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• W2144856874 abstract "A method for obtaining giant protoplasts ofEscherichia coli (the spheroplast incubation (SI) method: Kuroda et al. (Kuroda, T., Okuda, N., Saitoh, N., Hiyama, T., Terasaki, Y., Anazawa, H., Hirata, A., Mogi, T., Kusaka, I., Tsuchiya, T., and Yabe, I. (1998) J. Biol. Chem. 273, 16897–16904) was adapted to haploid cells of Saccharomyces cerevisiae. The yeast cell grew to become as large as 20 μm in diameter and to contain an oversized vacuole inside. A patch clamp technique in the whole cell/vacuole recording mode was applied for the vacuole isolated by osmotic shock. At zero membrane potential, ATP induced a strong current (as high as 100 pA; specific activity, 0.1 pA/μm2) toward the inside of the vacuole. Bafilomycin A1, a specific inhibitor of the V-type ATPase, strongly inhibited the activity (K i = 10 nm). Complete inhibition at higher concentrations indicated that any other ATP-driven transport systems were not expressed under the present incubation conditions. This current was not observed in the vacuoles prepared from a mutant that disrupted a catalytic subunit of the V-type ATPase (RH105(Δvma1::TRP)). TheK m value for the ATP dose response of the current was 159 μm and the H+/ATP ratio estimated from the reversible potential of the V-I curve was 3.5 ± 0.3. These values agreed well with those previously estimated by measuring the V-type ATPase activity biochemically. This method can potentially be applied to any type of ion channel, ion pump, and ion transporter inS. cerevisiae, and can also be used to investigate gene functions in various organisms by using yeast cells as hosts for homologous and heterogeneous expression systems. A method for obtaining giant protoplasts ofEscherichia coli (the spheroplast incubation (SI) method: Kuroda et al. (Kuroda, T., Okuda, N., Saitoh, N., Hiyama, T., Terasaki, Y., Anazawa, H., Hirata, A., Mogi, T., Kusaka, I., Tsuchiya, T., and Yabe, I. (1998) J. Biol. Chem. 273, 16897–16904) was adapted to haploid cells of Saccharomyces cerevisiae. The yeast cell grew to become as large as 20 μm in diameter and to contain an oversized vacuole inside. A patch clamp technique in the whole cell/vacuole recording mode was applied for the vacuole isolated by osmotic shock. At zero membrane potential, ATP induced a strong current (as high as 100 pA; specific activity, 0.1 pA/μm2) toward the inside of the vacuole. Bafilomycin A1, a specific inhibitor of the V-type ATPase, strongly inhibited the activity (K i = 10 nm). Complete inhibition at higher concentrations indicated that any other ATP-driven transport systems were not expressed under the present incubation conditions. This current was not observed in the vacuoles prepared from a mutant that disrupted a catalytic subunit of the V-type ATPase (RH105(Δvma1::TRP)). TheK m value for the ATP dose response of the current was 159 μm and the H+/ATP ratio estimated from the reversible potential of the V-I curve was 3.5 ± 0.3. These values agreed well with those previously estimated by measuring the V-type ATPase activity biochemically. This method can potentially be applied to any type of ion channel, ion pump, and ion transporter inS. cerevisiae, and can also be used to investigate gene functions in various organisms by using yeast cells as hosts for homologous and heterogeneous expression systems. spheroplast incubation method 2-deoxy-d-glucose vacuolar proton-translocating ATPase dithiothreitol 2-(N-morpholino)ethanesulfonic acid (3-morpholino)propanesulfonic acid 5(and 6)-carboxy-2′,7′-dichlorofluorescein propidium iodide ohm For the evaluation of ion transport systems, the patch clamp method developed by Nehr and Sackman (1Nehr E. Sakman B. Nature. 1976; 260: 799-802Crossref PubMed Scopus (1531) Google Scholar) in 1976 is one of the most direct and quantitative assay techniques which can be conducted under conditions similar to those in vivo. This technique, therefore, could potentially be one of the most powerful assay tools for the identification of transporter genes as well. Some transporter genes have been identified by introducing them into a heterogeneous expression system of Xenopus oocyte as a host for patch clamp recording (or two-electrode voltage clamp recording). Those genes had to be ones that Xenopus oocyte cells did not express or scarcely expressed on the Xenopus genome. For example, the genes for neurotransmitter receptor channels (2Mishina M. Kurosaki T. Tobimatsu T. Morimoto Y. Noda M. Yamamoto T. Treao M. Lindstrom J. Takahashi T. Kuno M. Numa S. Nature. 1984; 307: 604-608Crossref PubMed Scopus (248) Google Scholar, 3Masu Y. Nakayama K. Tamaki H. Harada Y. Kuno M. Nakanishi S. Nature. 1987; 329: 836-838Crossref PubMed Scopus (553) Google Scholar) and carrier-type ion transporters such as the Na+/Ca2+antiporter (4Hilgemann D.W. Nicoll D.A. Phillipson K.D. Nature. 1991; 352: 715-718Crossref PubMed Scopus (178) Google Scholar) were successfully identified using this particular method. Since the gene manipulation technique can be applied much more easily to eukaryotic unicellular yeast cells than to animal cells, Kung and associates (5Gustin M.C. Matinac B. Saimi Y. Culbertson M.R. Kung C. Science. 1986; 233: 1195-1197Crossref PubMed Scopus (104) Google Scholar) first tried to identify some transporter genes inSaccharomyces cerevisiae. Thus far, only one gene of an outward-rectifying K+ channel, TOK1(=DUK1=YKC1=YORK, Refs. 6Ketchum K.A. Joiner W.J. Sellers A.J. Kaczmarek L.K. Goldstein S.A.N. Nature. 1995; 376: 690-695Crossref PubMed Scopus (365) Google Scholar, 7Lesage F. Guillemare E. Fink M. Duprat F. Lazdunski M. Romey G. Barhanin J. J. Biol. Chem. 1996; 271: 4183-4187Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 8Zhou X-L. Vaillant B. Loukin S. H. Kung C. Saimi Y. FEBS Lett. 1995; 373: 170-176Crossref PubMed Scopus (76) Google Scholar, 9Reid J.D. Lukas W. Shafaatian R. Bertl A. Scheumann-Kettner C. Guy H.R. North R.A. Recept. Channels. 1996; 4: 51-62PubMed Google Scholar, 10Bertl A. Bihler H. Reid J.D. Kettner C. Slayman C.L. J. Membr. Biol. 1998; 162: 67-80Crossref PubMed Scopus (48) Google Scholar, 11Loukin S.H. Vaillant B. Zhou X.-L. Spalding E.P. Kung C. Saimi Y. EMBO J. 1997; 16: 4817-4825Crossref PubMed Scopus (53) Google Scholar, 12Vergani P. Miosga T. Jarvis S.M. Blatt M.R. FEBS Lett. 1997; 405: 337-344Crossref PubMed Scopus (31) Google Scholar) has been identified. For successful, quantitative patch clamp assays of ion pumps and carrier-type transporters that do not accompany high ionic currents, the sizes of the protoplasts are crucial; the small size of the cell used for the above study allowed identification only of ion channels with high ionic currents. For that reason, Bertl and co-workers (13Bertl A. Slayman C.L. J. Exp. Biol. 1992; 172: 271-287Crossref PubMed Google Scholar) tried to prepare yeast protoplasts as large as 20 μm in diameter by digesting the cell wall using enzymes followed by prolonged incubation in an osmotically protective medium containing 200 mm KCl. It should be noted that they added no inhibitor of cell wall synthesis. They reported an ATP-induced current flow on the giant vacuole isolated from these oversized protoplasts (14Bertl A. Bieler H. Kettner C. Slayman C.L. Pflugers Arch. Eur. J. Physiol. 1998; 436: 999-1013Crossref PubMed Scopus (49) Google Scholar). They appeared to have used polyploid cells, which were much larger than haploid cells to begin with, in order to obtain giant protoplasts as large as 20 μm. Polyploid cells, however, are not suitable for obtaining a disrupted mutant through genetic manipulation, because all the target genes on the multiple chromosomes have to be disrupted in order to obtain a stable phenotype. Recently, we succeeded in converting Escherichia coli cells into giant protoplasts that are suitable for patch clamp experiments (15Kuroda T. Okuda N. Saitoh N. Hiyama T. Terasaki Y. Anazawa H. Hirata A. Mogi T. Kusaka I. Tsuchiya T. Yabe I. J. Biol. Chem. 1998; 273: 16897-16904Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). This spheroplast incubation method (SI method)1 was a modification of the giant cell preparation method originally developed forBacillus megaterium by Kusaka (16Kusaka I. J. Bacteriol. 1967; 96: 884-888Crossref Google Scholar) in which giant protoplasts are formed after prolonged incubation of spheroplasts formed by treating cells with lysozyme in the presence of both penicillin G, an inhibitor of peptidoglycan synthesis, and an osmo-protectant. We have decided to apply a similar technique to haploid cells ofS. cerevisiae, and chose 2-deoxy-d-glucose (2-DG), a hydrophilic inhibitor of cell wall synthesis, since Biely and co-workers (17Bieley P. Kratky Z. Kovarik J. Bauer S. J. Bacteriol. 1971; 107: 121-129Crossref PubMed Google Scholar) found that a small amount of 2-DG, an analogue of glucose, specifically inhibits cell-wall synthesis without a significant effect on protein synthesis. In the present paper, we describe a method for preparing giant protoplasts of S. cerevisiae as large as 20 μm from haploid cells of the wild type and also a mutant that lacked the V-type ATPase activity. By using the whole cell/vacuole patch clamp technique upon giant vacuoles derived from the protoplasts, an ATP-induced and bafilomycin A1-sensitive pump current was measured and quantitatively investigated. The mutant, in which the gene for one of the crucial subunits of the V-type ATPase was disrupted, failed to show this activity. The feasibility of extending the present technique to other ion transporter genes from organisms other than yeast will also be discussed. The S. cerevisiaestrains used were X2180–1A (MAT a gal2 CUP1), YPH499 (MAT a leu2 ura3 trp1 lys2 his3 ade2), and YPH500 (MATα leu2 ura3 trp1 lys2 his3 ade2). RH105 (Δvma1::TRP1 derivative of YPH500) was constructed as in Ref. 18Hirata R. Anraku Y. Biochem. Biophys. Res. Commun. 1992; 188: 40-47Crossref PubMed Scopus (56) Google Scholar. Innoculum from a stock culture on agar medium was grown in 4 ml of YPD medium (2% glucose, 1% peptone, and 1% yeast extract, pH 7.0) for 7 h at 30 °C. Two hundred microliters of the culture were transferred to a fresh 4-ml aliquot of YPD medium and cultivated for 4 h until the cell reached an early logarithmic phase. Cells were harvested by low-speed centrifugation for a few minutes, resuspended in 2 ml of A buffer (5 mm EGTA, 0.1 m Tris-HCl, pH 7.2, and 5 mm DTT) using a vortex mixer, and then incubated on a shaker at 30 strokes/min for 10 min. The cells were again harvested by centrifugation as described above, washed once with distilled water (supplemented with 1 mm DTT), and then resuspended in 2 ml of B buffer (1 m sorbitol, 1 mm DTT, and 0.1m Tris-HCl, pH 7.2). Zymolyase was added to the suspension to give a final concentration of 1 mg/ml. The suspension was again shaken for 30 min. After confirming under a microscope that the cells had been fully converted to spherical cells (spheroplasts), they were harvested by centrifugation. The pellet was carefully resuspended in 2 ml of A medium (YPD medium supplemented with 1 m sorbitol and 0.05% 2-DG). One-hundred and fifty microliters of the suspension were diluted with 4 ml of A medium and incubated at 20 °C overnight on a shaker at 30 strokes/min. Since RH105, which is a catalytic subunit-disrupted mutant of the V-type ATPase, does not grow well at neutral pH but grows normally at lower pH (19Nelson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3503-3507Crossref PubMed Scopus (245) Google Scholar), the YPD medium and the A medium were supplemented with 50 mm MES-MOPS to lower the pH to 5.5. These media were further supplemented with leucine (20 mg/liter), uracil (20 mg/liter), lysine (30 mg/liter), histidine (20 mg/liter), and adenosine (20 mg/liter), since RH105 requires these nutrients for growth. Cells were all grown to about 10 μm after this incubation. After 2 ml of 0.5 m KCl was added, the cells were harvested by centrifugation, and resuspended in 2 ml of A medium. The suspension was divided into four 1.5-ml Eppendorf tubes (400 μl each) and stored at 10 °C until use. The stock can be stored for several hours without loss of activity. Prior to the next vacuole isolation step, the suspension was shaken for 3 h at 30 °C; spheroplasts had grown to 20–30 μm by this time. Two-hundred microliters of 0.5 m KCl were added to each tube, which was then centrifuged in an Eppendorf-type centrifuge at 5,000 rpm for 5 min. The pellet was suspended in 1 ml of C buffer (A buffer supplemented with 0.8 m sorbitol and 0.1 mKCl), and incubated for 10 min at 30 °C on a shaker at 30 strokes/min. The cells were harvested as described above, resuspended in 1 ml of D buffer (0.8 m sorbitol, 0.1 m KCl, 1 mm DTT, and 0.1 m Tris-HCl, pH 7.2) supplemented with 1 mg/ml Zymolyase, and then incubated for 30 min at 30 °C on a shaker at 30 strokes/min. Microscopic observation confirmed that the cells were fully converted to spheroplasts. For vacuole staining, spheroplasts were incubated in the YPD medium supplemented with 1m sorbitol, 50 mm citric acid, and 10 μm CDCFDA (20Manolson M.F. Proteau D. Preston R.A. Stenbit P.A. Roberts B.T. Hoyt M.A. Preuss D. Mulholland J. Botstein D. Jones E.W. J. Biol. Chem. 1992; 267: 14294-14303Abstract Full Text PDF PubMed Google Scholar), and were shaken at 30 strokes/min for 30 min at 30 °C. A vacuole was observed as a bright yellow fluorescent sphere inside the cell. This result indicates that the CDCFDA added to the medium was transported through the cytoplasmic membrane into a vacuole, hydrolyzed by an esterase, and became the fluorescent in the acidic vacuolar lumen. For nuclei staining, propidium iodide (PI) was used as fluorescent dye according to Ref. 21Eilam Y. Chernichovsky D. J. Gen. Microbiol. 1988; 134: 1063-1070PubMed Google Scholar. A confocal laser scanning microscope (TCS4D, Leica Co.) was used for observation with excitation at 488 nm. For CDCFDA and PI, a 550-nm band path filter and a 550-nm long path filter were used for emission, respectively. For electron microscopy, giant cells (X2180-1A) were fixed by using a conventional glutaraldehyde/OsO4 method without Zymolyase treatment (22Hirata A. Tanaka K. J. Gen. Appl. Microbiol. 1982; 28: 263-274Crossref Scopus (54) Google Scholar); for intact cells (YPH499), a freeze-substitution method (23Sun G-H. Hirata A. Ohya Y. Anraku Y. J. Cell Biol. 1992; 119: 1625-1639Crossref PubMed Scopus (77) Google Scholar) was used. Ultrathin sections were stained with uranyl acetate and Reynold's lead citrate, except for the observation of cell wall, for which a silver proteinate method for carbohydrate staining (24Courtoy R. Simar L.J. J. Microsc. 1974; 100: 199-211Crossref PubMed Scopus (77) Google Scholar) was employed. The suspension of giant spheroplasts obtained above was concentrated 5-fold by centrifugation. Ten microliters of the concentrate were transferred to the recording chamber of the patch clamp apparatus, diluted there with 200 μl of E buffer (0.1 m sorbitol, 0.1 m KCl, 5 mm EGTA, 20 mm Tris-MES, pH 7.5), and allowed to stand for 5 min. Under the microscope, it was observed that more than half of the cells were broken and released vacuoles into the medium. Vacuoles of more than 20 μm were selected for patch clamp experiments. As released vacuoles tend to stick to the glass wall of the chamber, unbroken spheroplasts, broken cytoplasmic membranes, organelles, and other debris can easily be washed away by pouring F buffer (0.1 m sorbitol, 0.1 m KCl, 2 mm MgCl2, 1 mm EGTA, 0.15 mm CaCl2 (10 nm free Ca2+), and 10 mm Tris-MES, pH 7.5) through a capillary tube with six-way valves as described below. Experiments were performed basically as described by Hamill et al. (25Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pflugers Arch. Eur J. Physiol. 1981; 391: 85-100Crossref PubMed Scopus (15140) Google Scholar). Capillaries were made of 75-μl disposable glass micropipettes (Duramont, Bromall, PA) using a two-stage pulling apparatus (PP-83, Narishige, Tokyo), and the tips were heat-polished. The conductivity of the open capillaries filled with G buffer (0.1 m sorbitol, 0.1 m KCl, 2 mm MgCl2, 10 mm MES-Tris, pH 5.5, 2 mm CaCl2) ranged from 3 to 5 MΩ. To produce a whole cell/vacuole patch, after a tight seal was formed (10 GΩ), the patched part of the vacuole membrane was broken by applying a few pulses (±2.0 volts) with duration ranging from 1 to 10 ms. After that, the resistance became 1 to 5 GΩ. Five minutes were usually long enough to exchange the medium inside the vacuole with another medium through the capillary by diffusion. The external medium was changed by a tandem 6-way valve system as described previously (15Kuroda T. Okuda N. Saitoh N. Hiyama T. Terasaki Y. Anazawa H. Hirata A. Mogi T. Kusaka I. Tsuchiya T. Yabe I. J. Biol. Chem. 1998; 273: 16897-16904Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Membrane currents were amplified by a patch/whole cell clamp amplifier (CEZ-2300, Nihon Kohden Co., Tokyo), and recorded on a digital audio tape recorder (DTC 55ES, Sony Corp., Tokyo). Stored data were subsequently processed and analyzed by using a personal computer (PC-9801DX, NEC Inc., Tokyo) and a software program (QP-120J, Nihon Kohden Co., Tokyo). Sign conventions throughout this report define the vacuolar interior as Ref. 26Bertl A. Blumwald E. Coronado R. Eisenberg R. Findlay G. Gradmann D. Hille B. Kohler K. Kolb H.-A. MacRobbie E. Meissner G. Miller C. Neher E. Palade P. Pnatoja O. Sanders D. Schroeder J. Slayman C. Spanswick R. Walker A. Williams A. Science. 1992; 258: 873-874Crossref PubMed Scopus (173) Google Scholar, so that positive membrane voltages mean that the cytoplasmic electric potential is positive to the vacuolar potential, and a positive current represents positive charges moving from the cytoplasm to the inside of a vacuole. Experiments were performed at room temperatures (20–23 °C). Peptone and yeast extract were purchased from Difco; sorbitol, EGTA, and DTT from Sigma; 2-deoxy-d-glucose from Wako Pure Chemicals, Osaka; CDCFDA, from Molecular Probes Ltd.; Zymolyase (20T) from Seikagaku Kogyo Co Ltd., Tokyo. Other reagents were all of analytical grade. As shown in Fig.1, A and B, the haploid cells of S. cerevisiae were all converted to giant cells as large as 20 μm, five times larger than untreated cells. Most of these cells were spherical; some had buds that appeared to have stopped developing further. In order to confirm that the spherical cells were protoplasts without the cell wall, electron microscopy was conducted (Fig. 2). The magnified images of the cell envelope revealed that the cells (protoplasts) lacked cell wall carbohydrates that would be stained by the silver proteinate method (Fig. 2 C). The envelope of intact cells was well stained (27Hirata A. Shimoda C. Arch. Microbiol. 1992; 158: 249-255Crossref PubMed Scopus (25) Google Scholar) and showed a thick layered structure with β-glucan and mannan-protein layers (Fig. 2 D). Further treatment with Zymolyase resulted in total conversion of the cells into the spherical shape. The ratio of 2-DG/glucose was found to be critical: ratios neither lower nor higher than 1/40 were effective in producing the giant protoplasts. The otherwise normal growth indicates that 2-DG at this critical concentration does act as an inhibitor of cell wall synthesis but does not significantly affect energy-generating glycolysis (17Bieley P. Kratky Z. Kovarik J. Bauer S. J. Bacteriol. 1971; 107: 121-129Crossref PubMed Google Scholar). It appears that the yeast cells were converted to giant protoplasts merely by the inhibition of cell wall synthesis. Although much needs to be done to elucidate the mechanism, fluorescence microscopy of the cells stained with PI for DNA (Fig.3) may provide some insight into this problem. These pictures show that the cells became multinuclear when budding was arrested either in the middle (Fig. 3, A andB) or at the start (Fig. 3, C and D). These observations are consistent with previous results with a mutant cell in which the cell morphogenesis checkpoint system was genetically impaired: nuclear divisions continued while the corresponding synchronous budding cycle (cytoplasmic division) was halted (28Sakaguchi S. Miyamoto S. Iida H. Suzuki T. Ohya Y. Anraku Y. Protoplasma. 1995; 189: 142-148Crossref Scopus (2) Google Scholar, 29Lew D.J. Reed S.I. J. Cell Biol. 1995; 129: 739-749Crossref PubMed Scopus (224) Google Scholar). It should be emphasized that these giant protoplasts, although they have multiple nuclei, derive originally from haploid cells and thus have identical nuclei.Figure 2Electron micrographs of a giant cell. A, an ultrathin section of a giant cell (X2180-1A) fixed by conventional methods and stained with uranyl acetate and Reynold's lead citrate. M, mitochondrion; V, vacuole;N, nucleus. B, intact cell (YPH499) fixed by the freeze-substitution method and stained with uranyl acetate and Reynold's lead citrate. C, a magnified image of the part indicated by an arrow in A. The ultrathin section used here was the next serial section to the one used in A, and stained for carbohydrate by using the silver proteinate method.D, a magnified image of the part indicated by anarrow in B. The ultrathin section used here was the next serial section to the one used in B, and stained for carbohydrate as in C. The arrows inC and D indicate cytoplasmic membranes. The same magnifications are used for A and B as well as for C and D.View Large Image Figure ViewerDownload (PPT)Figure 3Fluorescent images of PI stained giant cells. A and C , externally focused fluorescent images of the optical sections generated by the laser-scanning microscope (Leica TCS4D); B andD , side views of A and C, respectively. The same magnifications were used for A andB as well as for C and D. The strain used here was YPH500.View Large Image Figure ViewerDownload (PPT) As seen in a micrograph (Fig. 1 B), the giant protoplast was occupied by a huge organelle that appeared to be a vacuole. This organelle was indeed confirmed to be a vacuole, since it was stained with a vacuole-specific CDCFDA (Fig. 1 C) and did not show any electron-dense material inside the vacuole in an ultrathin section under the electron microscope (Fig. 2 A). By slightly lowering the osmotic pressure of the medium, the cell membrane of the giant protoplast was broken and, as a result, the intact vacuole was readily released into the medium (Fig. 1 D). The vacuole thus released swelled up and became as large as 30 μm, adhering well to the surface of the glass slide along with small pieces of broken cytoplasmic membrane and small spherical bodies acting like glue. The adhesion was stable enough for buffer washing through the capillary, thus enabling quick exchange of the medium simply by using the six-way valve system. The patch clamp technique in the whole cell/vacuole recording mode was applied to this vacuole. Two types of ion-transporter activities were detected. Fig. 4 shows an anion-specific transporter. With equimolar potassium (100 mm) on both sides of the membrane, the voltage versus current curve (V-I curve) changed little when 90% of the anion outside was replaced with fluoride (open circles); on the other hand, glutamate (triangles) and nitrate (squares) drastically changed the curve. With the Goldman equation and using the reversal potentials of these anions, permeability ratios were calculated to be 2.8 ± 0.2: 1:0.5 ± 0.3:0.1 ± 0.015 for NO3−:Cl−:F−:glutamate−. These results are consistent with previous reports that indicate that there is an electrogenic anion transporter on the vacuole membrane (30Wada Y. Ohsumi Y. Anraku Y. Biochim. Biophys. Acta. 1992; 1101: 296-302Crossref PubMed Scopus (35) Google Scholar), and that glutamate is accumulated mainly in the cytoplasm (31Kitamoto K. Hoshizawa K. Ohsumi Y. Anraku Y. J. Bacteriol. 1988; 170: 2683-2686Crossref PubMed Scopus (145) Google Scholar) and nitrate in the vacuole (32Martinola E. Heck U. Wiemken A. Nature. 1981; 289: 292-294Crossref Scopus (247) Google Scholar). A distinct ion-transport activity is shown in Fig.5. When 1 mm ATP was poured onto the surface of the vacuole through the capillary with the membrane potential being kept at zero, a huge current as high as 100 pA (0.1 pA/μm2) flowing into the vacuolar lumen was observed (Fig. 5 A). After a transient overshoot, the current gradually reached a steady-state level. Washing the medium outside the vacuole with an ATP-free medium brought the current down to the baseline level. Bafilomycin A1, which is a potent and specific inhibitor of V-type ATPase (33Bowman E.J. Siebers A. Altendorf K. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7972-7976Crossref PubMed Scopus (1599) Google Scholar), strongly inhibited this ATP-induced pump current (Fig. 5 B). At 10 μm, the pump was completely inhibited (data not shown); 50% inhibition was achieved at 10 nm. In order to confirm that this ATP-induced current is generated by V-type ATPase, mutant cells that lacked the V-type ATPase activity were also converted to giant cells using the SI method. The RH105 strain (Δvma1::TRP) was cultivated and converted to giant protoplasts in a similar way, except that the pH was kept at 5.5 as stated under “Experimental Procedures.” As expected, while no significant difference was observed in terms of the above described anion-specific transporter activity between the wild type (YPH500, Fig.6 B) and the mutant (RH105, Fig. 6 C), the ATP-induced current was detected only in the wild type and could not be detected in the RH105 mutant (Fig.6 A). It was thus confirmed that no other ATP-dependent pump such as Ca2+-ATPase (34Cunningham K.W. Fink G.R. Mol. Cell. Biol. 1996; 16: 2226-2237Crossref PubMed Scopus (380) Google Scholar) was induced under our present experimental conditions. It should be noted here that in this particular experiment (Fig.6 A) an ATP-regenerating system consisting of creatine phosphate and creatine kinase was supplemented in the medium in order to minimize the ADP concentration, because ADP is known to inhibit the activity (35Uchida E. Ohsumi Y. Anraku Y. J. Biol. Chem. 1985; 260: 1090-1095Abstract Full Text PDF PubMed Google Scholar). As a result, the overshoot observed in earlier experiments (Fig. 5) no longer appeared. Experiments hereafter were all conducted under these conditions. The ATP-induced current was measured at different ATP concentrations (Fig. 7 A). A double reciprocal plot of the current against the ATP concentration is shown in Fig. 7 B. The apparent K m value for ATP was calculated to be 0.159 mm, which is in good agreement with the K m value (0.2 mm) obtained by measurements of ATP hydrolysis for the yeast V-type ATPase (36Kakinuma Y. Ohsumi Y. Anraku Y. J. Biol. Chem. 1981; 256: 10859-10863Abstract Full Text PDF PubMed Google Scholar). Since the two V-I curves, one for the ATP-induced current and the other for the ATP-independent current (Fig.8 A) included a component due to the anion-specific transporter and a small leak current, the subtracted difference was plotted in Fig. 8 B in order to obtain the “true” V-I curve for the ATP-dependent pump. This curve has two distinct characteristics: (a) the H+-pump activity saturates at sufficiently high membrane potentials (>40 mV); (b) it does not become negative even at sufficiently low potentials (−70 mV). The former observation (a) suggests that the H+-pump is accelerated with an increase of the membrane potential until the rate becomes limited by a step independent of the potential such as ATP hydrolysis. The latter (b) indicates that the H+-pump (V-type ATPase) does not work in the negative direction which is necessary, if any, for the synthesis of ATP. This is consistent with the previous result that indicated that the V-type ATPase has no ATP-synthase activity (37Nelson N. Biochim. Biophys. Acta. 1992; 1100: 109-124Crossref PubMed Scopus (157) Google Scholar). Based on the reversal potential of −70 ± 5 mV and ΔpH of 2.0 units, the electrochemical potential difference of H+(ΔμH+) was calculated to be −190 ± 5 mV, which is very close to the value obtained with vacuolar membrane vesicles isolated from normal size yeast cells (36Kakinuma Y. Ohsumi Y. Anraku Y. J. Biol. Chem. 1981; 256: 10859-10863Abstract Full Text PDF PubMed Google Scholar). The free energy difference of ATP in equilibrium with this system (ΔG ATP = −15.3 Kcal/mol) was calculated by adopting a value for Δ GATPo = −9 Kcal/mol (38Alberty R.A. J. Biol. Chem. 1968; 243: 1337-1343Abstract Full Text PDF PubMed Google Scholar) and RT ln ([ADP] [Pi]/[ATP] = −6.3 Kcal/mol, which was estimated from the equilibrium constant for the creatine kinase reaction (39LoPresti P. Cohn M. Biochim. Biophys. Acta. 1989; 998: 317-320Crossref PubMed Scopus (18) Google Scholar). From the reversible ΔμH+ and ΔG ATP, the H+/ATP ratio was estimated to be 3.5 ± 0.3. In a previous report, the H+/ATP ratio for a plant vacuolar H+-pump was directly calculated to be 2 from the amounts of transported H+ and hydrolyzed ATP (40Bennett A.B. Spanswick R.M. Plant Physiol. 1984; 74: 545-548Crossref PubMed Google Scholar), which has since been assumed to be a fixed value. A recent result from patch clamp experiments with the vacuole of a plant cell, where inside and outside pH was widely varied, showed that the H+/ATP ratio varied from 1.75 to 3.28 depending on the pH inside and outside of the vacuole (41Davies J.M. Hunt I. Sanders D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8547-8551Crossref PubMed Scopus (107) Google Scholar). When the outside medium was basic (pH 8.0) and ΔpH was large (4.7), the H+/ATP ratio was 1.75; when the outside medium was neutral (pH 7.0) and Δp" @default.
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• W2144856874 title "Patch Clamp Studies on V-type ATPase of Vacuolar Membrane of Haploid Saccharomyces cerevisiae" @default.
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