Activity of Plasma Membrane V-ATPases Is Critical for the Invasion of MDA-MB231 Breast Cancer Cells^*

Materials and Antibodies

DMEM, FBS, penicillin-streptomycin, PBS, 0.05% trypsin-EDTA, Lipofectamine 2000, Blasticidin S, the Vivid Colors^TM pcDNA^TM6.2/N-EmGFP-GW/TOPO® mammalian expression vector, the mouse monoclonal antibody recognizing the V5 epitope, the Alexa Fluor® 488-conjugated goat anti-rabbit secondary antibody, the Alexa Fluor® 488-conjugated goat anti-mouse secondary antibody, the Alexa Fluor® 568 phalloidin antibody, the Alexa Fluor® 594 phalloidin antibody, and ProLong® Gold were purchased from Invitrogen. Aprotinin, leupeptin, and pepstatin were purchased from Roche Molecular Biochemicals. Precast polyacrylamide mini-protean Tris-glycine-extended gels, Tween 20, SDS, nitrocellulose membranes, and horseradish peroxidase-conjugated goat anti-mouse IgG were purchased from Bio-Rad. The chemiluminescence substrate for horseradish peroxidase was purchased from General Electric, and the signal was detected using Kodak BioMax Light film. A mouse monoclonal antibody that recognizes the V-ATPase V₁A subunit was purchased from Abnova, and mouse monoclonal antibodies against the V-ATPase V₀d subunit and the β1 subunit of the (Na⁺,K⁺)-ATPase (clone M17-P5-F11) were purchased from Abcam. A mouse monoclonal antibody recognizing α-tubulin was purchased from Genscript. The rabbit polyclonal antibody recognizing the V-ATPase V₁E subunit was obtained from Dr. Moshe Reuveni at the Department of Ornamental Horticulture of the Agricultural Research Organization Volcani Center (Bet-Dagan, Israel). SNARF-1 was purchased from Life Science Molecular Probes. Fluoroblok inserts with 8-μm pores were purchased from BD Biosciences, and Matrigel^TM was purchased from Corning. Zymolyase 20T was purchased from Seikagaku American, Inc. PMSF, the mouse monoclonal antibody against vinculin, calcein AM, streptavidin, concanamycin A, and all other chemicals were purchased from Sigma.

Cell Culture

The human breast cancer cell line MDA-MB231 was purchased from American Type Culture Collection. MB231 cells were grown in Falcon^TM T-75 flasks in DMEM with phenol red, 25 mm d-glucose, 4 mm l-glutamine, and 1 mm sodium pyruvate supplemented with 10% FBS, 60 μg/ml penicillin, and 125 μg/ml streptomycin. Cells were grown in a 95% air, 5% CO₂ humidified environment at 37 °C.

Plasmid Transfection

cDNA encoding the human c subunit was amplified by PCR and cloned into the Vivid Colors^TM pcDNA^TM6.2/N-EmGFP-GW/TOPO® mammalian expression vector to allow for C-terminal expression of the V5 epitope. GFP was removed from the plasmid, and successful deletion of GFP and insertion of the human c subunit cDNA were verified by sequencing. 15 μg of the plasmid was transfected into MB231 cells using Lipofectamine 2000 in accordance with the manufacturer's recommendations. Stable transfection was achieved by treatment of cells with 7.5 μg/ml blasticidin S for 21 days, beginning 3 days post-transfection. Cells were subsequently maintained under selective conditions.

Cell Lysis and Western Blotting

Cells were harvested by trypsinization, resuspended in lysis buffer with protease inhibitors (PBS-EDTA containing 137 mm NaCl, 1.2 mm KH₂PO₄, 15.3 mm Na₂HPO₄, 2.7 mm KCl, 2 mm EDTA, pH 7.2, 2 μg/ml aprotinin, 5 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 mm PMSF), and lysed by sonication. Cell lysates were centrifuged for 5 min at 4 °C at 500 × g to remove cellular debris. Protein concentrations were determined using the Lowry method (25). SDS sample buffer was added to the lysates, and proteins were separated by SDS-PAGE on 4–15% gradient acrylamide gels. The presence of the V5 epitope, subunit V₁A, subunit V₀d, α-tubulin, or vinculin was detected by Western blotting using mouse monoclonal antibodies followed by a horseradish peroxidase-conjugated secondary antibody. Blots were developed using chemiluminescent detection reagents. Western blots were quantified using ImageJ software with corrections made for loading.

Immunocytochemistry

A mouse monoclonal antibody raised against the V5 epitope was used to localize the V5-tagged subunit c in transfected cells, and a rabbit polyclonal antibody against the V-ATPase subunit V₁E was used to localize the V-ATPase in untransfected and transfected cells. Cells were plated onto 24 × 24-mm coverslips coated with poly-d-lysine in 6-well plates. Approximately 24 h later, cells were washed, fixed with 4% paraformaldehyde, and either permeabilized with 0.1% Triton-X-100 or incubated in PBS for nonpermeabilized cells. Nonspecific binding was blocked by incubation with 1% bovine serum albumin in PBS for 1 h. Cells were then incubated with the anti-V5 antibody at a 1:5000 dilution or the anti-V₁E antibody at a 1:1000 dilution overnight at 4 °C, washed with PBS, and then incubated with Alexa Fluor® 594 phalloidin or Alexa Fluor® 568 phalloidin to stain F-actin (1:250 dilution) and the Alexa Fluor® 488-conjugated goat anti-mouse secondary antibody (1:1000 dilution) or the Alexa Fluor® 488-conjugated goat anti-rabbit secondary antibody (1:500 dilution). After 2 h of incubation at room temperature, the cells were washed with PBS. The cells were prepared for viewing using ProLong® Gold mounting medium and allowed to cure at room temperature for 24 h. The stained cells were imaged using a Leica TCS SPE confocal microscope. The results from three separate experiments were analyzed. To quantify plasma membrane V-ATPase staining, 90 cells from the three separate experiments were counted, and the percentage of cells showing plasma membrane V-ATPase was determined.

Cytosolic pH Measurements

Cells were grown on rectangular coverslips (8 × 22 mm) and treated overnight with or without the anti-V5 antibody. Cells were then loaded with SNARF-1 (5-[and-6] carboxy-SNARF-1-AM) to measure cytosolic pH. Cells were transferred to a cell perfusion system and were continuously perfused at 3.0 ml/min at 37 °C with cell superfusion buffer containing 1.3 mm CaCl₂, 1 mm MgSO₄, 5.4 mm KCl, 0.44 mm KH₂PO₄, 110 mm NaCl, 0.35 mm NaH₂PO₄, 5 mm glucose, 2 mm glutamine, and 20 mm HEPES at pH^ex 7.4. The emission spectra of SNARF-1 (644/584 nm) were acquired using 534 nm as the excitation wavelength. The conversion of ratio values to cytosolic pH (pH_cyt) was performed as previously described (18). The fluorescence was monitored with a SLM-8100/DMX spectrofluorometer (Spectronics Instruments, Rochester, NY) equipped for sample perfusion. The sample temperature was maintained at 37 °C by keeping both the water jacket and perfusion media at 37 °C using an iso-temperature immersion circulator water bath (Lauda model RM20; Brinkmann Instruments, Westbury, NY). All measurements were performed using 4-nm-bandpass slits and an external rhodamine standard as a reference. Fluorescence data were converted to ASCII format for analyses.

In Situ Calibration of SNARF-1

In situ calibration curves were generated as described previously (18). Briefly, the cells attached to coverslips were perfused with High K⁺ buffer (10 mm NaCl, 146 mm KCl, 10 mm HEPES, 10 mm MES, 10 mm Bicine, 2 μm valinomycin, 6.8 μm nigericin, 5 mm glucose, 2 mm glutamine, pH 5.5–8.0 adjusted with NaOH). The buffer contains high K⁺ to approximate the intracellular K⁺ concentration. Nigericin is an ionophore that exchanges H⁺ and K⁺ across the membrane, rendering the pH_cyt equal to the extracellular pH (pH_ex). Valinomycin is an ionophore that moves K⁺ across the plasma membrane and, together with nigericin, helps to equilibrate pH_cyt and pH_ex. The pH of the buffers was determined using a Beckman pH meter with a glass electrode (Corning Inc., Horseheads, NY) calibrated at 37 °C with commercially available standard solutions (VWR Scientific, San Francisco, CA). The ratios (r = 644/584) of SNARF-1 were converted to pH using a modified Henderson-Hasselbalch equation (26). The equation was solved using nonlinear least squares analysis with Sigma Plot to obtain the values of pK_a, R_min, and R_max for SNARF-1 in these cells.

Plasma Membrane Proton Flux Measurements

The initial rate of pH_cyt recovery from an acid load induced by K⁺-acetate was measured as the dpH/dt, which is the slope of the linear regression curve relating time and pH_cyt. Cells loaded with SNARF-1 were perfused with cell superfusion buffer until the steady-state pH_cyt was reached. The cells were then perfused with 50 mm K⁺-acetate in HCO₃⁻ and Na⁺-free cell superfusion buffer to eliminate the contribution of potential HCO₃⁻ transporters and Na⁺/H⁺ exchanger. The perfusion with K⁺-acetate will cause a rapid intracellular acidification followed by a subsequent pH_cyt recovery. We then evaluated the pH_cyt recovery from this acidification within the first 3 min. The individual pH_cyt data points were then used to plot a linear regression curve relating time and ΔpH_cyt. To quantify the pH_cyt recovery, we expressed the recoveries as proton flux (J_H+), which is given by the apparent H⁺ buffering (B_H+ capacity multiplied by the dpH/dt) (27).

Cell Fractionation and Analysis of V-ATPase Assembly

Cell fractionation was performed as previously described (28). Cells were harvested by trypsinization, washed in cold PBS, and resuspended in homogenization buffer (250 mm sucrose, 1 mm EDTA, 10 mm HEPES, 2 μg/ml aprotinin, 2 μg/ml leupeptin, 2 μg/ml pepstatin, and 1 mm PMSF). Cells were then homogenized using a ball-bearing homogenizer that allows 10 μm of clearance. Cells were passed through the homogenizer 20 times, and the lysate was centrifuged at 500 × g for 10 min at 4 °C to pellet nuclei and cell debris. The supernatant was then centrifuged at 100,000 × g for 30 min at 4 °C to separate the membrane and cytosolic fractions. The cytosolic fraction was concentrated using an Amicon Ultra filter with a 10-kDa cut-off, and the membrane pellet was resusupended in homogenization buffer. Protein concentration was determined by the Lowry method (25). Protein from each fraction was separated by SDS-PAGE, and a Western blot was performed as described above. The anti-V_od antibody and the anti-vinculin antibody were used as loading controls for the membrane fraction and the cytosolic fraction, respectively, and the anti-V₁A antibody was used to assess the relative amounts of the V₁ domain in the cytosolic and membrane fractions. Western blots were quantitated using ImageJ software with corrections made for loading. The ratio of the amounts of the V₁A subunit in the membrane fraction versus the cytosolic fraction was calculated and served as a measurement of V-ATPase assembly. The ratio for each experimental condition tested was divided by the ratio observed for untreated MB231 cells to normalize the results. To examine whether treatment with the anti-V5 antibody altered V-ATPase assembly, cells were treated with or without anti-V5 at a concentration of 1:500 for 8 h prior to harvesting.

Invasion and Migration Assays

Assays for in vitro invasion and migration were performed as described previously (17–19). Fluoroblok inserts with an 8-μm pore size membrane were placed into the wells of a 24-well plate (29). To evaluate invasion, Matrigel^TM was diluted to a final concentration of 0.2 μg/μl, and a total of 11 μg was coated onto the membrane in each well. The membrane was allowed to dry overnight under vacuum at room temperature. Matrigel^TM-coated membranes were rehydrated with 60 μl of DMEM plus phenol red, 25 mm d-glucose, 4 mm l-glutamine, and 1 mm sodium pyruvate (termed “Medium”) for at least 2 h. For evaluation of the effects of the anti-V5 antibody on in vitro invasion and migration, cells were harvested by trypsinization and brought to a concentration of 1.75 × 10⁵ cells/ml in Medium containing DMSO, 100 nm of the V-ATPase-specific inhibitor concanamycin A (positive control), or the monoclonal anti-V5 antibody at a dilution of 1:500. Cells were incubated for 15 min on ice, and then 5.0 × 10⁴ cells were seeded onto the Matrigel^TM-coated membranes (invasion) or uncoated membranes (migration). Inserts were then placed into wells of a 24-well plate that contained 500 μl of Medium plus 5% FBS to serve as a chemoattractant. After 8 h at 37 °C, membranes were placed into wells containing 4 μg/ml calcein AM in PBS and incubated for 5 min at 37 °C in 5% CO₂. Cells that had migrated or invaded to the trans-membrane were quantitated using a Zeiss Axiovert 10 fluorescence microscope, and an average of 10 images were taken per well. To confirm that antibody binding to an extracellular epitope does not by itself affect invasiveness, we performed an overnight in vitro Matrigel^TM invasion assay with MB231 cells treated with or without an antibody against the extracellular epitope of the β1 subunit of the (Na⁺,K⁺)-ATPase (1:250 dilution). The number of invading cells was averaged over three wells per condition with all experiments performed at least three times. For evaluation of the effects of streptavidin-bound, biotin-conjugated bafilomycin on in vitro invasion and migration, 1500 nm biotin-conjugated bafilomycin (received as a gift from Dr. Markus Huss, Universität Osnabrück in Osnabrück, Germany) and 750 nm streptavidin were allowed to bind by rotating the compounds together for 1 h at 4 °C. Invasion and migration assays were performed as described above, with cells being brought to a concentration of 1.75 × 10⁵ cells/ml in Medium containing DMSO with 750 nm streptavidin, 100 nm concanamycin A, or 1500 nm biotin-conjugated bafilomycin with 750 nm streptavidin and incubated for 15 min at 37 °C before being seeded onto the coated or uncoated membranes.

Yeast Culture Conditions and Isolation of Vacuolar Membrane Vesicles

The yeast strain YPH500 (MATα, ura3-52, lys2-801amber, ade2-101ochre, trp1-Δ63, his3-Δ200, leu2-Δ1) (30) was maintained in YPD (1% yeast extract, 2% bacto-peptone, 2% glucose) buffered to pH 5.5. Vacuolar membranes were isolated as previously described (31). Briefly, YPH500 was grown overnight to A₆₀₀ of 1.2–1.5 in 1 liter of YPD, pH 5.5. The cells were then pelleted, washed with water, and resuspended in 100 ml of 100 mm Tris-HCl, pH 9.4, with 10 mm dithiothreitol. Cells were then incubated for 20 min at 30 °C at 100 rpm before washing in 50 ml of YPD medium containing 0.7 m sorbitol, 2 mm dithiothreitol, and 100 mm MES-Tris, pH 7.5. After resuspension in 50 ml of YPD containing 0.7 m sorbitol, 2 mm dithiothreitol, 100 mm MES-Tris, pH 7.5, and 20 mg of Zymolyase 20T, yeast were incubated for 1 h at 30 °C at 100 rpm. The spheroplasts were osmotically lysed, and vacuoles were isolated on two consecutive Ficoll gradients and diluted in transport buffer (15 mm MES-Tris, pH 7.0, 4.8% glycerol), and protein content was measured using the Lowry method (25).

Assessment of ATP-dependent Proton Transport Activity in Yeast

ATP-dependent proton transport was measured as described previously (31, 32). Fluorescence quenching of the fluorescence probe 9-amino-6-chloro-2-methoxy-acridine in transport buffer (25 mm MES-Tris, pH 7.2, 5 mm MgCl₂) was measured in the presence or absence of DMSO with 0.5 μm streptavidin, 1 μm biotin-conjugated bafilomycin, or 1 μm biotin-conjugated bafilomycin bound to 0.5 μm streptavidin. Biotin-conjugated bafilomycin and streptavidin were allowed to bind by rotating the compounds together for 1 h at 4 °C before starting experiments.

Statistical Analysis

The graphed data are averages from the indicated number of independent experiments. A Student's t test was used to calculate significance. p < 0.05 was considered significant.

MDA-MB231 Cells Stably Transfected with a V5-tagged Construct of Subunit c of the V-ATPase Express the V5 Epitope at the Plasma Membrane

To determine whether plasma membrane V-ATPases are critical to the invasiveness of MDA-MB231 cells, cells were first stably transfected with a construct expressing the proteolipid c subunit of the V-ATPase tagged with the V5 epitope at the C terminus (termed c-V5). The C terminus of subunit c has previously been shown to be present on the luminal surface of the protein such that, for a V-ATPase complex present at the plasma membrane, this epitope would be exposed on the extracellular surface of the membrane (33). Furthermore, because the c subunit is present in several copies per V-ATPase, selection of this subunit for epitope tagging enhances the likelihood that the epitope will be incorporated into most, if not all, of the V-ATPases in the cell (7, 34). Expression of the c-V5 construct in the MB231 transfected cells was verified by Western blotting of whole cell lysates (Fig. 1A). We next wanted to determine whether c-V5-containing V-ATPases were present at the plasma membrane. To address this, immunocytochemistry was conducted using an anti-V5 antibody in cells that either were or were not permeabilized by treatment with 0.1% Triton X-100. As shown in Fig. 1B, permeabilized cells were readily stained with phalloidin, which stains the intracellular marker F-actin, whereas nonpermeabilized cells showed no phalloidin staining, confirming that cells remained impermeable to antibodies under the conditions of fixation employed. Staining with the anti-V5 antibody was observed in both permeabilized and nonpermeabilized c-V5-expressing cells. In permeabilized cells, both diffuse and punctate staining for V5 was observed, with significant perinuclear staining, consistent with localization to intracellular compartments such as the Golgi and lysosomes. By contrast, in nonpermeabilized cells, only diffuse staining with the anti-V5 antibody was observed, indicating the presence of V5-tagged V-ATPase complexes diffusely distributed over the surface of cells (Fig. 1B). No staining with the V5 antibody was observed in untransfected MB231 cells (data not shown), indicating that the antibody specifically targets the V5 epitope.

Treatment of c-V5-expressing Cells with an Anti-V5 Antibody Causes Acidification of the Cytoplasm

To investigate the effect of addition of the anti-V5 antibody on cytosolic pH (pH_cyt) in cells expressing the c-V5 construct, the fluorescence pH indicator SNARF-1 was employed. Transfected and untransfected MB231 cells were loaded with SNARF-1, and cytosolic pH was determined by measuring the ratio of fluorescence intensity at 644 and 584 nm upon excitation at 534 nm, as described under “Experimental Procedures.” As shown in Fig. 2, c-V5-expressing cells had a more alkaline cytosolic pH than untransfected cells. Addition of the anti-V5 antibody decreased the pH_cyt of c-V5-expressing cells but did not affect the pH_cyt of untransfected cells.

To determine whether the higher cytosolic pH observed in c-V5-expressing cells was due to an increase in total V-ATPase expression, we examined the protein levels of subunit A (part of the V₁ domain) and subunit d (part of the V₀ domain) by Western blotting of whole cell lysates using anti-A and anti-d subunit antibodies. As shown in Fig. 3A, no difference in expression levels of subunit A or subunit d was observed between untransfected and transfected MB231 cells, indicating that expression of the c-V5 protein did not alter total V-ATPase expression. Next, we assessed whether the more alkaline cytosolic pH observed in c-V5-expressing cells was due to an alteration in V-ATPase assembly. To test this, untransfected and transfected cells were homogenized, and the cytosolic and membrane fractions were isolated by high speed centrifugation followed by Western blotting using antibodies against subunits A and d (28). A change in assembly would be reflected as a shift in subunit A between the cytosolic and membrane fractions. No difference in assembly was observed between untransfected and transfected cells (Fig. 3B). Finally, we assessed whether the more alkaline pH in transfected cells was a result of increased V-ATPase expression at the plasma membrane. To address this, immunocytochemistry was performed using an anti-V₁E antibody on permeabilized cells. This subunit is present in all of the V-ATPases in the cell (7). Phalloidin was used to stain actin and outline the cell periphery. Colocalization of subunit E and phalloidin at the cell periphery was used to identify plasma membrane V-ATPases, as previously described (18, 19). As shown in Fig. 3C, untransfected and transfected cells exhibited a similar localization of V-ATPases, and a similar fraction of cells showed staining for V-ATPases at the cell periphery. Although it remains unclear why c-V5-transfected cells experience a more alkaline cytosolic pH, our results suggest that expression of c-V5 does not alter V-ATPase expression, assembly, or localization in MB231 cells.

Treatment of c-V5-expressing Cells with an Anti-V5 Antibody Inhibits Proton Flux across the Plasma Membrane

To assess whether addition of the anti-V5 antibody inhibits proton flux across the plasma membrane of c-V5-expressing cells, the rate of pH_cyt recovery following an acute acid load induced by K⁺-acetate was monitored as described under “Experimental Procedures.” Cells loaded with SNARF-1 were first perfused with buffer in the absence of HCO₃⁻ and Na⁺ to eliminate the contributions of HCO₃⁻ transporters and Na⁺/H⁺ exchangers to proton flux. Cells were subsequently equilibrated with 50 mm K⁺-acetate, resulting in a rapid intracellular acidification followed by a subsequent pH_cyt recovery. The rate of proton extrusion (J_H+) during the first 3 min of pH_cyt recovery was measured. As shown in Fig. 4A, no difference in proton flux across the plasma membrane was observed between untransfected and transfected cells. Upon addition of the anti-V5 antibody, proton flux was significantly reduced in c-V5-expressing cells but not in untransfected cells. These results suggest that the anti-V5 antibody inhibits proton flux across the plasma membrane of c-V5-transfected cells by blocking the activity of V-ATPases.

Next, we determined whether the anti-V5 antibody inhibits V-ATPase activity in c-V5-expressing cells by reducing assembly of the two functional domains. We incubated untransfected and transfected cells in the presence or absence of the anti-V5 antibody for 8 h and assessed V-ATPase assembly as described above. As shown in Fig. 4B, treatment of transfected cells with the anti-V5 antibody did not cause a decrease in V-ATPase assembly. The small—and not significant—increase in assembly observed after antibody treatment may be the result of reduced disassembly of the V-ATPase complex caused by inhibition of activity, because dissociation of the V-ATPase has been shown to be activity-dependent (35).

Treatment of c-V5-expressing MB231 Cells with an Anti-V5 Antibody Inhibits in Vitro Invasion and Migration

We next determined what effect addition of the anti-V5 antibody would have on the invasiveness of the c-V5-expressing cells. To address this, invasion was measured using an in vitro Matrigel^TM assay. Cells were incubated in the presence or absence of the anti-V5 antibody, plated on Matrigel^TM-coated wells and induced to invade using a chemoattractant (fetal bovine serum) on the trans-side of the well. After 8 h, cells on the trans-side were stained with calcein AM and counted as described under “Experimental Procedures.” As shown in Fig. 5A, transfected cells displayed a similar degree of invasiveness as untransfected cells, indicating that expression of the c-V5 construct has no effect on MB231 cell invasion. Both cell lines show reduced invasion after treatment with the membrane-permeable, V-ATPase-specific inhibitor concanamycin A, as previously described (17–19). By contrast, whereas addition of the anti-V5 antibody had no effect on invasion of untransfected cells, invasion by the c-V5-expressing cells was significantly inhibited after antibody treatment (Fig. 5A). To confirm that the binding of an antibody to an extracellular epitope does not by itself reduce invasiveness, we treated MB231 cells with an antibody against an extracellular epitope of the β1 subunit of the (Na⁺,K⁺)-ATPase, which is found on the plasma membrane of all cells. Anti-β1 treatment did not alter MB231 cell invasiveness compared with untreated cells (data not shown). This confirms that the reduction in invasiveness observed upon treatment of cells with the anti-V5 antibody is a specific effect. Overall, our results suggest that inhibition of plasma membrane V-ATPases in c-V5-expressing MB231 cells is sufficient to block their invasion.

Because invasiveness of cells also requires cell migration, we assessed the effect of the anti-V5 antibody on the ability of the c-V5-expressing cells to migrate using an in vitro migration assay. As shown in Fig. 5B, both transfected and untransfected cells display reduced migration after treatment with a V-ATPase inhibitor, as previously observed (17). However, treatment with the anti-V5 antibody reduced migration of only the c-V5-expressing cells.

Treatment of MB231 Cells with a Membrane-impermeable Form of the V-ATPase-specific Inhibitor Bafilomycin Inhibits in Vitro Invasion and Migration

To confirm the role of plasma membrane V-ATPases in tumor cell invasion, we next employed an alternative method to specifically inhibit plasma membrane V-ATPases in MB231 cells. We utilized a biotin-conjugated form of bafilomycin, a membrane-permeable and specific inhibitor of the V-ATPase, and allowed this compound to bind to streptavidin to prevent its ability to cross the plasma membrane. First, we sought to confirm that biotin-bafilomycin is membrane-impermeable when bound to streptavidin. Although sufficient quantities of streptavidin-biotin-bafilomycin were not available to perform the cytoplasmic pH and proton flux experiments described above, we assessed ATP-dependent proton transport in vacuolar membranes isolated from yeast in the presence and absence of this compound. Bafilomycin binds to the V-ATPase at a site within the ring of c subunits (36). We expected that when bound to streptavidin, biotin-bafilomycin would be able to inhibit activity from the luminal (or extracellular) surface but because of steric interference between streptavidin and the V₁ domain would be unable to inhibit the pump from the cytoplasmic surface. We isolated vacuoles from the yeast strain YPH500 and measured ATP-dependent proton transport by 9-amino-6-chloro-2-methoxyacridine fluorescence quenching, as described previously (31, 32), in the presence of biotin bafilomycin or streptavidin-bound biotin bafilomycin. As shown in Fig. 6A, the addition of streptavidin alone had no significant effect on proton transport activity relative to vacuoles treated with DMSO. Treatment with 1 μm biotin-bafilomycin completely ablated ATP-dependent proton transport activity, consistent with V-ATPase inhibition. When biotin-bafilomycin was bound to streptavidin, its ability to inhibit proton transport was prevented. This suggests that biotin bafilomycin is membrane-impermeable when bound to streptavidin.

We next treated MB231 cells with the membrane-impermeable, streptavidin-bound biotin-bafilomycin to determine whether inhibition of plasma membrane V-ATPases reduces invasion. After confirming that treatment with biotin-bafilomycin does not induce MB231 cell death (data not shown), we performed in vitro invasion assays and allowed MB231 cells to invade in the presence or absence of streptavidin-bound biotin-bafilomycin for 8 h. As shown in Fig. 6B, we found that the in vitro invasiveness of these cells is significantly reduced after treatment with this compound. This reduction in invasiveness is similar to what is observed after treatment with a membrane-permeable inhibitor. The in vitro migratory capability of MB231 cells was also decreased following treatment with streptavidin-biotin-bafilomycin (Fig. 6C). These results thus further support the role of plasma membrane V-ATPases in the invasiveness of breast cancer cells.