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• W2079844556 abstract "The vacuole of the yeast Saccharomyces cerevisiae is a major storage compartment for phosphate. We have measured phosphate transport across the vacuolar membrane. Isolated intact vacuoles take up large amounts of added [32P]phosphate by counterflow exchange with phosphate present in the vacuoles at the time of their isolation. The bidirectional phosphate transporter has an intrinsic dissociation constant for phosphate of 0.4 mm. Exchange mediated by this carrier is faster than unidirectional efflux of phosphate from the vacuoles. The transporter is highly selective for phosphate; of other anions tested, only arsenate is also a substrate. Transport is strongly pH-dependent with increasing activity at lower pH. Similar phosphate transport behavior was observed in right-side-out vacuolar membrane vesicles. The vacuole of the yeast Saccharomyces cerevisiae is a major storage compartment for phosphate. We have measured phosphate transport across the vacuolar membrane. Isolated intact vacuoles take up large amounts of added [32P]phosphate by counterflow exchange with phosphate present in the vacuoles at the time of their isolation. The bidirectional phosphate transporter has an intrinsic dissociation constant for phosphate of 0.4 mm. Exchange mediated by this carrier is faster than unidirectional efflux of phosphate from the vacuoles. The transporter is highly selective for phosphate; of other anions tested, only arsenate is also a substrate. Transport is strongly pH-dependent with increasing activity at lower pH. Similar phosphate transport behavior was observed in right-side-out vacuolar membrane vesicles. Phosphate is an important nutrient, and phosphate metabolism in the yeast Saccharomyces cerevisiae has been extensively studied. This system has provided a model for understanding how a cell makes a coordinated response to environmental changes (1Lenburg M.E. O'Shea E.K. Trends Biochem. Sci. 1996; 21: 383-387Abstract Full Text PDF PubMed Scopus (221) Google Scholar). Phosphate is often present in only low amounts in the environment (2Harold F.M. Bacteriol. Rev. 1966; 30: 772-794Crossref PubMed Google Scholar), and as for other microorganisms, yeast has evolved complex mechanisms to deal with changes in phosphate availability.One aspect of phosphate metabolism in yeast which has received substantial attention is the question of how the cell obtains phosphate from its surroundings. Several secreted phosphatases which release free phosphate in the extracellular space have been identified (3Vogel K. Hinnen A. Mol. Microbiol. 1990; 4: 2013-2017Crossref PubMed Scopus (70) Google Scholar). Uptake of free phosphate from outside the cell is mediated by a number of plasma membrane transport systems. One has a high affinity for phosphate and is encoded by the PHO84 gene, whose expression is derepressed under conditions of phosphate starvation (4Bun-Ya M. Nishimura M. Harashima M. Oshima Y. Mol. Cell. Biol. 1991; 11: 3229-3238Crossref PubMed Scopus (331) Google Scholar). Others include a sodium/phosphate cotransporter and a low affinity, constitutive transport system (5Tamai Y. Toh-E A. Oshima Y. J. Bacteriol. 1985; 164: 964-968Crossref PubMed Google Scholar, 6Borst-Pauwels G.W.F.H. Biochim. Biophys. Acta. 1981; 650: 88-127Crossref PubMed Scopus (257) Google Scholar).Once phosphate has been taken up by the yeast cell, a second important consideration is its intracellular compartmentalization. In this respect the yeast vacuole plays a major role. The vacuole is the site of storage of large amounts of phosphate and polyphosphate, a linear polymer of phosphate in anhydrous linkage (7Okorokov L.A. Lichko L.P. Kulaev I.S. J. Bacteriol. 1980; 144: 661-665Crossref PubMed Google Scholar, 8Urech K. Dürr M. Boller T. Wiemken A. Schwencke J. Arch. Microbiol. 1978; 116: 275-278Crossref PubMed Scopus (101) Google Scholar, 9Kornberg A. J. Bacteriol. 1995; 177: 491-496Crossref PubMed Scopus (466) Google Scholar). These vacuolar pools are either augmented or depleted depending on changes in phosphate availability (2Harold F.M. Bacteriol. Rev. 1966; 30: 772-794Crossref PubMed Google Scholar, 10Nicolay K. Scheffers W.A. Bruinenberg P.M. Kaptein R. Arch. Microbiol. 1983; 134: 270-275Crossref PubMed Scopus (37) Google Scholar, 11Gillies R.J. Ugurbil K. den Hollander J.A. Shulman R.G. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2125-2129Crossref PubMed Scopus (110) Google Scholar). This process clearly entails net movements of phosphate across the vacuolar membrane. However, in contrast to the situation with the plasma membrane, no phosphate transport system in the vacuolar membrane has yet been characterized.A variety of different substances are concentrated in the yeast vacuole (12Klionsky D.J. Herman P.K. Emr S.D. Microbiol. Rev. 1990; 54: 266-292Crossref PubMed Google Scholar), and numerous vacuolar transport systems have been described. These include transporters of protons (13Kakinuma Y. Ohsumi Y. Anraku Y. J. Biol. Chem. 1981; 256: 10859-10863Abstract Full Text PDF PubMed Google Scholar), Ca2+ (14Ohsumi Y. Anraku Y. J. Biol. Chem. 1983; 258: 5614-5617Abstract Full Text PDF PubMed Google Scholar, 15Cunningham K.W. Fink G.R. J. Exp. Biol. 1994; 196: 157-166Crossref PubMed Google Scholar), amino acids (16Sato T. Ohsumi Y. Anraku Y. J. Biol. Chem. 1984; 259: 11505-11508Abstract Full Text PDF PubMed Google Scholar, 17Boller T. Dürr M. Wiemken A. Eur. J. Biochem. 1975; 54: 81-91Crossref PubMed Scopus (57) Google Scholar), K+ and Na+ (18Bertl A. Slayman C.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7824-7828Crossref PubMed Scopus (101) Google Scholar, 19Wada Y. Ohsumi Y. Tanifuji M. Kasai M. Anraku Y. J. Biol. Chem. 1987; 262: 17260-17263Abstract Full Text PDF PubMed Google Scholar), polyamines (20Kakinuma Y. Masuda N. Igarashi K. Biochim. Biophys. Acta. 1992; 1107: 126-130Crossref PubMed Scopus (21) Google Scholar), glutathione S-conjugates (21Li Z.S. Szczypka M. Lu Y.-P. Thiele D.J. Rea P.A. J. Biol. Chem. 1996; 271: 6509-6517Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar),S-adenosylmethionine (22Schwencke J. De Robichon Szulmajster H. Eur. J. Biochem. 1976; 65: 49-60Crossref PubMed Scopus (48) Google Scholar), purines (23Nagy M. Biochim. Biophys. Acta. 1979; 558: 221-232Crossref PubMed Scopus (11) Google Scholar), and chloride (24Wada Y. Ohsumi Y. Anraku Y. Biochim. Biophys. Acta. 1992; 1101: 296-302Crossref PubMed Scopus (33) Google Scholar). Phosphate and polyphosphate are major vacuolar anions, with a large buffering capacity. Thus, understanding vacuolar phosphate transport is also important for understanding the storage functions of the vacuole in terms of overall charge balance, vacuolar acidification, and osmoregulation.We have measured transport of phosphate across the vacuolar membrane by performing transport assays with isolated intact vacuoles and vacuolar membrane vesicles. Counterflow behavior is observed indicative of a reversible phosphate transporter with a millimolar affinity for phosphate. This transporter is highly specific for phosphate.DISCUSSIONWe report here the characterization of a phosphate transporter in the vacuolar membrane of yeast. This transporter mediates bidirectional transport and has a millimolar affinity for phosphate. We were able to observe a large uptake of [32P]phosphate due to the presence in isolated yeast vacuoles of a pool of phosphate that provides a large although transient driving force for uptake via exchange.In the yeast cell, the direction of net movement of phosphate across the vacuolar membrane depends on physiological conditions. When metabolic requirements for phosphate exceed what can be obtained from outside the cell, vacuolar polyphosphate pools are mobilized to replenish cytoplasmic phosphate (10Nicolay K. Scheffers W.A. Bruinenberg P.M. Kaptein R. Arch. Microbiol. 1983; 134: 270-275Crossref PubMed Scopus (37) Google Scholar, 11Gillies R.J. Ugurbil K. den Hollander J.A. Shulman R.G. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2125-2129Crossref PubMed Scopus (110) Google Scholar, 34Bostian K.A. Lemire J.M. Halvorson H.O. Mol. Cell. Biol. 1983; 3: 839-853Crossref PubMed Scopus (45) Google Scholar). Several exopolyphosphatases have been identified in the vacuole (35Wurst H. Shiba T. Kornberg A. J. Bacteriol. 1995; 177: 898-906Crossref PubMed Scopus (162) Google Scholar, 36Andreeva N.A. Lichko L.P. Kulakovskaia T.V. Okorokov L.A. Biokhimiia. 1993; 58: 1053-1061PubMed Google Scholar), where they can act to release phosphate from polyphosphate by hydrolysis. The released phosphate then moves out of the vacuole (10Nicolay K. Scheffers W.A. Bruinenberg P.M. Kaptein R. Arch. Microbiol. 1983; 134: 270-275Crossref PubMed Scopus (37) Google Scholar). This phosphate efflux may be mediated by the transporter that we have identified, although as previously noted, it is possible that the efflux and exchange activities we have observed are due to two distinct transporters. In the absence of any specific inhibitors of these activities, this possibility cannot be excluded.Conversely, under conditions where phosphate and metabolic energy are available, and especially when phosphate is added to cells previously starved for phosphate, polyphosphate is synthesized (2Harold F.M. Bacteriol. Rev. 1966; 30: 772-794Crossref PubMed Google Scholar, 37Bourne R.M. Biochim. Biophys. Acta. 1990; 1055: 1-9Crossref PubMed Scopus (12) Google Scholar). However, the mechanism of polyphosphate synthesis and the vacuolar transport processes required for this synthesis are not clear. Polyphosphate synthesis requires a high energy phosphate donor rather than simply orthophosphate. This donor has not been definitively identified (38Booth J.W. Guidotti G. J. Biol. Chem. 1995; 270: 19377-19382Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), but it presumably must be transported into the vacuole during periods of polyphosphate synthesis.In vivo studies of phosphate metabolism in yeast conducted using 31P-nuclear magnetic resonance (NMR) (10Nicolay K. Scheffers W.A. Bruinenberg P.M. Kaptein R. Arch. Microbiol. 1983; 134: 270-275Crossref PubMed Scopus (37) Google Scholar, 11Gillies R.J. Ugurbil K. den Hollander J.A. Shulman R.G. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2125-2129Crossref PubMed Scopus (110) Google Scholar, 33Bourne R.M. Biochim. Biophys. Acta. 1991; 1067: 81-88Crossref PubMed Scopus (8) Google Scholar,37Bourne R.M. Biochim. Biophys. Acta. 1990; 1055: 1-9Crossref PubMed Scopus (12) Google Scholar), as well as studies using differential extraction techniques to distinguish vacuolar and cytoplasmic ion pools (7Okorokov L.A. Lichko L.P. Kulaev I.S. J. Bacteriol. 1980; 144: 661-665Crossref PubMed Google Scholar) have suggested that a large vacuole-to-cytoplasm gradient of phosphate concentration can exist. However, we have found no evidence for active ATP-driven transport of phosphate across the vacuolar membrane similar to that seen for other substances located in the vacuole.To satisfy conditions of electroneutrality, the efflux of phosphate from vacuoles that we observe must be accompanied by either cation efflux or inward movement of another anion. Since the only anion added to the outside of the vacuoles is the large buffer anion PIPES, the latter possibility seems unlikely. Rather, a vacuolar cation presumably moves out with phosphate, either through the same transporter or in parallel through a separate transporter, e.g. the cation channel previously identified in the vacuolar membrane (18Bertl A. Slayman C.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7824-7828Crossref PubMed Scopus (101) Google Scholar, 19Wada Y. Ohsumi Y. Tanifuji M. Kasai M. Anraku Y. J. Biol. Chem. 1987; 262: 17260-17263Abstract Full Text PDF PubMed Google Scholar). The fact that we could not observe any membrane potential-driven phosphate uptake suggests that the phosphate carrier itself may perform electroneutral transport.It should be noted that the vacuoles used in our experiments were all obtained from cells grown in medium containing ample free phosphate. Thus, the transporter does not appear to require phosphate starvation for its induction, like Pho84p, but rather is present constitutively.There is a striking stimulation of counterflow uptake as the pH is lowered from 7.3 to 5.5. This may suggest that the univalent form of phosphate is the substrate of this carrier, as is the case for the plasma membrane phosphate transporters (6Borst-Pauwels G.W.F.H. Biochim. Biophys. Acta. 1981; 650: 88-127Crossref PubMed Scopus (257) Google Scholar).Most of the transporters identified in the vacuolar membrane to date have been studied using vacuolar membrane vesicles. The majority of these transporters mediate active uptake driven by the H+-ATPase; this is probably a reflection of the fact that passive transport is difficult to observe in vesicles, given their small internal volume and the fact that they are largely devoid of vacuolar contents (32Ohsumi Y. Anraku Y. J. Biol. Chem. 1981; 256: 2079-2082Abstract Full Text PDF PubMed Google Scholar). With intact vacuoles, in contrast, passive transport systems can be discovered and analyzed more easily (25Boller T. Dürr M. Wiemken A. Methods Enzymol. 1989; 174: 504-518Crossref PubMed Scopus (5) Google Scholar). Thus, using intact vacuoles we were able to observe and characterize a transporter which mediates passive transport of phosphate across the vacuolar membrane. Phosphate is an important nutrient, and phosphate metabolism in the yeast Saccharomyces cerevisiae has been extensively studied. This system has provided a model for understanding how a cell makes a coordinated response to environmental changes (1Lenburg M.E. O'Shea E.K. Trends Biochem. Sci. 1996; 21: 383-387Abstract Full Text PDF PubMed Scopus (221) Google Scholar). Phosphate is often present in only low amounts in the environment (2Harold F.M. Bacteriol. Rev. 1966; 30: 772-794Crossref PubMed Google Scholar), and as for other microorganisms, yeast has evolved complex mechanisms to deal with changes in phosphate availability. One aspect of phosphate metabolism in yeast which has received substantial attention is the question of how the cell obtains phosphate from its surroundings. Several secreted phosphatases which release free phosphate in the extracellular space have been identified (3Vogel K. Hinnen A. Mol. Microbiol. 1990; 4: 2013-2017Crossref PubMed Scopus (70) Google Scholar). Uptake of free phosphate from outside the cell is mediated by a number of plasma membrane transport systems. One has a high affinity for phosphate and is encoded by the PHO84 gene, whose expression is derepressed under conditions of phosphate starvation (4Bun-Ya M. Nishimura M. Harashima M. Oshima Y. Mol. Cell. Biol. 1991; 11: 3229-3238Crossref PubMed Scopus (331) Google Scholar). Others include a sodium/phosphate cotransporter and a low affinity, constitutive transport system (5Tamai Y. Toh-E A. Oshima Y. J. Bacteriol. 1985; 164: 964-968Crossref PubMed Google Scholar, 6Borst-Pauwels G.W.F.H. Biochim. Biophys. Acta. 1981; 650: 88-127Crossref PubMed Scopus (257) Google Scholar). Once phosphate has been taken up by the yeast cell, a second important consideration is its intracellular compartmentalization. In this respect the yeast vacuole plays a major role. The vacuole is the site of storage of large amounts of phosphate and polyphosphate, a linear polymer of phosphate in anhydrous linkage (7Okorokov L.A. Lichko L.P. Kulaev I.S. J. Bacteriol. 1980; 144: 661-665Crossref PubMed Google Scholar, 8Urech K. Dürr M. Boller T. Wiemken A. Schwencke J. Arch. Microbiol. 1978; 116: 275-278Crossref PubMed Scopus (101) Google Scholar, 9Kornberg A. J. Bacteriol. 1995; 177: 491-496Crossref PubMed Scopus (466) Google Scholar). These vacuolar pools are either augmented or depleted depending on changes in phosphate availability (2Harold F.M. Bacteriol. Rev. 1966; 30: 772-794Crossref PubMed Google Scholar, 10Nicolay K. Scheffers W.A. Bruinenberg P.M. Kaptein R. Arch. Microbiol. 1983; 134: 270-275Crossref PubMed Scopus (37) Google Scholar, 11Gillies R.J. Ugurbil K. den Hollander J.A. Shulman R.G. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2125-2129Crossref PubMed Scopus (110) Google Scholar). This process clearly entails net movements of phosphate across the vacuolar membrane. However, in contrast to the situation with the plasma membrane, no phosphate transport system in the vacuolar membrane has yet been characterized. A variety of different substances are concentrated in the yeast vacuole (12Klionsky D.J. Herman P.K. Emr S.D. Microbiol. Rev. 1990; 54: 266-292Crossref PubMed Google Scholar), and numerous vacuolar transport systems have been described. These include transporters of protons (13Kakinuma Y. Ohsumi Y. Anraku Y. J. Biol. Chem. 1981; 256: 10859-10863Abstract Full Text PDF PubMed Google Scholar), Ca2+ (14Ohsumi Y. Anraku Y. J. Biol. Chem. 1983; 258: 5614-5617Abstract Full Text PDF PubMed Google Scholar, 15Cunningham K.W. Fink G.R. J. Exp. Biol. 1994; 196: 157-166Crossref PubMed Google Scholar), amino acids (16Sato T. Ohsumi Y. Anraku Y. J. Biol. Chem. 1984; 259: 11505-11508Abstract Full Text PDF PubMed Google Scholar, 17Boller T. Dürr M. Wiemken A. Eur. J. Biochem. 1975; 54: 81-91Crossref PubMed Scopus (57) Google Scholar), K+ and Na+ (18Bertl A. Slayman C.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7824-7828Crossref PubMed Scopus (101) Google Scholar, 19Wada Y. Ohsumi Y. Tanifuji M. Kasai M. Anraku Y. J. Biol. Chem. 1987; 262: 17260-17263Abstract Full Text PDF PubMed Google Scholar), polyamines (20Kakinuma Y. Masuda N. Igarashi K. Biochim. Biophys. Acta. 1992; 1107: 126-130Crossref PubMed Scopus (21) Google Scholar), glutathione S-conjugates (21Li Z.S. Szczypka M. Lu Y.-P. Thiele D.J. Rea P.A. J. Biol. Chem. 1996; 271: 6509-6517Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar),S-adenosylmethionine (22Schwencke J. De Robichon Szulmajster H. Eur. J. Biochem. 1976; 65: 49-60Crossref PubMed Scopus (48) Google Scholar), purines (23Nagy M. Biochim. Biophys. Acta. 1979; 558: 221-232Crossref PubMed Scopus (11) Google Scholar), and chloride (24Wada Y. Ohsumi Y. Anraku Y. Biochim. Biophys. Acta. 1992; 1101: 296-302Crossref PubMed Scopus (33) Google Scholar). Phosphate and polyphosphate are major vacuolar anions, with a large buffering capacity. Thus, understanding vacuolar phosphate transport is also important for understanding the storage functions of the vacuole in terms of overall charge balance, vacuolar acidification, and osmoregulation. We have measured transport of phosphate across the vacuolar membrane by performing transport assays with isolated intact vacuoles and vacuolar membrane vesicles. Counterflow behavior is observed indicative of a reversible phosphate transporter with a millimolar affinity for phosphate. This transporter is highly specific for phosphate. DISCUSSIONWe report here the characterization of a phosphate transporter in the vacuolar membrane of yeast. This transporter mediates bidirectional transport and has a millimolar affinity for phosphate. We were able to observe a large uptake of [32P]phosphate due to the presence in isolated yeast vacuoles of a pool of phosphate that provides a large although transient driving force for uptake via exchange.In the yeast cell, the direction of net movement of phosphate across the vacuolar membrane depends on physiological conditions. When metabolic requirements for phosphate exceed what can be obtained from outside the cell, vacuolar polyphosphate pools are mobilized to replenish cytoplasmic phosphate (10Nicolay K. Scheffers W.A. Bruinenberg P.M. Kaptein R. Arch. Microbiol. 1983; 134: 270-275Crossref PubMed Scopus (37) Google Scholar, 11Gillies R.J. Ugurbil K. den Hollander J.A. Shulman R.G. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2125-2129Crossref PubMed Scopus (110) Google Scholar, 34Bostian K.A. Lemire J.M. Halvorson H.O. Mol. Cell. Biol. 1983; 3: 839-853Crossref PubMed Scopus (45) Google Scholar). Several exopolyphosphatases have been identified in the vacuole (35Wurst H. Shiba T. Kornberg A. J. Bacteriol. 1995; 177: 898-906Crossref PubMed Scopus (162) Google Scholar, 36Andreeva N.A. Lichko L.P. Kulakovskaia T.V. Okorokov L.A. Biokhimiia. 1993; 58: 1053-1061PubMed Google Scholar), where they can act to release phosphate from polyphosphate by hydrolysis. The released phosphate then moves out of the vacuole (10Nicolay K. Scheffers W.A. Bruinenberg P.M. Kaptein R. Arch. Microbiol. 1983; 134: 270-275Crossref PubMed Scopus (37) Google Scholar). This phosphate efflux may be mediated by the transporter that we have identified, although as previously noted, it is possible that the efflux and exchange activities we have observed are due to two distinct transporters. In the absence of any specific inhibitors of these activities, this possibility cannot be excluded.Conversely, under conditions where phosphate and metabolic energy are available, and especially when phosphate is added to cells previously starved for phosphate, polyphosphate is synthesized (2Harold F.M. Bacteriol. Rev. 1966; 30: 772-794Crossref PubMed Google Scholar, 37Bourne R.M. Biochim. Biophys. Acta. 1990; 1055: 1-9Crossref PubMed Scopus (12) Google Scholar). However, the mechanism of polyphosphate synthesis and the vacuolar transport processes required for this synthesis are not clear. Polyphosphate synthesis requires a high energy phosphate donor rather than simply orthophosphate. This donor has not been definitively identified (38Booth J.W. Guidotti G. J. Biol. Chem. 1995; 270: 19377-19382Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), but it presumably must be transported into the vacuole during periods of polyphosphate synthesis.In vivo studies of phosphate metabolism in yeast conducted using 31P-nuclear magnetic resonance (NMR) (10Nicolay K. Scheffers W.A. Bruinenberg P.M. Kaptein R. Arch. Microbiol. 1983; 134: 270-275Crossref PubMed Scopus (37) Google Scholar, 11Gillies R.J. Ugurbil K. den Hollander J.A. Shulman R.G. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2125-2129Crossref PubMed Scopus (110) Google Scholar, 33Bourne R.M. Biochim. Biophys. Acta. 1991; 1067: 81-88Crossref PubMed Scopus (8) Google Scholar,37Bourne R.M. Biochim. Biophys. Acta. 1990; 1055: 1-9Crossref PubMed Scopus (12) Google Scholar), as well as studies using differential extraction techniques to distinguish vacuolar and cytoplasmic ion pools (7Okorokov L.A. Lichko L.P. Kulaev I.S. J. Bacteriol. 1980; 144: 661-665Crossref PubMed Google Scholar) have suggested that a large vacuole-to-cytoplasm gradient of phosphate concentration can exist. However, we have found no evidence for active ATP-driven transport of phosphate across the vacuolar membrane similar to that seen for other substances located in the vacuole.To satisfy conditions of electroneutrality, the efflux of phosphate from vacuoles that we observe must be accompanied by either cation efflux or inward movement of another anion. Since the only anion added to the outside of the vacuoles is the large buffer anion PIPES, the latter possibility seems unlikely. Rather, a vacuolar cation presumably moves out with phosphate, either through the same transporter or in parallel through a separate transporter, e.g. the cation channel previously identified in the vacuolar membrane (18Bertl A. Slayman C.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7824-7828Crossref PubMed Scopus (101) Google Scholar, 19Wada Y. Ohsumi Y. Tanifuji M. Kasai M. Anraku Y. J. Biol. Chem. 1987; 262: 17260-17263Abstract Full Text PDF PubMed Google Scholar). The fact that we could not observe any membrane potential-driven phosphate uptake suggests that the phosphate carrier itself may perform electroneutral transport.It should be noted that the vacuoles used in our experiments were all obtained from cells grown in medium containing ample free phosphate. Thus, the transporter does not appear to require phosphate starvation for its induction, like Pho84p, but rather is present constitutively.There is a striking stimulation of counterflow uptake as the pH is lowered from 7.3 to 5.5. This may suggest that the univalent form of phosphate is the substrate of this carrier, as is the case for the plasma membrane phosphate transporters (6Borst-Pauwels G.W.F.H. Biochim. Biophys. Acta. 1981; 650: 88-127Crossref PubMed Scopus (257) Google Scholar).Most of the transporters identified in the vacuolar membrane to date have been studied using vacuolar membrane vesicles. The majority of these transporters mediate active uptake driven by the H+-ATPase; this is probably a reflection of the fact that passive transport is difficult to observe in vesicles, given their small internal volume and the fact that they are largely devoid of vacuolar contents (32Ohsumi Y. Anraku Y. J. Biol. Chem. 1981; 256: 2079-2082Abstract Full Text PDF PubMed Google Scholar). With intact vacuoles, in contrast, passive transport systems can be discovered and analyzed more easily (25Boller T. Dürr M. Wiemken A. Methods Enzymol. 1989; 174: 504-518Crossref PubMed Scopus (5) Google Scholar). Thus, using intact vacuoles we were able to observe and characterize a transporter which mediates passive transport of phosphate across the vacuolar membrane. We report here the characterization of a phosphate transporter in the vacuolar membrane of yeast. This transporter mediates bidirectional transport and has a millimolar affinity for phosphate. We were able to observe a large uptake of [32P]phosphate due to the presence in isolated yeast vacuoles of a pool of phosphate that provides a large although transient driving force for uptake via exchange. In the yeast cell, the direction of net movement of phosphate across the vacuolar membrane depends on physiological conditions. When metabolic requirements for phosphate exceed what can be obtained from outside the cell, vacuolar polyphosphate pools are mobilized to replenish cytoplasmic phosphate (10Nicolay K. Scheffers W.A. Bruinenberg P.M. Kaptein R. Arch. Microbiol. 1983; 134: 270-275Crossref PubMed Scopus (37) Google Scholar, 11Gillies R.J. Ugurbil K. den Hollander J.A. Shulman R.G. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2125-2129Crossref PubMed Scopus (110) Google Scholar, 34Bostian K.A. Lemire J.M. Halvorson H.O. Mol. Cell. Biol. 1983; 3: 839-853Crossref PubMed Scopus (45) Google Scholar). Several exopolyphosphatases have been identified in the vacuole (35Wurst H. Shiba T. Kornberg A. J. Bacteriol. 1995; 177: 898-906Crossref PubMed Scopus (162) Google Scholar, 36Andreeva N.A. Lichko L.P. Kulakovskaia T.V. Okorokov L.A. Biokhimiia. 1993; 58: 1053-1061PubMed Google Scholar), where they can act to release phosphate from polyphosphate by hydrolysis. The released phosphate then moves out of the vacuole (10Nicolay K. Scheffers W.A. Bruinenberg P.M. Kaptein R. Arch. Microbiol. 1983; 134: 270-275Crossref PubMed Scopus (37) Google Scholar). This phosphate efflux may be mediated by the transporter that we have identified, although as previously noted, it is possible that the efflux and exchange activities we have observed are due to two distinct transporters. In the absence of any specific inhibitors of these activities, this possibility cannot be excluded. Conversely, under conditions where phosphate and metabolic energy are available, and especially when phosphate is added to cells previously starved for phosphate, polyphosphate is synthesized (2Harold F.M. Bacteriol. Rev. 1966; 30: 772-794Crossref PubMed Google Scholar, 37Bourne R.M. Biochim. Biophys. Acta. 1990; 1055: 1-9Crossref PubMed Scopus (12) Google Scholar). However, the mechanism of polyphosphate synthesis and the vacuolar transport processes required for this synthesis are not clear. Polyphosphate synthesis requires a high energy phosphate donor rather than simply orthophosphate. This donor has not been definitively identified (38Booth J.W. Guidotti G. J. Biol. Chem. 1995; 270: 19377-19382Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), but it presumably must be transported into the vacuole during periods of polyphosphate synthesis. In vivo studies of phosphate metabolism in yeast conducted using 31P-nuclear magnetic resonance (NMR) (10Nicolay K. Scheffers W.A. Bruinenberg P.M. Kaptein R. Arch. Microbiol. 1983; 134: 270-275Crossref PubMed Scopus (37) Google Scholar, 11Gillies R.J. Ugurbil K. den Hollander J.A. Shulman R.G. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2125-2129Crossref PubMed Scopus (110) Google Scholar, 33Bourne R.M. Biochim. Biophys. Acta. 1991; 1067: 81-88Crossref PubMed Scopus (8) Google Scholar,37Bourne R.M. Biochim. Biophys. Acta. 1990; 1055: 1-9Crossref PubMed Scopus (12) Google Scholar), as well as studies using differential extraction techniques to distinguish vacuolar and cytoplasmic ion pools (7Okorokov L.A. Lichko L.P. Kulaev I.S. J. Bacteriol. 1980; 144: 661-665Crossref PubMed Google Scholar) have suggested that a large vacuole-to-cytoplasm gradient of phosphate concentration can exist. However, we have found no evidence for active ATP-driven transport of phosphate across the vacuolar membrane similar to that seen for other substances located in the vacuole. To satisfy conditions of electroneutrality, the efflux of phosphate from vacuoles that we observe must be accompanied by either cation efflux or inward movement of another anion. Since the only anion added to the outside of the vacuoles is the large buffer anion PIPES, the latter possibility seems unlikely. Rather, a vacuolar cation presumably moves out with phosphate, either through the same transporter or in parallel through a separate transporter, e.g. the cation channel previously identified in the vacuolar membrane (18Bertl A. Slayman C.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7824-7828Crossref PubMed Scopus (101) Google Scholar, 19Wada Y. Ohsumi Y. Tanifuji M. Kasai M. Anraku Y. J. Biol. Chem. 1987; 262: 17260-17263Abstract Full Text PDF PubMed Google Scholar). The fact that we could not observe any membrane potential-driven phosphate uptake suggests that the phosphate carrier itself may perform electroneutral transport. It should be noted that the vacuoles used in our experiments were all obtained from cells grown in medium containing ample free phosphate. Thus, the transporter does not appear to require phosphate starvation for its induction, like Pho84p, but rather is present constitutively. There is a striking stimulation of counterflow uptake as the pH is lowered from 7.3 to 5.5. This may suggest that the univalent form of phosphate is the substrate of this carrier, as is the case for the plasma membrane phosphate transporters (6Borst-Pauwels G.W.F.H. Biochim. Biophys. Acta. 1981; 650: 88-127Crossref PubMed Scopus (257) Google Scholar). Most of the transporters identified in the vacuolar membrane to date have been studied using vacuolar membrane vesicles. The majority of these transporters mediate active uptake driven by the H+-ATPase; this is probably a reflection of the fact that passive transport is difficult to observe in vesicles, given their small internal volume and the fact that they are largely devoid of vacuolar contents (32Ohsumi Y. Anraku Y. J. Biol. Chem. 1981; 256: 2079-2082Abstract Full Text PDF PubMed Google Scholar). With intact vacuoles, in contrast, passive transport systems can be discovered and analyzed more easily (25Boller T. Dürr M. Wiemken A. Methods Enzymol. 1989; 174: 504-518Crossref PubMed Scopus (5) Google Scholar). Thus, using intact vacuoles we were able to observe and characterize a transporter which mediates passive transport of phosphate across the vacuolar membrane. We are grateful to Dr. Hans-Peter Feidler of the University of Tübingen for his kind gift of bafilomycin A1 and Dr. Anthony Morielli for critical reading of the manuscript." @default.
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