The Saccharomyces cerevisiae High Affinity Phosphate Transporter Encoded by PHO84 Also Functions in Manganese Homeostasis
2003; Elsevier BV; Volume: 278; Issue: 43 Linguagem: Inglês
10.1074/jbc.m307413200
ISSN1083-351X
AutoresLaran T. Jensen, Mispa Ajua-Alemanji, Valeria Culotta,
Tópico(s)Plant Micronutrient Interactions and Effects
ResumoIn the bakers' yeast Saccharomyces cerevisiae, high affinity manganese uptake and intracellular distribution involve two members of the Nramp family of genes, SMF1 and SMF2. In a search for other genes involved in manganese homeostasis, PHO84 was identified. The PHO84 gene encodes a high affinity inorganic phosphate transporter, and we find that its disruption results in a manganese-resistant phenotype. Resistance to zinc, cobalt, and copper ions was also demonstrated for pho84Δ yeast. When challenged with high concentrations of metals, pho84Δ yeast have reduced metal ion accumulation, suggesting that resistance is due to reduced uptake of metal ions. Pho84p accounted for virtually all the manganese accumulated under metal surplus conditions, demonstrating that this transporter is the major source of excess manganese accumulation. The manganese taken in via Pho84p is indeed biologically active and can not only cause toxicity but can also be incorporated into manganese-requiring enzymes. Pho84p is essential for activating manganese enzymes in smf2Δ mutants that rely on low affinity manganese transport systems. A role for Pho84p in manganese accumulation was also identified in a standard laboratory growth medium when high affinity manganese uptake is active. Under these conditions, cells lacking both Pho84p and the high affinity Smf1p transporter accumulated low levels of manganese, although there was no major effect on activity of manganese-requiring enzymes. We conclude that Pho84p plays a role in manganese homeostasis predominantly under manganese surplus conditions and appears to be functioning as a low affinity metal transporter. In the bakers' yeast Saccharomyces cerevisiae, high affinity manganese uptake and intracellular distribution involve two members of the Nramp family of genes, SMF1 and SMF2. In a search for other genes involved in manganese homeostasis, PHO84 was identified. The PHO84 gene encodes a high affinity inorganic phosphate transporter, and we find that its disruption results in a manganese-resistant phenotype. Resistance to zinc, cobalt, and copper ions was also demonstrated for pho84Δ yeast. When challenged with high concentrations of metals, pho84Δ yeast have reduced metal ion accumulation, suggesting that resistance is due to reduced uptake of metal ions. Pho84p accounted for virtually all the manganese accumulated under metal surplus conditions, demonstrating that this transporter is the major source of excess manganese accumulation. The manganese taken in via Pho84p is indeed biologically active and can not only cause toxicity but can also be incorporated into manganese-requiring enzymes. Pho84p is essential for activating manganese enzymes in smf2Δ mutants that rely on low affinity manganese transport systems. A role for Pho84p in manganese accumulation was also identified in a standard laboratory growth medium when high affinity manganese uptake is active. Under these conditions, cells lacking both Pho84p and the high affinity Smf1p transporter accumulated low levels of manganese, although there was no major effect on activity of manganese-requiring enzymes. We conclude that Pho84p plays a role in manganese homeostasis predominantly under manganese surplus conditions and appears to be functioning as a low affinity metal transporter. Manganese is a biologically important metal that is required by many enzymes for activity. The enzymes that rely on manganese for activity range from carboxylases and phosphatases in the cytosol (1Wedler F.C. Klimis-Tavantzis D.J. Manganese in Health and Disease. CRC Press Inc., Boca Raton, FL1994: 1-38Google Scholar, 2Jabalquinto A.M. Laivenieks M. Zeikus J.G. Cardemil E. J. 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In humans manganese is a potent neurotoxin, and industrial uses of manganese have led to cases of “manganism,” which is characterized by disturbances in mental processes and symptoms similar to those of Parkinson's disease (14Pal P.K. Samii A. Calne D.B. Neurotoxicology. 1999; 20: 227-238PubMed Google Scholar, 15Finley J.W. Davis C.D. Biofactors. 1999; 10: 15-24Crossref PubMed Scopus (109) Google Scholar, 16Kaiser J. Science. 2003; 300: 926-928Crossref PubMed Scopus (92) Google Scholar). It is therefore critical that cells maintain manganese under tight homeostatic control. The bakers' yeast Saccharomyces cerevisiae has served as an excellent model system in which to study the homeostasis of manganese. Two of the manganese transporters identified in this organism are Smf1p and Smf2p (17West A.H. Clark D.J. Martin J. Neupert W. Hart F.U. Horwich A.L. J. Biol. Chem. 1992; 267: 24625-24633Abstract Full Text PDF PubMed Google Scholar, 18Cohen A. Nelson H. Nelson N. J. Biol. Chem. 2000; 275: 33388-33394Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 19Portnoy M.E. Liu X.F. Culotta V.C. Mol. Cell. Biol. 2000; 20: 7893-7902Crossref PubMed Scopus (182) Google Scholar), members of the Nramp family of divalent metal transporters that are conserved from bacteria to humans (20Cellier M. Prive G. Belouchi A. Kwan T. Rodrigues V. Chia W. Gros P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10089-10093Crossref PubMed Scopus (297) Google Scholar). Smf1p is localized to the plasma membrane when manganese is limiting (21Liu X.F. Culotta V.C. J. Biol. Chem. 1999; 274: 4863-4868Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) and has the capacity for high affinity uptake of manganese (22Supek F. Supekova L. Nelson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5105-5110Crossref PubMed Scopus (288) Google Scholar). However, when cells are not starved for manganese, as is the case in standard laboratory growth medium, Smf1p does not appear to be essential for manganese accumulation. In smf1Δ cells lacking this transporter there is no major deficiency in the uptake of manganese, and the activity of manganese-requiring enzymes is not significantly decreased, indicating that other cell surface manganese transporters must be active (23Luk E. Culotta V.C. J. Biol. Chem. 2001; 276: 47556-47562Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). However, the identity of such transporter(s) is currently unknown. The other Nramp manganese transporter of S. cerevisiae, Smf2p, does not directly function in the uptake of extracellular manganese but is localized to intracellular vesicles (19Portnoy M.E. Liu X.F. Culotta V.C. Mol. Cell. Biol. 2000; 20: 7893-7902Crossref PubMed Scopus (182) Google Scholar) and appears to function in the distribution of manganese within the cell (23Luk E. Culotta V.C. J. Biol. Chem. 2001; 276: 47556-47562Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Yeast lacking SMF2 display a severe reduction in whole cell manganese and activity of manganese-requiring enzymes. The reduction in whole cell manganese of smf2Δ mutants has been proposed to result from a feedback inhibition of cell surface manganese uptake (23Luk E. Culotta V.C. J. Biol. Chem. 2001; 276: 47556-47562Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). A separate set of manganese transporter(s) may become active when cells accumulate elevated or toxic levels of the metal (“manganese surplus” state). This is certainly the case for zinc, copper, and iron uptake in yeast. Although high affinity transporters contribute to zinc, copper, and iron uptake when these metals are present at moderate levels (24Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (453) Google Scholar, 25Dancis A. Haile D. Yuan D.S. Klausner R.D. J. Biol. Chem. 1994; 269: 25660-25667Abstract Full Text PDF PubMed Google Scholar, 26Stearman R. Yuan D. Yamaguchi-Iwan Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (582) Google Scholar), upon exposure to elevated concentrations of these metals the low affinity transporter(s) become the primary source of metal for the cell (27Zhao H. Eide D. J. Biol. Chem. 1996; 271: 23203-23210Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 28Dix D. Bridgham J.T. Broderius M.A. Byersdorfer C.A. Eide D.J. J. Biol. Chem. 1994; 269: 26092-26099Abstract Full Text PDF PubMed Google Scholar, 29Dix D. Bridgham J. Broderius M. Eide D. J. Biol. Chem. 1997; 272: 11770-11777Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 30Hassett R. Dix D.R. Eide D.J. Kosman D.J. Biochem. J. 2000; 351: 477-484Crossref PubMed Scopus (98) Google Scholar). In the current study we provide evidence that the apparent low affinity uptake of manganese in yeast is accomplished by the action of Pho84p, a cell surface transporter of phosphate (31Bun-Ya M. Nishimura M. Harashima S. Oshima Y. Mol. Cell. Biol. 1991; 11: 3229-3238Crossref PubMed Scopus (340) Google Scholar, 32Petersson J. Pattison J. Kruckeberg A.L. Berden J.A. Persson B.L. FEBS Lett. 1999; 462: 37-42Crossref PubMed Scopus (53) Google Scholar). Yeast lacking PHO84 are resistant to high concentrations of manganese and have altered metal ion accumulation. Our results indicate that Pho84p works with the high affinity transporter Smf1p to help maintain cellular manganese levels. This is the first example of an eukaryotic phosphate transporter functioning in metal ion transport. Strains, Culture Conditions, and Plasmids—Strains used in this study were derived from either BY4741 (Mat a, leu2Δ0, met15Δ0, ura3Δ0, his3Δ1) or AA255 (Mat a, ade2, his3Δ200, leu2–3112, lys2Δ201, ura3–52). Yeast strains containing single deletions in PHO84, PHO87, PHO88, PHO89, PHO90, and PHO91 were derived from BY4741 and purchased from Research Genetics. Strains XL112 (smf1Δ), XL117 (smf2Δ), and XL131 (smf1Δ smf2Δ) were derived from AA255 and described previously (19Portnoy M.E. Liu X.F. Culotta V.C. Mol. Cell. Biol. 2000; 20: 7893-7902Crossref PubMed Scopus (182) Google Scholar). Disruptions of PHO84 in strains AA255, XL112, and XL117 were generated with plasmid pLJ246, resulting in strains LJ328 (pho84Δ), LJ329 (pho84Δ smf1Δ), and LJ330 (smf2Δ pho84Δ); gene deletions were verified by in vivo PCR. Yeast transformations were performed using the lithium acetate procedure (33Gietz R.D. Schiestl R.H. Yeast. 1991; 7: 253-263Crossref PubMed Scopus (368) Google Scholar). Cells were propagated without shaking at 30 °C either in an enriched yeast extract, peptone-based medium supplemented with 2% dextrose (YPD) 1The abbreviations used are: YPD, yeast extract, peptone, and dextrose; YPLG, yeast extract, peptone, lactate, and glucose. or in minimal synthetic dextrose medium (34Sherman F. Fink G.R. Lawrence C.W. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1978Google Scholar). Cultures for invertase activity assays were grown in a yeast extract, peptone-based medium with 2% lactate, and 0.1% glucose (YPLG). The PHO84 disruption plasmid was generated by PCR amplifying the upstream (from –837 to –15) and downstream sequences (from 2008 to 2672) introducing BamHI and NotI or XhoI and BamHI sites, respectively. The PHO84 PCR products were digested with the indicated enzymes and ligated in a trimolecular reaction into pRS305 (LEU2) digested with XhoI and NotI, resulting in pLJ246. Transformation of yeast strains with pLJ246 digested with BamHI resulted in deletion of PHO84 sequences from –14 to 2007. Metal Measurements—Yeast cells were grown to an A 600 of 2 in YPD medium or the same medium supplemented with the indicated metal ions. The cultures were harvested and washed with TE (10 mm Tris-Cl, and 1 mm EDTA, pH 8), then deionized water, and resuspended in deionized water as described (35Jensen L. Culotta V.C. Mol. Cell. Biol. 2000; 20: 3918-3927Crossref PubMed Scopus (155) Google Scholar). Metal analysis of whole yeast cells was carried out on a PerkinElmer Life Sciences AAnalyst 600 graphite furnace atomic absorption spectrometer according to the manufacturer's specifications. Biochemical Assays—Superoxide dismutase enzymatic activity was assayed in cells grown in YPD medium to an A 600 of 2. For invertase activity gels, cells were grown in YPD to an A 600 of 1, washed with deionized water, resuspended in YPLG medium at an A 600 of 0.3, and grown for 5 h. To prepare cell lysates for electrophoresis on both denaturing and non-denaturing gels, cells were harvested, and spheroplasts were generated as described previously (35Jensen L. Culotta V.C. Mol. Cell. Biol. 2000; 20: 3918-3927Crossref PubMed Scopus (155) Google Scholar). Spheroplasts were lysed with 30 strokes of a microcentrifuge tube pestle in 10 mm HEPES, 1 mm EDTA, and 0.1% Tween 20, pH 7.5, containing protease inhibitors. Extracts were filtered through a 0.45-μm membrane, and protein content was quantitated by the method of Bradford (36Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217508) Google Scholar). Analysis of superoxide dismutase activity by non-denaturing gel electrophoresis and staining with nitro blue tetrazolium and immunoblot analysis of Sod2p levels were performed as described previously (23Luk E. Culotta V.C. J. Biol. Chem. 2001; 276: 47556-47562Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 37Flohe L. Otting F. Packer L. Methods in Enzymology: Oxygen Radicals in Biological Systems. Vol. 105. Academic Press, New York1984: 93-104Google Scholar). Invertase activity gels were analyzed by non-denaturing gel electrophoresis and staining with tetrazolium red as described previously (23Luk E. Culotta V.C. J. Biol. Chem. 2001; 276: 47556-47562Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 38Gabriel O. Wong S.F. Anal. Biochem. 1969; 27: 545-554Crossref PubMed Scopus (188) Google Scholar). Disruption of PHO84 but Not Other Phosphate Transporters Genes Causes Resistance to Manganese—Through an analysis of the Research Genetics collection of yeast deletion mutants, we noted that yeast lacking PHO84 were resistant to high concentrations of manganese. PHO84 encodes an inorganic phosphate transporter that localizes to the cell surface of S. cerevisiae and appears to be a major source of cellular phosphate (31Bun-Ya M. Nishimura M. Harashima S. Oshima Y. Mol. Cell. Biol. 1991; 11: 3229-3238Crossref PubMed Scopus (340) Google Scholar, 32Petersson J. Pattison J. Kruckeberg A.L. Berden J.A. Persson B.L. FEBS Lett. 1999; 462: 37-42Crossref PubMed Scopus (53) Google Scholar). However, pho84Δ mutants are viable because these mutants can obtain phosphate from other transporters. S. cerevisiae is known to express at least six phosphate transporters that contribute to cellular phosphate under various conditions (39Wykoff D.D. O'Shea E.K. Genetics. 2001; 159: 1491-1499Crossref PubMed Google Scholar). To determine whether manganese resistance was associated with the deletion of other phosphate transporters, we tested all six corresponding mutants in a manganese toxicity growth test. As seen in Fig. 1, of the six potential phosphate transporters only disruption of PHO84 resulted in increased resistance to manganese. The identical manganese-resistant phenotype of the pho84Δ strain was observed in two independent genetic backgrounds, i.e. the wild type strain used in the Research Genetics collection (BY4741) (Fig. 1) and an unrelated wild type strain AA255 (not shown). This confirmed that the manganese resistance was in fact due to loss of PHO84. Yeast Lacking PHO84 Are Resistant to Other Metal Ions—We tested whether pho84Δ cells were resistant to other metal ions. As seen in Fig. 2, deletion of PHO84 was not specific to manganese and conferred resistance to zinc and cobalt and a slight resistance to copper. As one possible explanation for the metal resistance, pho84Δ mutants might accumulate lowered concentrations of these metals. We therefore monitored the total cellular concentration of manganese, zinc, cobalt, and copper in cells grown under conditions of moderate metal exposure (e.g. standard laboratory YPD growth medium not supplemented with metals) or exposed to concentrations of these metals that inhibited wild type cell growth by ≈50% (e.g. metal surplus conditions). When grown in a standard laboratory medium without metal supplementation, the cellular levels of zinc and copper were not affected by disruption of PHO84; however, we did observe nearly a 2-fold decrease in manganese and a 4-fold decrease in cobalt levels in the pho84Δ strain relative to wild type (Fig. 3A). Under metal surplus conditions, accumulations of manganese, zinc, cobalt, and copper in the pho84Δ strain were all reduced compared with the wild type (Fig. 3B). The largest effect was observed with manganese in which an 80-fold decrease in metal accumulation was obtained with pho84Δ mutants. Metal accumulation was down 4- to 5-fold for both zinc and cobalt in the pho84Δ strain, and copper showed the smallest difference with a ∼2-fold decrease in pho84Δ yeast. These results indicate that reduced metal accumulation and presumably reduced metal ion uptake is the mechanism of metal ion resistance in pho84Δ yeast. However, Pho84p may also be contributing to the accumulation of manganese and cobalt in a standard laboratory medium in which these metals are present at moderate concentrations.Fig. 3Metal accumulation in pho84 Δ yeast under moderate and surplus metal conditions. Strains AA255 (WT) and LJ328 (pho84Δ) were grown to mid-log phase in YPD medium (A) or the same medium supplemented with 100 μm MnCl2, 750 μm ZnCl2, 250 μm CoSO4, or 2 mm CuSO4 (B) (concentrations of metal that inhibit wild type cell growth by 50%; data not shown), and whole cell metal content was measured by atomic absorption spectrometry. The data (± S.D.) are from at least two independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) PHO84 Is Responsible for Increased Manganese Accumulation When Cells Are Exposed to Elevated Concentrations of the Metal—Using wild type and pho84Δ strains, we sought to determine at what concentration of manganese in the growth medium does Pho84p contribute to manganese accumulation. Both strains were grown in YPD medium containing increasing concentrations of MnCl2, and whole cell manganese was measured by atomic absorption spectrophotometry. The steady state accumulation of manganese under these conditions is shown in Fig. 4. Interestingly, intracellular manganese remained relatively constant until the medium manganese concentration reached ≈5 μm. At this manganese concentration a manganese surplus state ensued in which the wild type strain began to accumulate excess manganese at a level that was proportional to the level of extracellular metal. This increase in cellular manganese was PHO84-dependent, as manganese levels in the pho84Δ strain were unchanged with concentrations of up to 25 μm MnCl2 (Fig. 4). The results clearly show that, under conditions of manganese surplus, virtually all the over-accumulation of manganese is dependent on PHO84. PHO84 and SMF1 Both Contribute to Manganese Acquisition—To date, the only reported high affinity manganese transporter at the cell surface in yeast is Smf1p (22Supek F. Supekova L. Nelson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5105-5110Crossref PubMed Scopus (288) Google Scholar). However, yeast lacking SMF1 only displays a mild reduction in manganese accumulation and does not display symptoms of manganese starvation such as lowered activity in the manganese-containing enzymes Mn-Sod2p and the manganese-requiring mannosyltransferases (23Luk E. Culotta V.C. J. Biol. Chem. 2001; 276: 47556-47562Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Clearly other routes of manganese acquisition are present beyond this high affinity transporter. To test the role of Pho84p in these other pathways of manganese acquisition, we created a strain lacking both SMF1 and PHO84. Under standard laboratory conditions (YPD medium not supplemented with manganese), a single disruptant of either SMF1 or PHO84 was associated with similar reductions in manganese content (≈30 or ≈40% decrease, respectively). Disruption of both SMF1 and PHO84 was found to be additive and further decreased manganese accumulation ≈80% compared with wild type (Fig 5A). This additivity demonstrates that Smf1p and Pho84p are functioning in separate pathways for the accumulation of manganese. The in vivo manganese status of cells grown under standard laboratory conditions was monitored using the activity of two distinct manganese-requiring enzymes, namely Mn-Sod2p in the mitochondria and mannosyltransferases in the Golgi. The activity of manganese-dependent mannosyltransferases was measured indirectly by observing the level of invertase glycosylation, which can be measured by electrophoretic shifts on non-denaturing gels (38Gabriel O. Wong S.F. Anal. Biochem. 1969; 27: 545-554Crossref PubMed Scopus (188) Google Scholar). For example, smf2Δ mutants, which accumulate very low levels of manganese, show an increased mobility of invertase, indicative of poor glycosylation (Fig. 5C) and (23Luk E. Culotta V.C. J. Biol. Chem. 2001; 276: 47556-47562Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). We noted that even with the dramatic decrease in manganese content, the smf1Δ pho84Δ strain did not exhibit symptoms of manganese starvation. As is seen in Fig. 5, B and C, smf1Δ pho84Δ cells grown in a standard laboratory medium contained near wild type levels of Mn-Sod2p activity, and the ability of mannosyltransferases to modify invertase was not impaired. In fact, invertase from cells disrupted for PHO84 showed slower mobility, indicating that invertase from these strains was modified to a somewhat greater extent than that from the wild type sample; however, the cause of this mobility difference is not clear. Therefore, even though the smf1Δ pho84Δ strains accumulate low levels of manganese in standard laboratory medium, there is no obvious impairment in the delivery of manganese to enzymes in the mitochondria or the Golgi. By comparison, smf2Δ strains. which also accumulate low manganese, exhibit a noticeable deficiency in manganese-requiring enzymes (Fig. 5, B and C) and (23Luk E. Culotta V.C. J. Biol. Chem. 2001; 276: 47556-47562Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Although the rationale for this difference is unknown, it may reflect differential compartmentalization and availability of the manganese in the two types of mutants. Rescue of Mn-Sod2p Activity by Manganese Is Impaired in smf2Δ pho84Δ Yeast—The aforementioned experiments demonstrate that Pho84p is not critical for activation of manganese enzymes in a standard laboratory medium when high affinity manganese uptake systems are operative. However, the situation may be different when cells rely on more low affinity transport systems for manganese acquisition (e.g. manganese surplus conditions). Low affinity transport systems are certainly important in the case of smf2 mutants of yeast, as these cells show severe symptoms of manganese starvation unless the medium is supplemented with elevated manganese (23Luk E. Culotta V.C. J. Biol. Chem. 2001; 276: 47556-47562Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The addition of excess manganese to the growth medium, in this case 5–25 μm manganese, corrects the smf2Δ defect and completely restores the activity of Mn-Sod2p (Fig. 6A). Whole cell manganese content was also returned to wild type levels in the smf2Δ strain with the addition of 5 μm manganese, and excess manganese accumulation was observed upon exposure to higher manganese concentrations (Fig. 6B). This important low affinity pathway does not involve Smf1p, because deletion of SMF1 did not block the rescue of the manganese starvation phenotype by excess manganese in smf2Δ yeast (Fig. 6A, right panel). Thus, this manganese uptake must be occurring through an alternative manganese transporter, perhaps Pho84p. To determine whether this was the case, a strain containing a double disruption of SMF2 and PHO84 was generated. Measuring Mn-Sod2p activity and whole cell manganese content of smf2Δ pho84Δ yeast revealed that PHO84 is critical in alleviation of the manganese starvation phenotype when excess manganese is added to the growth medium (Fig. 6A). Whereas smf2Δ cells require the addition of 5 μm MnCl2 to restore activity of Mn-Sod2p, yeast containing double disruptions of SMF2 and PHO84 require 50 μm MnCl2 to achieve similar levels of Sod2p activity. Even with 50 μm manganese added, the whole cell manganese content of smf2Δ pho84Δ is still only ∼60% that of untreated wild type yeast (Fig. 6B). Taken together, these results clearly demonstrate that Pho84p is a major component of the low affinity manganese transport system. This manganese taken up via Pho84p is indeed biologically active. The metal not only contributes to cell toxicity but can enter manganese utilization pathways and be incorporated into manganese-requiring enzymes such as Sod2p. The results presented here provide strong evidence that PHO84 encoding an inorganic phosphate transporter also has a role in metal ion transport. Unlike dedicated metal transporters whose activity is tightly regulated by metal concentration (21Liu X.F. Culotta V.C. J. Biol. 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Biol. 1997; 17: 5044-5052Crossref PubMed Scopus (223) Google Scholar), the uptake of excess metals by Pho84p appears to escape this regulation, perhaps because of its primary function in phosphate transport. As we observed, even in the presence of toxic concentrations of metal ions, PHO84-dependent metal transport continues. Disruption of PHO84 has the strongest effect on manganese accumulation under conditions of manganese surplus, and Pho84p appears to be the primary low affinity transporter of manganese. We find that the manganese transported by Pho84p is biologically active and can both cause toxicity and be incorporated into manganese-requiring enzymes. The intracellular manganese content of yeast is remarkably constant over a certain range of extracellular manganese levels, e.g. up to 5 μm in the case of enriched YPD medium. This buffering of intracellular manganese levels is likely maintained, at least in part, through the action of Pmr1p, a manganese-transporting P-type ATPase (44Antebi A. Fink G.R. Mol. Biol. Cell. 1992; 3: 633-654Crossref PubMed Scopus (378) Google Scholar) that facilitates manganese export from the cell through the secretory pathway (45Durr G. Strayle J. Plemper R. Elbs S. Klee S.K. Catty P. Wolf D.H. Rudolph H.K. Mol. Biol. Cell. 1998; 9: 1149-1162Crossref PubMed Scopus (349) Google Scholar, 46Mandal D. Woolf T.B. Rao R. J. Biol. Chem. 2000; 275: 23933-23938Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). It seems that when the external manganese concentration reaches a certain threshold (e.g. 5 μm in YPD medium), Pho84p becomes effective in causing manganese accumulation, and the export of manganese through Pmr1p is not sufficient to offset the Pho84p-transported manganese. We suggest that Smf1p and Pho84p together comprise a manganese uptake system that functions over a wide array of external metal concentrations. This is similar to what has been described for the metal transport systems for iron, copper, and zinc, which contain both high (24Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (453) Google Scholar, 25Dancis A. Haile D. Yuan D.S. Klausner R.D. J. Biol. Chem. 1994; 269: 25660-25667Abstract Full Text PDF PubMed Google Scholar, 26Stearman R. Yuan D. Yamaguchi-Iwan Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Crossref PubMed Scopus (582) Google Scholar) and low affinity transporters (27Zhao H. Eide D. J. Biol. Chem. 1996; 271: 23203-23210Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 28Dix D. Bridgham J.T. Broderius M.A. Byersdorfer C.A. Eide D.J. J. Biol. Chem. 1994; 269: 26092-26099Abstract Full Text PDF PubMed Google Scholar, 30Hassett R. Dix D.R. Eide D.J. Kosman D.J. Biochem. J. 2000; 351: 477-484Crossref PubMed Scopus (98) Google Scholar). Although Pho84p seems to function primarily under manganese surplus or low affinity conditions, our results indicate that PHO84 as well as SMF1 contribute to the acquisition of manganese under conditions of moderate metal exposure (e.g. growth in a standard laboratory medium). Yeast lacking both SMF1 and PHO84 accumulated low levels of manganese, although no major effect on activity of manganese-requiring enzymes was observed in a standard laboratory medium. Our results suggest that under standard conditions the transport of cobalt is also reduced in pho84Δ yeast, indicating that, like manganese, Pho84p contributes to cobalt accumulation under a wide range of environmental metal concentrations. Yet, this is not the case for all metals, because with copper and zinc the pho84Δ mutations only affected uptake with metal surplus and not under standard laboratory conditions. The different levels of PHO84-dependent metal accumulation observed with manganese, cobalt, zinc, and copper may be the result of metal availability in the medium or the competing influence of other specific transporters for these metals. Presumably, Pho84p is co-transporting divalent metal ions and phosphate in vivo in the form of metal-phosphate complexes. Evidence for such a transport mechanism has come from studies of recombinant Pho84p in reconstituted proteoliposomes (47Fristedt U. van der Rest M. Poolman B. Konings W.N. Persson B.L. Biochemistry. 1999; 38: 16010-16015Crossref PubMed Scopus (31) Google Scholar). Recombinant Pho84p was found to require the presence of metal ions such as manganese and cobalt for phosphate transport, and a MeHPO4 metal-phosphate complex was the proposed substrate. Not all metal phosphate complexes are good substrates for Pho84p, as the addition of magnesium was shown to actually have an inhibitory effect on phosphate transport with recombinant Pho84p (47Fristedt U. van der Rest M. Poolman B. Konings W.N. Persson B.L. Biochemistry. 1999; 38: 16010-16015Crossref PubMed Scopus (31) Google Scholar). Consistent with this, we found an inhibitory effect of magnesium on the Pho84p-dependent uptake of manganese in vivo (data not shown). The preference of Pho84p for metal-phosphate complexes containing manganese (and also cobalt) is consistent with our proposal that Pho84p functions as a low affinity transporter for these metals in vivo. A phosphate transporter that utilizes a metal-phosphate complex as a substrate is not unique to yeast. Bacterial phosphate transporters from Escherichia coli and Acinetobacter johnsonii have been described that require the presence of divalent cations for phosphate transport in reconstituted proteoliposomes (48van Veen H.W. Abee T. Kortstee G.J. Konings W.N. Zehnder A.J. Biochemistry. 1994; 33: 1766-1770Crossref PubMed Scopus (105) Google Scholar, 49van Veen H.W. Abee T. Kortstee G.J. Konings W.N. Zehnder A.J. J. Biol. Chem. 1993; 268: 19377-19383Abstract Full Text PDF PubMed Google Scholar). These bacterial phosphate transporters show a striking biochemical similarity to Pho84p. The substrate for both Pho84p and the bacterial phosphate transporters are thought to be neutral metal-phosphate complex, and each of these transporters shows a preference for MnHPO4 (47Fristedt U. van der Rest M. Poolman B. Konings W.N. Persson B.L. Biochemistry. 1999; 38: 16010-16015Crossref PubMed Scopus (31) Google Scholar, 48van Veen H.W. Abee T. Kortstee G.J. Konings W.N. Zehnder A.J. Biochemistry. 1994; 33: 1766-1770Crossref PubMed Scopus (105) Google Scholar, 49van Veen H.W. Abee T. Kortstee G.J. Konings W.N. Zehnder A.J. J. Biol. Chem. 1993; 268: 19377-19383Abstract Full Text PDF PubMed Google Scholar). Pho84p is homologous to transporters from mammalian, plant, fungal, and bacterial sources and include transporters for both phosphate and sugars (50Henderson P.J. Curr. Opin. Cell Biol. 1993; 5: 708-721Crossref PubMed Scopus (130) Google Scholar, 51Nelissen B. Mordant P. Jonniaux J.L. De Wachter R. Goffeau A. FEBS Lett. 1995; 377: 232-236Crossref PubMed Scopus (74) Google Scholar, 52Andre B. Yeast. 1995; 11: 1575-1611Crossref PubMed Scopus (207) Google Scholar, 53Harrison M.J. van Buuren M.L. Nature. 1995; 378: 626-629Crossref PubMed Scopus (431) Google Scholar, 54Muchhal U.S. Pardo J.M. Raghothama K.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10519-10523Crossref PubMed Scopus (364) Google Scholar). Although Pho84p is the first eukaryotic phosphate transporter described with a role in metal transport, it is likely that this type of system is present in higher organisms, including humans. Exposure to high concentrations of manganese has been shown to result in the development of manganism, a neurological disorder similar to Parkinson's Disease that is characterized by high concentrations of manganese in the brain (14Pal P.K. Samii A. Calne D.B. Neurotoxicology. 1999; 20: 227-238PubMed Google Scholar, 15Finley J.W. Davis C.D. Biofactors. 1999; 10: 15-24Crossref PubMed Scopus (109) Google Scholar, 16Kaiser J. Science. 2003; 300: 926-928Crossref PubMed Scopus (92) Google Scholar). It is possible that neuronally expressed homologues to Pho84p contribute to manganese accumulation under metal surplus conditions. The unexpected connection between phosphate and manganese metabolism revealed here with studies in yeast may have implications in the understanding of manganese toxicity and diseases of manganese exposure in humans.
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