chemicals) for 20 min on ice, and the difference between the ATPase activities of vesicles with and without preincubation with inhibitor was taken as the vacuolar ATPase activity. (Concanamycin A is a highly specific inhibitor of V-type ATPases(23) .)


RESULTS

To determine whether the state of assembly of the yeast vacuolar H-ATPase is altered in response to changes in extracellular conditions, yeast spheroplasts were biosynthetically labeled with TranS-label for 45 min and then shifted to different media containing unlabeled methionine for a 15-min ``chase'' time prior to immunoprecipitation. Fig. 1A shows that after labeling for 45 min, all of the previously identified subunits of the yeast vacuolar H-ATPase can be co-precipitated with monoclonal antibody 8B1 against the 69-kDa catalytic subunit of the enzyme (lane1), and there is little change in the distribution of subunits immunoprecipitated after a subsequent 15-min chase period in the same medium (lane2). This indicates that assembly of the yeast vacuolar H-ATPase is almost completed during the 45-min labeling time, and that the assembled complexes are stable through the subsequent 15-min chase period. Very similar complexes were obtained from cells chased in medium buffered to pH 5 (lane5), buffered to pH 7.5 (lane3), or containing 50 mM CaCl (lane4). These conditions were chosen because yeast mutants lacking vacuolar H-ATPase activity fail to grow in medium buffered to pH 7.5 or containing 100 mM CaCl but are able to grow on medium buffered to pH 5, suggesting that cellular requirements for the vacuolar H-ATPase differ under these different growth conditions (19, 24, 25). However, these results indicate that the different media do not grossly affect assembled vacuolar H-ATPase complexes. In contrast, assembly of the yeast vacuolar H-ATPase was significantly affected by transferring the labeled cells to medium lacking glucose for 15 min (lane6). Complexes co-precipitated from cells chased in medium without glucose showed greatly reduced levels of the 100-, 42-, 36-, and 17-kDa subunits. (Although there appears to be some reduction in the level of the 32-kDa subunit in Fig. 1A, this was not seen as consistently. The level of the 32-kDa subunit in the experiments shown in Fig. 2A and 5A was more typical.) The 60-, 32-, and 27-kDa proteins that remain associated with the 69-kDa subunit after incubation in medium lacking glucose have been implicated as members of the peripheral V sector of the vacuolar H-ATPase, along with the 42-kDa subunit (reviewed in Ref. 4). The 100- and 17-kDa subunits have been shown to be integral membrane proteins(15, 16, 17) , and the 36-kDa subunit appears to be closely associated with these subunits even though it is not an integral membrane subunit (14, 26).


Figure 1: Disassembly of the yeast vacuolar H-ATPase. Yeast cells were converted to spheroplasts and labeled with Tran[S]-label for 45 min as described under ``Experimental Procedures.'' Labeled spheroplasts were then lysed immediately (lane1) or chased for 15 min in SD-Met (lane2), YEPD buffered to pH 7.5 (lane 3), YEPD containing 100 mM CaCl (lane 4), YEPD buffered to pH 5 (lane 5), or YEP buffered to pH 5 (lane 6) before lysis. Chase media contained 1.2 M sorbitol. Monoclonal antibodies 8B1, against a V subunit (A), and 10D7, against a V subunit (B), were used to isolate complexes containing vacuolar H-ATPase subunits, and immunoprecipitated proteins were visualized by autoradiography. Positions of C-labeled molecular mass markers are indicated on the left (from top: 200, 97, 68, 43, 29, 18.4, and 14.3 kDa), and previously identified subunits of the yeast vacuolar H-ATPase are indicated by arrows on the right.




Figure 2: Disassembly and reassembly of the vacuolar H-ATPase do not require new protein synthesis. Yeast cells were converted to spheroplasts and labeled with TranS-label for 45 min as described under ``Experimental Procedures.'' Labeled spheroplasts were then lysed immediately (lane1), or chased for 20 min in YEPD buffered to pH 5 (lane2), 40 min in YEPD buffered to pH 5 (lanes 3 and 6), 20 min in YEP buffered to pH 5 (lanes4 and 7), or 20 min in YEP buffered to pH 5, followed by an additional 20 min with 2% glucose added (lanes5 and 8). In lanes 6-8, the chase medium contained 100 µg/ml cycloheximide. All of the chase media contained 1.2 M sorbitol. Monoclonal antibodies 8B1 (A) and 10D7 (B) were used, and immunoprecipitated proteins were visualized by autoradiography. Molecular mass markers and H-ATPase subunits are identified as in Fig. 1.



Fig. 1B shows the complexes immunoprecipitated from the same biosynthetically labeled spheroplasts by the 10D7 monoclonal antibody, which recognizes an epitope on the 100-kDa subunit that appears to be accessible only when the V subunits are not bound(15) . This antibody has been used to localize (15) and immunoprecipitate (14) V complexes under conditions where the peripheral V complex cannot bind to the membrane. The 10D7 antibody immunoprecipitates a complex containing the 100-, 36-, and 17-kDa subunits, in addition to several other proteins that have not been characterized, from wild-type cells suggesting that the cells contain a pool of assembled V complexes that are not associated with the V subunits (Fig. 1B, lanes1-5). After the chase period in no glucose medium (lane6), there is an increase in the amount of 100-, 36-, and 17-kDa subunits immunoprecipitated by the 10D7 antibody. The results in Fig. 1indicate that: 1) incubation in medium lacking glucose has caused dissociation of the peripheral V sector of the yeast vacuolar H-ATPase from the integral membrane V complex, and 2) after dissociation, the subunits of each sector, with the exception of the 42-kDa subunit, remain assembled.

The dissociation of the peripheral V sector would be expected to reduce the ATPase activity of vacuolar membranes. To assess this directly, vacuolar membrane vesicles were isolated from spheroplasts incubated in the presence or absence of glucose immediately before lysis. Vesicles prepared from spheroplasts incubated in medium containing 2% glucose contained 3.9 units/mg of vacuolar ATPase activity (where 1 unit = 1 µmol of ATP hydrolyzed/min and vacuolar ATPase activity is taken as the ATPase activity inhibited by 100 nM concanamycin A). The spheroplasts incubated in the absence of glucose yielded membranes containing only 1.5 units/mg vacuolar ATPase activity. This suggests that incubation of spheroplasts in the absence of glucose results in a loss of 61.5% of the vacuolar ATPase activity from the membranes.

The presence of relatively stable, assembled V and V sectors in the cells incubated in medium lacking glucose (Fig. 1) suggested that the disassembled complexes might remain competent for reassembly. To investigate whether disassembly of the vacuolar H-ATPase is a reversible process, we incubated labeled spheroplasts first with medium lacking glucose for 20 min and then added back glucose for 20 min and examined the immunoprecipitated complexes. As shown in Fig. 2(A and B, lane4), the assembled vacuolar H-ATPase complexes were extensively dissociated after 20 min in medium without glucose. However, if glucose was then added back to the cells for 20 min (Fig. 2, A and B, lane5), the complexes obtained were almost indistinguishable from those immunoprecipitated from cells incubated with glucose throughout the chase period (Fig. 2, A and B, lanes2 and 3). Neither disassembly nor reassembly required incubation of the spheroplasts at pH 5. Disassembly occurred in unbuffered medium lacking glucose, and reassembly occurred when glucose was added back to unbuffered medium.

All of the labeled subunits are present in complexes reassembled during the chase period, so new synthesis of subunits is probably not necessary for reassembly. Nonetheless, it is still possible that other proteins involved in disassembly or reassembly must be newly synthesized. To address this possibility, we included 100 µg/ml cycloheximide in the chase medium (Fig. 2, A and B, lanes 6-8) and examined the effects on the immunoprecipitated complexes. (This level of cycloheximide was shown previously to rapidly inhibit yeast protein synthesis(27) .) Comparison of lanes3 and 6 in Fig. 2shows that cycloheximide does not appear to have a direct effect on the assembled vacuolar H-ATPase complexes, and lanes7 and 8 demonstrate that both disassembly and reassembly can occur in the absence of new protein synthesis.

In order to obtain a quantitative estimate of the extent of disassembly and reassembly of the vacuolar H-ATPase in the presence and absence of glucose, the distribution of the 100-, 36-, and 17-kDa subunits between the complexes immunoprecipitated by the 8B1 antibody (assembled VV complexes) and the complexes immunoprecipitated by the 10D7 antibody (V subunits only) was measured using a phosphorimager. The results are shown in . In cells chased for 20 min in the presence of 2% glucose, 75-80% of the total quantity of these V subunits precipitated by both antibodies was co-precipitated with the V subunits, suggesting that most of the V subunits are found as part of fully assembled ATPase complexes. However, when the cells were subjected to a 20-min chase in the absence of glucose, only 22-33% of the subunits were immunoprecipitated by the 8B1 antibody and and 67-78% were precipitated by the 10D7 antibody, indicating that the three subunits are found predominantly in V complexes not containing V subunits. Readdition of glucose restored the distribution seen before cells were deprived of glucose.

Disassembly and reassembly of the yeast vacuolar H-ATPase were also visualized by indirect immunofluorescence microscopy, and the results are shown in Fig. 3and Fig. 4. Yeast vacuoles appear as depressions when cells are visualized under Nomarski optics ( Fig. 3and Fig. 4, A, C, and E; Ref. 20). Indirect immunofluorescence microscopy with the 13D11 antibody, which recognizes the 60-kDa peripheral subunit of the enzyme, shows colocalization of the V sector with the vacuolar membrane in yeast cells incubated in the presence of 2% glucose (Fig. 3B). After the cells have been deprived of glucose for 20 min, the same antibody shows diffuse staining of the cytoplasm, in addition to some staining of the vacuolar membrane (Fig. 3D). Restoration of glucose to the cells results in loss of cytoplasmic staining and restoration of bright vacuolar membrane staining (Fig. 3F). These results are entirely consistent with the results from immunoprecipitations described in Fig. 1and Fig. 2. Further evidence of disassembly and reassembly of the vacuolar H-ATPase complex was obtained from indirect immunofluorescence microscopy using the 10D7 monoclonal antibody (Fig. 4, B, D, and F). This antibody was previously shown to give little or no staining of wild-type cells grown in the presence of 2% glucose(15) , and in this experiment, there appears to be very little staining of cells incubated with glucose throughout (Fig. 4B) or cells deprived of glucose that subsequently had the glucose restored (Fig. 4F). However, the antibody did recognize the vacuolar membrane in cells deprived of glucose (Fig. 4D), suggesting that the epitope on the 100-kDa subunit is available in these cells. Previous experiments on mutants that fail to assemble the peripheral subunits onto the vacuolar membrane gave similar results(15) , indicating that exposure of the 10D7 antigen reflects disassembly of the peripheral membrane subunits from the integral membrane complex. The microscopy results in Fig. 3and 4 also eliminate any possibility that disassembly and reassembly occurs only in yeast spheroplasts, since whole cells were incubated in the presence and absence of glucose and fixed before they were converted to spheroplasts in these experiments.


Figure 3: Indirect immunofluorescence microscopy of the 60-kDa subunit in yeast cells treated under varied conditions. Cells were incubated in YEPD for 40 min (A and B), YEP for 20 min (C and D), or YEP for 20 min, followed by an additional 20 min with 2% glucose present (E and F) as described under ``Experimental Procedures.'' Cells were fixed and then stained with monoclonal antibody 13D11 to visualize the V sector. Identical fields were viewed under Nomarski optics (A, C, and E) or fluorescein fluorescence optics (B, D, and F).




Figure 4: Indirect immunofluorescence microscopy of the 100-kDa subunit in yeast cells treated under varied conditions. Cells were prepared as in Fig. 3, but were stained with monoclonal antibody 10D7 to visualize the V sector when it is not complexed with the V subunits. Identical fields were viewed under Nomarski optics (A, C, and E) or fluorescein fluorescence optics (B, D, and F).



The reversible dissociation of the yeast vacuolar H-ATPase in medium lacking any carbon source suggested that there might also be rapid changes in the ATPase structure when cells were shifted between different carbon sources. Yeast cells grown and biosynthetically labeled for 50 min in 2% glucose were shifted to three different carbon sources for a 20-min chase period, and the results are shown in Fig. 5. Incubation of the labeled complexes in 2% raffinose (lane4), 2% galactose (lane6), and 3% glycerol, 2% ethanol (lane8) resulted in disassembly of the vacuolar H-ATPase. Under all of these conditions, the 100-, 42-, 36-, and 17-kDa subunits appear to be lost from the complexes immunoprecipitated by the 8B1 antibody, and increased amounts of the 100-, 36-, and 17-kDa subunits appear in the V sectors precipitated by the 10D7 antibody. Readdition of glucose (to a final concentration of 2%) for 20 min could once again stimulate reassembly of the vacuolar H-ATPase from previously synthesized V and V sectors in all of these carbon sources (Fig. 5, lanes5, 7, and 9).


Figure 5: Disassembly and reassembly of the yeast vacuolar H-ATPase can occur in a variety of carbon sources. Yeast cells were converted to spheroplasts and labeled with Tran[S]-label for 50 min in medium containing 2% glucose. Labeled spheroplasts were lysed immediately (lane1) or chased for 20 min in YEPD (lanes2 and 3), YEP containing 2% raffinose (lanes4 and 5), YEP containing 2% galactose (lanes6 and 7), or YEP containing 3% glycerol/2% ethanol (lanes8 and 9). All chase media contained 1.2 M sorbitol. Glucose (final concentration 2%) was added to the samples shown in lanes3, 5, 7, and 9 after the initial 20-min incubation, and the incubation was continued for another 20 min. Monoclonal antibodies 8B1 (A) and 10D7 (B) were used in the immunoprecipitations, and precipitated proteins were visualized by autoradiography. Molecular mass markers and H-ATPase subunits are indicated as in Fig. 1.



The kinetics of disassembly of the vacuolar H-ATPase were examined by varying the chase time in medium lacking glucose. At the shortest chase times examined (2 min at 30 °C without glucose), the immunoprecipitated complexes were almost identical to those seen after 20 min in medium without glucose (data not shown). Similarly, the kinetics of reassembly were examined by immunoprecipitation of complexes at various times after restoration of glucose to labeled spheroplasts that had been deprived of glucose for 20 min. Reassembly also appeared to be complete after a 2-min incubation in glucose (data not shown). Although processing of the spheroplasts may have added as much as 2 min to the time between removal or addition of glucose and solubilization, both disassembly from glucose deprivation and reassembly with glucose readdition are essentially complete in less than 5 min.

In Fig. 6, the kinetics and extent of disassembly under several different conditions are compared. Immunoprecipitation data were quantitated using a phosphorimager, and the ratio of the 100- and 69-kDa subunits immunoprecipitated by the 8B1 antibody is shown as a measure of assembly and disassembly. The topcurve represents spheroplasts chased in glucose, and these data demonstrate that a small amount of assembly of the 100-kDa subunit with the 69-kDa subunit appears to continue during the chase period. However, incubation of the labeled spheroplasts in the absence of any carbon source or in the presence of galactose (2%) or glycerol/ethanol (3%/2%) induced a rapid dissociation of the 100-kDa subunit from the immune complexes precipitated by the 8B1 antibody. The 17- and 36-kDa subunits were lost at a similar rate under these conditions (data not shown). The disassembly may have been slightly slower in galactose than in the other carbon sources, but for each condition, almost all of the disassembly was complete by the 20-min chase point. (The apparent increase in the 100/69-kDa ratio at the 20-min time point in medium lacking glucose was not seen in other experiments.) It is also notable that the final extent of disassembly is similar under each of the different conditions. Complexes immunoprecipitated with the 8B1 antibody after a 40-min chase period in media that cause disassembly (i.e. no carbon source, galactose, and glycerol/ethanol) contain only 34 ± 2% as much 100-kDa subunit, 33 ± 6% as much 36-kDa subunit, and 25 ± 6% as much 17-kDa subunit as those immunoprecipitated after a chase period in 2% glucose.


Figure 6: Kinetics of vacuolar H-ATPase disassembly in different carbon sources. Spheroplasts were labeled for 45 min in medium containing 2% glucose (t = 0 for all samples), then shifted to YEPD (), YEP (), YEP containing 2% galactose (), or YEP containing 3% glycerol/2% ethanol (▾), all containing 1.2 M sorbitol, for an additional chase period at 30 °C. Aliquots were withdrawn after 5, 20, and 40 min of chase, and fully or partially assembled complexes of the vacuolar H-ATPase were immunoprecipitated with the 8B1 antibody as described under ``Experimental Procedures.'' The amount of radioactivity present in the 100-kDa and 69-kDa bands after SDS-polyacrylamide gel electrophoresis was quantitated using a phosphorimager, and the ratio of the two bands is expressed as a function of chase time.



The microscopy results shown in Fig. 3and Fig. 4indicate that disassembly and reassembly in the absence of glucose are not restricted to the vacuolar H-ATPase complexes synthesized within the 45-min labeling time of the immunoprecipitation experiments. This conclusion was further supported by Western blot analysis of the distribution of the 69- and 60-kDa subunits between supernatant and pellet fractions obtained from spheroplasts that had been incubated under varied conditions, lysed, and cross-linked with DSP as described under ``Experimental Procedures'' (Fig. 7). The peripheral subunits were found predominantly in the pellets from spheroplasts incubated in glucose before lysis (samples1 and 3), even if the spheroplasts had been subjected to a period of glucose deprivation prior to glucose readdition (sample3). However, if the spheroplasts were incubated in medium lacking glucose immediately before lysis (sample2), the peripheral subunits were found predominantly in the supernatant fraction.


Figure 7: Membrane attachment of peripheral subunits of the yeast vacuolar H-ATPase in cells incubated under varied conditions. Cells were grown overnight in YEPD (samples 1-3), YEP containing 2% raffinose (samples4 and 5), and YEP containing 3% glycerol/2% ethanol (sample6), then converted to spheroplasts. Spheroplasts were incubated for 20 min in YEPD (sample1), 20 min in YEP (sample2), 20 min in YEP followed by 20 min with glucose added to 2% (sample3), 20 min in YEP containing 2% raffinose (sample4), 20 min in YEP containing 2% raffinose followed by 20 min with 2% glucose added (sample5), and 20 min in YEP containing 3% glycerol/2% ethanol. All incubations of spheroplasts contained 1.2 M sorbitol. Spheroplasts were lysed, cross-linked, and fractionated into supernatant (S) and pellet (P) fractions as described under ``Experimental Procedures.'' The fractions were solubilized, subjected to SDS-polyacrylamide gel electrophoresis, and analyzed by Western blotting with monoclonal antibodies 8B1 (against the 69-kDa subunit) and 13D11 (against the 60-kDa subunit. Approximately 2.5 10 cells from each treatment were fractionated into supernatant and pellet fractions, and 1/50 of the total volume obtained from each fraction was loaded to obtain the blot shown.



All of the experiments described so far examine changes in assembly of vacuolar H-ATPase complexes that had initially been allowed to assemble in glucose-containing medium. However, the disassembly of the ATPase in a variety of different carbon sources suggests that assembly of the enzyme during growth on these carbon sources could also be affected. To address this, yeast cells were grown overnight in media containing 2% raffinose or 3% glycerol, 2% ethanol as their sole carbon source, and the distribution of the 69- and 60-kDa subunits between membrane (pellet) and supernatant fractions was determined by Western blot analysis (Fig. 7). In cells grown overnight in 2% raffinose (sample 4), the 69- and 60-kDa peripheral subunits appear to be predominantly in the supernatant fraction, indicating that they are either not assembled or not retained on the membrane efficiently. In cells grown overnight in glycerol/ethanol, approximately 50% of the peripheral subunits are found in the supernatant fraction (sample 6), suggesting that assembly or retention of the subunits on the membrane is less efficient than in glucose-grown cells but more efficient than in raffinose-grown cells.

The presence of the peripheral subunits in the supernatant from cells grown overnight in raffinose could result from a reduction in the amount of V sector relative to the V subunits so that there are no longer enough membrane-binding sites for the V subunits. However, when spheroplasts derived from cells grown in raffinose are treated for 20 min in glucose, the 69- and 60-kDa subunits are transferred almost quantitatively to the pellet fraction (Fig. 7, sample5). This suggests that there are existing V subunits or complexes present in the raffinose-grown cells that become available to bind V subunits after a relatively brief incubation in glucose. This possibility was further explored by biosynthetically labeling vacuolar H-ATPase complexes during growth on raffinose, and then examining the assembly of the 100-kDa subunit into VV complexes during a chase period in raffinose or glucose. Spheroplasts from cells grown overnight in 2% raffinose were labeled for 45 min with [S]methionine in the presence of 2% raffinose, then an excess of unlabeled methionine was added along with additional raffinose or glucose. shows the percentage of the total 100-, 36-, and 17-kDa subunits immunoprecipitated by the 8B1 antibody as part of VV complexes, as quantitated by phosphorimager analysis. There is very little change in the percentage of the V subunits coprecipitated by the 69-kDa subunit (8B1 antibody) during a 15-min chase in raffinose, suggesting that only about one-third of these subunits can assemble into complete VV complexes in raffinose, while the rest are in free V complexes. In contrast, addition of glucose for 15 min causes a significant redistribution of these subunits from free V complexes to fully assembled VV complexes. Although the 54-60% assembly of the V subunits into VV complexes is still lower than the 75-80% assembly shown in after incubation in glucose, it is clear that the free V complexes present in raffinose-grown cells must be able to be incorporated into fully assembled complexes when the proper signal is received.


DISCUSSION

The results reported here have clear implications for the structure of the yeast vacuolar H-ATPase and also suggest intriguing possibilities for enzyme regulation or redistribution of vacuolar H-ATPases in response to extracellular conditions.

Previous work has supported a model for V-type ATPases in which the V and V sectors may be structurally independent but are functionally interdependent(8, 9, 10, 11, 12, 13, 14) . Independent V and V complexes can clearly form in mutants lacking one subunit(14) , and a number of investigators have reported the presence of free V and V sectors in wild-type yeast cells as well as in other cell types(13, 14, 17, 28) . One possible explanation for these results is that fully assembled complexes are disassembled during biochemical purification. Although it is difficult to completely eliminate this possibility, the data presented here suggest that dissociation after lysis cannot account for all of the free V and V complexes. Clear differences in subunit distribution with different treatments are seen by a number of different methods, including immunoprecipitation under nondenaturing conditions, subcellular fractionation, and immunofluorescence microscopy, and in some cases, these differences have been supported by demonstrating functional differences in ATPase activity of isolated vacuoles. In order to minimize dissociation of the complex during purification, a cross-linking reagent was also included in both the immunoprecipitation and cell fractionation experiments. In the presence of the cross-linker, both the subunit composition of the immunoprecipitated complexes and the distribution of the V subunits between soluble and membrane fractions resemble those obtained previously in the absence of cross-linker(14) , with the exception of the 42-kDa subunit, which could only be co-precipitated in the presence of cross-linker. However, the amount of the V subunits coprecipitated with the V subunits and the distribution of subunits in experiments like that shown in Fig. 7were found to be more consistent between different experiments when the cross-linker was included. In the absence of cross-linker, there were some experiments when very little of the V subunits were coprecipitated by the 8B1 antibody and some experiments where almost all of the peripheral subunits were found in the supernatants from cell lysates, regardless of whether the cells had been incubated in glucose. This lack of consistency may well have been caused by variable loss of the 42-kDa subunit in the absence of cross-linking, because experiments on the clathrin-coated vesicle ATPase have indicated that although VV complexes can form in the absence of the comparable 40-kDa subunit, these complexes are unstable(29) . By approaching the distribution of the V and V subunits with a number of different, independent methods and limiting the possibility for dissociation of complexes after lysis, the data presented here provide strong support for indications that the V and V sectors of the V-type ATPases can exist separately in intact cells.

The most significant new structural insight from these results is that the free V and V subcomplexes may, in fact, be in a dynamic equilibrium with fully assembled complexes. Under conditions that promote ATPase disassembly, the 69-, 60-, 32-, and 27-kDa V subunits are removed as a complex to the cytosol, while the 100-, 36-, and 17-kDa V subunits remain assembled in the membrane. The 42-kDa subunit appears to be lost from both sectors, in accord with previous genetic (14) and biochemical data (29). More remarkably, yeast cells can reassemble intact vacuolar H-ATPase complexes using previously synthesized subunits in response to changes in extracellular conditions. Therefore, following disassembly in the absence of glucose, the integral V sector, the peripheral V sector, and the 42-kDa subunit must all remain competent for assembly. Both the rapid kinetics of diassembly and reassembly and the occurrence of both processes in the presence of cycloheximide indicate that any enzymatic activity necessary for signaling dissociation and reassociation of the complex must be contained within the assembled vacuolar H-ATPase itself or in other proteins synthesized during the initial 45-50-min labeling time. The free V and V subcomplexes described in yeast cells (14, 17) and bovine clathrin-coated vesicles (13, 28) may be intermediates in an ongoing process of assembly and disassembly of the collection of V-ATPases in these cells. In a more dynamic structural picture of the V-type ATPases, it becomes extremely important to understand the features holding the V and V together and how these structural features might respond to extracellular signals.

Although there has been extensive research into the cellular responses to changes in carbon sources and the mechanisms for sensing glucose, a unified picture is only beginning to emerge(30, 31, 32) . Growth in glucose, the preferred carbon source for rapid fermentative growth, results in transcriptional repression of many of the gene products involved in utilization of other carbon sources(32) . Upon a shift from glucose to a less-preferred carbon source, cells adapt by reversing the process of glucose repression and in some cases actively inducing genes required for metabolism of the available sugar(32) . Both raffinose and glucose are ultimately used in fermentation, but raffinose mediates weak glucose repression and glucose repression is relieved during growth on galactose. Glycerol/ethanol is a non-fermentable carbon source, and thus requires a still different set of proteins for metabolism. Despite the variety of eventual outcomes of shifting to these different carbon sources, the immediate disassembly of the vacuolar H-ATPase seen upon a shift to any of these carbon sources is very similar to that seen in the absence of any carbon source. Readdition of glucose to each of these carbon sources elicited a similar, rapid reassociation of the V and V sectors, as well. A large number of transcriptional and post-translational changes also occur when cells are shifted from respiratory or gluconeogenic growth to rapid fermentative growth(30, 32) . The changes in assembly of the vacuolar H-ATPase when cells are shifted from glucose are rapid and can occur in the presence of cycloheximide, suggesting a post-translational mechanism. However, cells grown overnight in ethanol/glycerol or raffinose also showed reduced levels of ATPase assembly, suggesting both long term and rapid effects of carbon source on ATPase assembly.

The disassembly and reassembly processes identified here could potentially provide a mechanism for regulating acidification of the yeast vacuole or other acidic organelles. If membrane attachment is required for ATPase activity in vivo, as it is in vitro, then removing the catalytic subunit, along with the other peripheral subunits, from the vacuolar membrane, could both reduce proton pumping and prevent unproductive ATP hydrolysis. Recently, it has been shown that rapid changes in transepithelial voltage seen at specific developmental stages of tobacco hornworm larvae were caused by a specific loss of the V subunits from the insect gut plasma membrane V-ATPase, which resulted in inactivation of ATPase activity and proton pumping(33) . It is entirely possible that the same structural changes that induce disassembly and reassembly in the yeast system also cause the dissociation of the insect vacuolar H-ATPase, and it is intriguing to speculate whether the same intracellular signal could operate in both systems. It is also interesting that the yeast plasma membrane H-ATPase is activated post-translationally by addition of glucose to cells in gluconeogenic or respiratory growth and by intracellular acidification (one consequence of growth in a rapidly fermenting sugar such as glucose)(30, 34) , and that activation is paralleled by changes in phosphorylation of the enzyme(35) . Activation of the plasma membrane H-ATPase when growth in glucose is initiated has been invoked as a mechanism of regulating cytoplasmic pH(34) , and an increased number of assembled vacuolar H-ATPases induced by addition of glucose could also assist in raising cytoplasmic pH. In addition, if cells contained excess V complexes, or multiple forms of the V complex, as results in yeast and in other systems have indicated(13, 36, 37, 38) , the reassembly process could potentially redistribute the V complexes to different organelles or change the distribution of the catalytic subunits among biochemically distinct complexes. Characterization of the molecular triggers of disassembly and reassembly should provide new insights into the structural interactions within the vacuolar H-ATPase as well as the mechanisms for regulation of organelle acidification.

  
Table: Percentages of 100-, 36-, and 17-kDa subunits immunoprecipitated as part of VV versus V complexes following different treatments

Yeast spheroplasts were labeled with TranS-label for 50 min and then incubated for an additional chase period in the presence or absence of glucose, as indicated. Assembled and partially assembled complexes of the vacuolar H-ATPase were immunoprecipitated using the 8B1 and 10D7 antibodies and analyzed by SDS-polyacrylamide gel electrophoresis as described under ``Experimental Procedures.'' The total amount of radioactivity in each of the 100-, 36-, and 17-kDa subunit bands precipitated by the two antibodies was quantitated using a PhosphorImager. The percentage of each subunit co-precipitated as part of fully assembled VV complexes was calculated by comparing the quantity immunoprecipitated by the 8B1 antibody to the total immunoprecipitated by both antibodies. The percentage immunoprecipitated by the 10D7 antibody (not shown) represents the portion of the subunits present in free V complexes not assembled with V subunits.


  
Table: Glucose induces assembly of VV complexes in cells grown in raffinose

Yeast spheroplasts were labeled with TranS-label in medium containing 2% raffinose for 45 min and then incubated for a chase period of 5 or 15 min after addition 2% raffinose or 2% glucose. Assembled and partially assembled complexes of the vacuolar H-ATPase were immunoprecipitated using the 8B1 and 10D7 antibodies and analyzed by SDS-polyacrylamide gel electrophoresis as described under ``Experimental Procedures.'' The percentage of each subunit co-precipitated by the 8B1 antibody as part of fully assembled VV complexes was calculated as described in Table I.



FOOTNOTES

*
This work was supported by National Science Foundation Presidential Young Investigator Award MCB-9296244 and National Institutes of Health Grant R01-GM50322. 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.

The abbreviations used are: H-ATPase, proton-translocating ATPase; YEPD, yeast extract-peptone-2% dextrose medium; YEP, 1% yeast extract-2% peptone medium without dextrose; SD-Met, supplemented minimal medium lacking methionine; DSP, dithiobis(succinimidylpropionate); PBS, phosphate-buffered saline.


ACKNOWLEDGEMENTS

I thank David Turner for the use of his microscope, Jianzhong Liu for assistance with photography, and Christine Tachibana for a critical reading of the manuscript.


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