The Yeast Plasma Membrane Protein Alr1 Controls Mg2+ Homeostasis and Is Subject to Mg2+-dependent Control of Its Synthesis and Degradation
2001; Elsevier BV; Volume: 276; Issue: 19 Linguagem: Inglês
10.1074/jbc.m101504200
ISSN1083-351X
AutoresAnton Graschopf, Jochen A. Stadler, M.K. Hoellerer, Sandra Eder, Monika Sieghardt, Sepp D. Kohlwein, Rudolf J. Schweyen,
Tópico(s)Magnesium in Health and Disease
ResumoThe Saccharomyces cerevisiae ALR1(YOL130w) gene product Alr1p is the first known candidate for a Mg2+ transport system in eukaryotic cells and is distantly related to the bacterial CorA Mg2+ transporter family. Here we provide the first experimental evidence for the location of Alr1p in the yeast plasma membrane and for the tight control of its expression and turnover by Mg2+. Using well characterizednpi1 and end3 mutants deficient in the endocytic pathway, we demonstrate that Alr1 protein turnover is dependent on ubiquitination and endocytosis. Furthermore, cells lacking the vacuolar protease Pep4p accumulated Alr1p in the vacuole. Mutants lacking Alr1p (Δalr1) showed a 60% reduction of total intracellular Mg2+ compared with the wild type and failed to grow in standard media. When starved of Mg2+, mutant and wild-type cells had similar low levels of intracellular Mg2+; but upon addition of Mg2+, wild-type cells replenished the intracellular Mg2+ pool within a few hours, whereasΔalr1 mutant cells did not. Expression of the bacterial Mg2+ transporter CorA in the yeastΔalr1 mutant partially restored growth in standard media. The results are discussed in terms of Alr1p being a plasma membrane transporter with high selectivity for Mg2+. The Saccharomyces cerevisiae ALR1(YOL130w) gene product Alr1p is the first known candidate for a Mg2+ transport system in eukaryotic cells and is distantly related to the bacterial CorA Mg2+ transporter family. Here we provide the first experimental evidence for the location of Alr1p in the yeast plasma membrane and for the tight control of its expression and turnover by Mg2+. Using well characterizednpi1 and end3 mutants deficient in the endocytic pathway, we demonstrate that Alr1 protein turnover is dependent on ubiquitination and endocytosis. Furthermore, cells lacking the vacuolar protease Pep4p accumulated Alr1p in the vacuole. Mutants lacking Alr1p (Δalr1) showed a 60% reduction of total intracellular Mg2+ compared with the wild type and failed to grow in standard media. When starved of Mg2+, mutant and wild-type cells had similar low levels of intracellular Mg2+; but upon addition of Mg2+, wild-type cells replenished the intracellular Mg2+ pool within a few hours, whereasΔalr1 mutant cells did not. Expression of the bacterial Mg2+ transporter CorA in the yeastΔalr1 mutant partially restored growth in standard media. The results are discussed in terms of Alr1p being a plasma membrane transporter with high selectivity for Mg2+. polymerase chain reaction hemagglutinin green fluorescent protein high pressure liquid chromatography Mg2+ is the most abundant divalent cation in cells. It is essential for the activation of hundreds of enzymes, for the maintenance of active conformations of macromolecules, for charge compensation, and for the modification of various ion channels. Total cellular concentrations of Mg2+ are in the millimolar range; the vast majority is bound to negatively charged ligands, particularly phosphate, ATP, RNA, and DNA, leaving a only small fraction in the free ionized form (reviewed in Refs. 1Romani A. Scarpa A. Arch. Biochem. Biophys. 1992; 298: 1-12Crossref PubMed Scopus (321) Google Scholar and 2Günther T. Miner. Electrolyte Metab. 1993; 19: 259-265PubMed Google Scholar).Free ionized Mg2+ concentrations remain relatively unchanged in mammalian cells, whereas total concentrations can vary to a considerable extent, mostly depending on the intracellular ion milieu and on metabolic stimulation by hormones and other factors (1Romani A. Scarpa A. Arch. Biochem. Biophys. 1992; 298: 1-12Crossref PubMed Scopus (321) Google Scholar, 3Fatholahi M. LaNoue K. Romani A. Scarpa A. Arch. Biochem. Biophys. 2000; 374: 395-401Crossref PubMed Scopus (63) Google Scholar). Cells of the yeast Saccharomyces cerevisiae tightly control intracellular Mg2+ levels, which remain relatively constant in growth media containing 1–100 mm Mg2+. Yeast cells starved of Mg2+ stop growing and lose their viability when the intracellular Mg2+ concentration falls below a threshold level (4Beeler T. Bruce K. Dunn T. Biochim. Biophys. Acta. 1997; 1323: 310-318Crossref PubMed Scopus (39) Google Scholar).The physiology of Mg2+ transport has been studied in vertebrate and mammalian cell types and in plasma membrane vesicles during the past 40 years, mostly by observing extrusion from cells rather than uptake of the ion into cells. Most studies agree on the presence of a Mg2+/Na+ antiporter in the plasma membrane. Recent observations indicate the presence of up to three Mg2+ transporters. These act as antiporters, which exchange Mg2+ for sodium or calcium, or as cotransporters of Mg2+ and anions (reviewed in Refs. 1Romani A. Scarpa A. Arch. Biochem. Biophys. 1992; 298: 1-12Crossref PubMed Scopus (321) Google Scholar, 2Günther T. Miner. Electrolyte Metab. 1993; 19: 259-265PubMed Google Scholar, and 5Günther T. Vormann J. Biochim. Biophys. Acta. 1995; 1234: 105-110Crossref PubMed Scopus (40) Google Scholar, 6Tashiro M. Konishi M. Biophys. J. 1997; 73: 3371-3384Abstract Full Text PDF PubMed Scopus (28) Google Scholar, 7Cefaratti C. Romani A. Scarpa A. Am. J. Physiol. 1998; 275: C995-C1008Crossref PubMed Google Scholar, 8Cefaratti C. Romani A. Scarpa A. J. Biol. Chem. 2000; 275: 3772-3780Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 9Schweigel M. Vormann J. Martens H. Am. J. Physiol. 2000; 278: G400-G408Crossref PubMed Google Scholar). Further transport systems are to be expected in membranes of intracellular compartments that are likely to sequester and release Mg2+(10Nelson N. EMBO J. 1999; 18: 4361-4371Crossref PubMed Scopus (248) Google Scholar). Whereas none of these mammalian transporters have been specified in molecular terms yet, candidate genes encoding Mg2+transporters in the vacuolar membrane of the plant Arabidopsis thaliana and in the mitochondrial membrane of the yeast S. cerevisiae have been described recently (11Shaul O. Hilgemann D.W. de Almeira-Engler J. Van Montagu M. Inzé D. Galili G. EMBO J. 1999; 18: 3973-3980Crossref PubMed Scopus (206) Google Scholar, 12Bui D.M. Gregan J. Jarosch E. Ragnini A. Schweyen R.J. J. Biol. Chem. 1999; 274: 20438-20443Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 13Gregan J. Bui M.D. Pillich R. Fink M. Zsurka G. Schweyen R.J. Mol. Gen. Genet. 2001; 264: 773-781Crossref PubMed Scopus (54) Google Scholar).In bacteria, three proteins (CorA, MgtA, and MgtB) have been shown to be involved in Mg2+ transport across the plasma membrane. Members of the CorA family are virtually ubiquitous in eubacteria and archaea and form their constitutive Mg2+ influx system (reviewed in Ref. 14Smith R.L. Maguire M.E. Mol. Microbiol. 1998; 28: 217-226Crossref PubMed Scopus (148) Google Scholar). Although their sequences may have diverged considerably, all family members are characterized by two or three adjacent transmembrane domains near their carboxyl termini, one of which is followed by the motif (Y/F)GMN. Even distant homologs have been shown to be functionally equivalent Mg2+transporters. Whereas the CorA proteins form a family of their own, the bacterial MgtA and MgtB Mg2+ transport systems belong to the P-type ATPases. Unlike CorA, their expression is regulated via the two-component signal transduction system PhoPQ, which itself is subject to regulation by Mg2+ (reviewed in Ref. 14Smith R.L. Maguire M.E. Mol. Microbiol. 1998; 28: 217-226Crossref PubMed Scopus (148) Google Scholar).Recently, eukaryotic homologs of the bacterial CorA Mg2+transporters have been identified in the yeast S. cerevisiae. They are characterized by two predicted transmembrane domains and the sequence motif (Y/F)GMN in the short segment connecting them. Mrs2p and Lpe10p are related proteins of the inner mitochondrial membrane. Absence of one or the other of this pair renders cells mitochondrially defective and causes a 2-fold reduction of intramitochondrial Mg2+ concentrations. Consistently, overexpression of Mrs2p and Lpe10p leads to a moderate increase in the mitochondrial Mg2+ concentration (12Bui D.M. Gregan J. Jarosch E. Ragnini A. Schweyen R.J. J. Biol. Chem. 1999; 274: 20438-20443Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 13Gregan J. Bui M.D. Pillich R. Fink M. Zsurka G. Schweyen R.J. Mol. Gen. Genet. 2001; 264: 773-781Crossref PubMed Scopus (54) Google Scholar).Alr1p is essential for growth of yeast cells, except in media with high Mg2+ concentrations, and its overexpression confers resistance to aluminum (15MacDiarmid C.W. Gardner R.C. J. Biol. Chem. 1998; 273: 1727-1732Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). High expression of Alr1p correlates with an increase in the uptake of labeled cobalt, which is likely to be transported by Mg2+ transporters. These phenotypic features of Δalr1 mutants suggest that Alr1p is part of an essential Mg2+ transport system in the yeast plasma membrane. The yeast genome encodes a close homolog of this protein, named Alr2p. Growth of yeast cells is not dependent on the presence of this homolog; but when overexpressed, it can compensate for the absence of Alr1p (15MacDiarmid C.W. Gardner R.C. J. Biol. Chem. 1998; 273: 1727-1732Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar).Here we present the first evidence for Alr1p being a protein in the yeast plasma membrane whose expression and turnover via endocytosis and vacuolar decay are tightly controlled by Mg2+. InΔalr1 cells grown in standard media, the intracellular Mg2+ concentration is reduced by a factor of 2 compared with wild-type cells. When grown in Mg2+-depleted media, mutant and wild-type cells exhibit a comparable reduction of intracellular Mg2+, but mutant cells have a reduced ability to replenish Mg2+ pools from external sources, indicating that they have an impaired Mg2+ transport capacity.DISCUSSIONThe yeast protein Alr1 was the first candidate for a transporter of Mg2+ in eukaryotic cells (15MacDiarmid C.W. Gardner R.C. J. Biol. Chem. 1998; 273: 1727-1732Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The results presented here show its location in the plasma membrane and elucidate the specific role of Alr1p in cellular Mg2+ homeostasis as well as in the ion-specific expression and turnover of this protein.With two predicted transmembrane domains in its carboxyl-terminal part, the first of which is followed by the conserved motif (Y/F)GMN, Alr1p appears to be distantly related to the bacterial CorA proteins. Additional members of this family of putative Mg2+transporters are Mrs2p and Lpe10p in yeast mitochondria. This relationship is confirmed by the finding that growth defects caused byΔalr1, Δmrs2, orΔlpe10 mutations can be partially suppressed by expression of the bacterial CorA protein in yeast cells (Refs. 12Bui D.M. Gregan J. Jarosch E. Ragnini A. Schweyen R.J. J. Biol. Chem. 1999; 274: 20438-20443Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar and13Gregan J. Bui M.D. Pillich R. Fink M. Zsurka G. Schweyen R.J. Mol. Gen. Genet. 2001; 264: 773-781Crossref PubMed Scopus (54) Google Scholar and this work). Cell fractionation and fluorescence microscopy data reveal that Alr1p is located in the plasma membrane. This location is predominant in cells grown in limiting concentrations of Mg2+, whereas cells grown in medium containing standard or high Mg2+ concentrations show reduced total amounts of Alr1p, and the residual protein is found partly in the plasma membrane and partly in intracellular vesicles.Mg2+ plays a crucial role in Alr1 protein stability. Exposure of cells to even standard Mg2+ concentrations leads to a dramatic decrease in the stability of this protein. This is reminiscent of regulation of the plasma membrane manganese transporter Smf1p and contrary to the copper transporter Ctr1p and the zinc transporter Zrt1p, where turnover of these proteins is induced only by relatively high copper and zinc concentrations (35Gitan R.S. Luo H. Rodgers J. Broderius M. Eide D. J. Biol. Chem. 1998; 273: 28617-28624Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 38Liu X.F. Culotta V.C. J. Biol. Chem. 1999; 274: 4863-4868Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 39Ooi C.E. Rabinovich E. Dancis A. Bonifacino J.S. Klausner R.D. EMBO J. 1996; 15: 3515-3523Crossref PubMed Scopus (178) Google Scholar). Data obtained here with the npi1, end3, andpep4 mutants affecting ubiquitination, endocytosis, and vacuolar degradation, respectively, reveal that Alr1p is internalized via the endocytic pathway and delivered to the vacuole for degradation. The data do not exclude additional routes of degradation of Alr1p. Interestingly, these mutants also accumulate a modified form of Alr1p, which constitutes a minor fraction of Alr1p in wild-type cells. This modification apparently precedes ubiquitination and endocytosis of Alr1p. It still remains to be shown how Mg2+ triggers the initial steps of Alr1 protein turnover and in which form Alr1p enters the endocytic pathway. The mechanism of Alr1p turnover is reminiscent of substrate-triggered degradation of plasma membrane proteins such as Ste2p, Ste3p, Pdr5p, Fur4p, sugar permeases, and the Zn2+transporter Zrt1p, which are removed from the plasma membrane by endocytosis for vacuolar degradation (26Rotin D. Staub O. Haguenauer-Tsapis R. J. Membr. Biol. 2000; 176: 1-17Crossref PubMed Google Scholar, 29Benedetti H. Raths S. Crausaz F. Riezman H. Mol. Biol. Cell. 1994; 5: 1023-1037Crossref PubMed Scopus (237) Google Scholar, 31Berkower C. Loayza D. Michaelis S. Mol. Biol. Cell. 1994; 5: 1185-1198Crossref PubMed Scopus (88) Google Scholar, 32Medintz I. Jiang H. Han E.K. Cui W. Michels C.A. J. Bacteriol. 1996; 178: 2245-2254Crossref PubMed Google Scholar, 33Chiang H.L. Schekman R. Hamamoto S. J. Biol. Chem. 1996; 271: 9934-9941Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 34Volland C. Urban-Grimal D. Geraud G. Haguenauer-Tsapis R. J. Biol. Chem. 1994; 269: 9833-9841Abstract Full Text PDF PubMed Google Scholar, 35Gitan R.S. Luo H. Rodgers J. Broderius M. Eide D. J. Biol. Chem. 1998; 273: 28617-28624Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Rapid decay of these plasma membrane receptors and transporters is induced by their physiological substrates. Similarly, Alr1p degradation appears to be triggered by Mg2+, with high selectivity over other divalent metal ions. Only cobalt, which has been shown to be taken up by the Mg2+ transport systems (14Smith R.L. Maguire M.E. Mol. Microbiol. 1998; 28: 217-226Crossref PubMed Scopus (148) Google Scholar, 15MacDiarmid C.W. Gardner R.C. J. Biol. Chem. 1998; 273: 1727-1732Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), and manganese affect Alr1p stability, but only when present at non-physiologically high concentrations. In addition to this post-translational control,ALR1 mRNA steady-state levels are down-regulated in medium containing standard or high Mg2+ concentrations compared with Mg2+ limiting growth conditions. It still remains to be shown whether this regulation is exerted by metal ion-sensitive transcription factors, as in the case of the zinc transporters Zrt1p, Zrt2p, and Zrt3p (40Zhao H. Butler E. Rodgers J. Spizzo T. Duesterhoeft S. Eide D. J. Biol. Chem. 1998; 273: 28713-28720Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 41MacDiarmid C.W. Gaither L.A. Eide D. EMBO J. 2000; 19: 2845-2855Crossref PubMed Scopus (301) Google Scholar), or by mRNA turnover.Mg2+ appears to be the only ion whose intracellular concentration becomes growth-limiting in Δalr1cells. First, the total intracellular concentration of Mg2+(but not of other ions) is significantly reduced in this mutant. Second, out of many metal ions tested, only Mg2+ at non-physiologically high concentrations can efficiently restore growth of the deletion mutant (15MacDiarmid C.W. Gardner R.C. J. Biol. Chem. 1998; 273: 1727-1732Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Mg2+ uptake or homeostasis therefore appears to be the essential function of Alr1p when low or standard Mg2+ concentrations are provided. In growth media containing non-physiologically high Mg2+ concentrations, Alr1p is dispensable; Δalr1 cells regain growth; and wild-type cells severely reduce the amount of Alr1 protein in the plasma membrane. Uptake of Mg2+ under these conditions apparently is mediated by other uptake mechanisms.Consistent with previous studies on wild-type yeast cells (4Beeler T. Bruce K. Dunn T. Biochim. Biophys. Acta. 1997; 1323: 310-318Crossref PubMed Scopus (39) Google Scholar), the data reported here confirm a very tight control of total intracellular Mg2+ concentrations, allowing a 3–4-fold decrease only, when the external concentrations change by 4 orders of magnitude (from 100 mm to 10 μm). They were found to be kept rather constant at ∼3 mg/g (dry weight) with extracellular Mg2+ concentrations ranging from 1 to 200 mm. Only further studies will reveal if reduced influx or increased efflux (or both) accounts for the lack of cellular Mg2+accumulation in the presence of very high external concentrations. Low intracellular Mg2+ concentrations of ∼1 mg/g (dry weight) were detected both when wild-type cells were grown in essentially Mg2+-free medium (data not shown) and whenΔalr1 mutant cells were grown in medium containing ≤1 mm Mg2+. It remains to be shown whether this arrest is due to specific signals resulting from low intracellular Mg2+, leading to a defined state of cellular differentiation, as previously described for the fission yeastSchizosaccharomyces pombe (42Walker G.M. Magnesium. 1986; 5: 9-23PubMed Google Scholar), or whether the demand of a certain essential enzymatic function for Mg2+ can no longer be met, leading to growth arrest.Taken together, the data presented here consistently show that Alr1p expression is essential to maintain cellular Mg2+concentrations at levels suitable for growth of yeast cells. The suppression of the Δalr1 mutant phenotype by high Mg2+ (but not by other ions) and the control of expression and stability of Alr1p by Mg2+ suggest a specificity for Mg2+. Changes in steady-state levels of other ions appear to be secondary, reflecting charge compensation. The location of Alr1p in the plasma membrane of yeast cells and its apparent functional and structural homology to the bacterial Mg2+ transporter CorA protein tentatively classify Alr1p as a Mg2+ transporter of the yeast plasma membrane. It remains to be shown by which mechanisms Alr1p mediates uptake of Mg2+ into yeast cells and whether homologs of Alr1p exist in plasma membranes of higher eukaryotes. Mg2+ is the most abundant divalent cation in cells. It is essential for the activation of hundreds of enzymes, for the maintenance of active conformations of macromolecules, for charge compensation, and for the modification of various ion channels. Total cellular concentrations of Mg2+ are in the millimolar range; the vast majority is bound to negatively charged ligands, particularly phosphate, ATP, RNA, and DNA, leaving a only small fraction in the free ionized form (reviewed in Refs. 1Romani A. Scarpa A. Arch. Biochem. Biophys. 1992; 298: 1-12Crossref PubMed Scopus (321) Google Scholar and 2Günther T. Miner. Electrolyte Metab. 1993; 19: 259-265PubMed Google Scholar). Free ionized Mg2+ concentrations remain relatively unchanged in mammalian cells, whereas total concentrations can vary to a considerable extent, mostly depending on the intracellular ion milieu and on metabolic stimulation by hormones and other factors (1Romani A. Scarpa A. Arch. Biochem. Biophys. 1992; 298: 1-12Crossref PubMed Scopus (321) Google Scholar, 3Fatholahi M. LaNoue K. Romani A. Scarpa A. Arch. Biochem. Biophys. 2000; 374: 395-401Crossref PubMed Scopus (63) Google Scholar). Cells of the yeast Saccharomyces cerevisiae tightly control intracellular Mg2+ levels, which remain relatively constant in growth media containing 1–100 mm Mg2+. Yeast cells starved of Mg2+ stop growing and lose their viability when the intracellular Mg2+ concentration falls below a threshold level (4Beeler T. Bruce K. Dunn T. Biochim. Biophys. Acta. 1997; 1323: 310-318Crossref PubMed Scopus (39) Google Scholar). The physiology of Mg2+ transport has been studied in vertebrate and mammalian cell types and in plasma membrane vesicles during the past 40 years, mostly by observing extrusion from cells rather than uptake of the ion into cells. Most studies agree on the presence of a Mg2+/Na+ antiporter in the plasma membrane. Recent observations indicate the presence of up to three Mg2+ transporters. These act as antiporters, which exchange Mg2+ for sodium or calcium, or as cotransporters of Mg2+ and anions (reviewed in Refs. 1Romani A. Scarpa A. Arch. Biochem. Biophys. 1992; 298: 1-12Crossref PubMed Scopus (321) Google Scholar, 2Günther T. Miner. Electrolyte Metab. 1993; 19: 259-265PubMed Google Scholar, and 5Günther T. Vormann J. Biochim. Biophys. Acta. 1995; 1234: 105-110Crossref PubMed Scopus (40) Google Scholar, 6Tashiro M. Konishi M. Biophys. J. 1997; 73: 3371-3384Abstract Full Text PDF PubMed Scopus (28) Google Scholar, 7Cefaratti C. Romani A. Scarpa A. Am. J. Physiol. 1998; 275: C995-C1008Crossref PubMed Google Scholar, 8Cefaratti C. Romani A. Scarpa A. J. Biol. Chem. 2000; 275: 3772-3780Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 9Schweigel M. Vormann J. Martens H. Am. J. Physiol. 2000; 278: G400-G408Crossref PubMed Google Scholar). Further transport systems are to be expected in membranes of intracellular compartments that are likely to sequester and release Mg2+(10Nelson N. EMBO J. 1999; 18: 4361-4371Crossref PubMed Scopus (248) Google Scholar). Whereas none of these mammalian transporters have been specified in molecular terms yet, candidate genes encoding Mg2+transporters in the vacuolar membrane of the plant Arabidopsis thaliana and in the mitochondrial membrane of the yeast S. cerevisiae have been described recently (11Shaul O. Hilgemann D.W. de Almeira-Engler J. Van Montagu M. Inzé D. Galili G. EMBO J. 1999; 18: 3973-3980Crossref PubMed Scopus (206) Google Scholar, 12Bui D.M. Gregan J. Jarosch E. Ragnini A. Schweyen R.J. J. Biol. Chem. 1999; 274: 20438-20443Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 13Gregan J. Bui M.D. Pillich R. Fink M. Zsurka G. Schweyen R.J. Mol. Gen. Genet. 2001; 264: 773-781Crossref PubMed Scopus (54) Google Scholar). In bacteria, three proteins (CorA, MgtA, and MgtB) have been shown to be involved in Mg2+ transport across the plasma membrane. Members of the CorA family are virtually ubiquitous in eubacteria and archaea and form their constitutive Mg2+ influx system (reviewed in Ref. 14Smith R.L. Maguire M.E. Mol. Microbiol. 1998; 28: 217-226Crossref PubMed Scopus (148) Google Scholar). Although their sequences may have diverged considerably, all family members are characterized by two or three adjacent transmembrane domains near their carboxyl termini, one of which is followed by the motif (Y/F)GMN. Even distant homologs have been shown to be functionally equivalent Mg2+transporters. Whereas the CorA proteins form a family of their own, the bacterial MgtA and MgtB Mg2+ transport systems belong to the P-type ATPases. Unlike CorA, their expression is regulated via the two-component signal transduction system PhoPQ, which itself is subject to regulation by Mg2+ (reviewed in Ref. 14Smith R.L. Maguire M.E. Mol. Microbiol. 1998; 28: 217-226Crossref PubMed Scopus (148) Google Scholar). Recently, eukaryotic homologs of the bacterial CorA Mg2+transporters have been identified in the yeast S. cerevisiae. They are characterized by two predicted transmembrane domains and the sequence motif (Y/F)GMN in the short segment connecting them. Mrs2p and Lpe10p are related proteins of the inner mitochondrial membrane. Absence of one or the other of this pair renders cells mitochondrially defective and causes a 2-fold reduction of intramitochondrial Mg2+ concentrations. Consistently, overexpression of Mrs2p and Lpe10p leads to a moderate increase in the mitochondrial Mg2+ concentration (12Bui D.M. Gregan J. Jarosch E. Ragnini A. Schweyen R.J. J. Biol. Chem. 1999; 274: 20438-20443Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 13Gregan J. Bui M.D. Pillich R. Fink M. Zsurka G. Schweyen R.J. Mol. Gen. Genet. 2001; 264: 773-781Crossref PubMed Scopus (54) Google Scholar). Alr1p is essential for growth of yeast cells, except in media with high Mg2+ concentrations, and its overexpression confers resistance to aluminum (15MacDiarmid C.W. Gardner R.C. J. Biol. Chem. 1998; 273: 1727-1732Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). High expression of Alr1p correlates with an increase in the uptake of labeled cobalt, which is likely to be transported by Mg2+ transporters. These phenotypic features of Δalr1 mutants suggest that Alr1p is part of an essential Mg2+ transport system in the yeast plasma membrane. The yeast genome encodes a close homolog of this protein, named Alr2p. Growth of yeast cells is not dependent on the presence of this homolog; but when overexpressed, it can compensate for the absence of Alr1p (15MacDiarmid C.W. Gardner R.C. J. Biol. Chem. 1998; 273: 1727-1732Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Here we present the first evidence for Alr1p being a protein in the yeast plasma membrane whose expression and turnover via endocytosis and vacuolar decay are tightly controlled by Mg2+. InΔalr1 cells grown in standard media, the intracellular Mg2+ concentration is reduced by a factor of 2 compared with wild-type cells. When grown in Mg2+-depleted media, mutant and wild-type cells exhibit a comparable reduction of intracellular Mg2+, but mutant cells have a reduced ability to replenish Mg2+ pools from external sources, indicating that they have an impaired Mg2+ transport capacity. DISCUSSIONThe yeast protein Alr1 was the first candidate for a transporter of Mg2+ in eukaryotic cells (15MacDiarmid C.W. Gardner R.C. J. Biol. Chem. 1998; 273: 1727-1732Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The results presented here show its location in the plasma membrane and elucidate the specific role of Alr1p in cellular Mg2+ homeostasis as well as in the ion-specific expression and turnover of this protein.With two predicted transmembrane domains in its carboxyl-terminal part, the first of which is followed by the conserved motif (Y/F)GMN, Alr1p appears to be distantly related to the bacterial CorA proteins. Additional members of this family of putative Mg2+transporters are Mrs2p and Lpe10p in yeast mitochondria. This relationship is confirmed by the finding that growth defects caused byΔalr1, Δmrs2, orΔlpe10 mutations can be partially suppressed by expression of the bacterial CorA protein in yeast cells (Refs. 12Bui D.M. Gregan J. Jarosch E. Ragnini A. Schweyen R.J. J. Biol. Chem. 1999; 274: 20438-20443Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar and13Gregan J. Bui M.D. Pillich R. Fink M. Zsurka G. Schweyen R.J. Mol. Gen. Genet. 2001; 264: 773-781Crossref PubMed Scopus (54) Google Scholar and this work). Cell fractionation and fluorescence microscopy data reveal that Alr1p is located in the plasma membrane. This location is predominant in cells grown in limiting concentrations of Mg2+, whereas cells grown in medium containing standard or high Mg2+ concentrations show reduced total amounts of Alr1p, and the residual protein is found partly in the plasma membrane and partly in intracellular vesicles.Mg2+ plays a crucial role in Alr1 protein stability. Exposure of cells to even standard Mg2+ concentrations leads to a dramatic decrease in the stability of this protein. This is reminiscent of regulation of the plasma membrane manganese transporter Smf1p and contrary to the copper transporter Ctr1p and the zinc transporter Zrt1p, where turnover of these proteins is induced only by relatively high copper and zinc concentrations (35Gitan R.S. Luo H. Rodgers J. Broderius M. Eide D. J. Biol. Chem. 1998; 273: 28617-28624Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 38Liu X.F. Culotta V.C. J. Biol. Chem. 1999; 274: 4863-4868Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 39Ooi C.E. Rabinovich E. Dancis A. Bonifacino J.S. Klausner R.D. EMBO J. 1996; 15: 3515-3523Crossref PubMed Scopus (178) Google Scholar). Data obtained here with the npi1, end3, andpep4 mutants affecting ubiquitination, endocytosis, and vacuolar degradation, respectively, reveal that Alr1p is internalized via the endocytic pathway and delivered to the vacuole for degradation. The data do not exclude additional routes of degradation of Alr1p. Interestingly, these mutants also accumulate a modified form of Alr1p, which constitutes a minor fraction of Alr1p in wild-type cells. This modification apparently precedes ubiquitination and endocytosis of Alr1p. It still remains to be shown how Mg2+ triggers the initial steps of Alr1 protein turnover and in which form Alr1p enters the endocytic pathway. The mechanism of Alr1p turnover is reminiscent of substrate-triggered degradation of plasma membrane proteins such as Ste2p, Ste3p, Pdr5p, Fur4p, sugar permeases, and the Zn2+transporter Zrt1p, which are removed from the plasma membrane by endocytosis for vacuolar degradation (26Rotin D. Staub O. Haguenauer-Tsapis R. J. Membr. Biol. 2000; 176: 1-17Crossref PubMed Google Scholar, 29Benedetti H. Raths S. Crausaz F. Riezman H. Mol. Biol. Cell. 1994; 5: 1023-1037Crossref PubMed Scopus (237) Google Scholar, 31Berkower C. Loayza D. Michaelis S. Mol. Biol. Cell. 1994; 5: 1185-1198Crossref PubMed Scopus (88) Google Scholar, 32Medintz I. Jiang H. Han E.K. Cui W. Michels C.A. J. Bacteriol. 1996; 178: 2245-2254Crossref PubMed Google Scholar, 33Chiang H.L. Schekman R. Hamamoto S. J. Biol. Chem. 1996; 271: 9934-9941Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 34Volland C. Urban-Grimal D. Geraud G. Haguenauer-Tsapis R. J. Biol. Chem. 1994; 269: 9833-9841Abstract Full Text PDF PubMed Google Scholar, 35Gitan R.S. Luo H. Rodgers J. Broderius M. Eide D. J. Biol. Chem. 1998; 273: 28617-28624Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Rapid decay of these plasma membrane receptors and transporters is induced by their physiological substrates. Similarly, Alr1p degradation appears to be triggered by Mg2+, with high selectivity over other divalent metal ions. Only cobalt, which has been shown to be taken up by the Mg2+ transport systems (14Smith R.L. Maguire M.E. Mol. Microbiol. 1998; 28: 217-226Crossref PubMed Scopus (148) Google Scholar, 15MacDiarmid C.W. Gardner R.C. J. Biol. Chem. 1998; 273: 1727-1732Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), and manganese affect Alr1p stability, but only when present at non-physiologically high concentrations. In addition to this post-translational control,ALR1 mRNA steady-state levels are down-regulated in medium containing standard or high Mg2+ concentrations compared with Mg2+ limiting growth conditions. It still remains to be shown whether this regulation is exerted by metal ion-sensitive transcription factors, as in the case of the zinc transporters Zrt1p, Zrt2p, and Zrt3p (40Zhao H. Butler E. Rodgers J. Spizzo T. Duesterhoeft S. Eide D. J. Biol. Chem. 1998; 273: 28713-28720Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 41MacDiarmid C.W. Gaither L.A. Eide D. EMBO J. 2000; 19: 2845-2855Crossref PubMed Scopus (301) Google Scholar), or by mRNA turnover.Mg2+ appears to be the only ion whose intracellular concentration becomes growth-limiting in Δalr1cells. First, the total intracellular concentration of Mg2+(but not of other ions) is significantly reduced in this mutant. Second, out of many metal ions tested, only Mg2+ at non-physiologically high concentrations can efficiently restore growth of the deletion mutant (15MacDiarmid C.W. Gardner R.C. J. Biol. Chem. 1998; 273: 1727-1732Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Mg2+ uptake or homeostasis therefore appears to be the essential function of Alr1p when low or standard Mg2+ concentrations are provided. In growth media containing non-physiologically high Mg2+ concentrations, Alr1p is dispensable; Δalr1 cells regain growth; and wild-type cells severely reduce the amount of Alr1 protein in the plasma membrane. Uptake of Mg2+ under these conditions apparently is mediated by other uptake mechanisms.Consistent with previous studies on wild-type yeast cells (4Beeler T. Bruce K. Dunn T. Biochim. Biophys. Acta. 1997; 1323: 310-318Crossref PubMed Scopus (39) Google Scholar), the data reported here confirm a very tight control of total intracellular Mg2+ concentrations, allowing a 3–4-fold decrease only, when the external concentrations change by 4 orders of magnitude (from 100 mm to 10 μm). They were found to be kept rather constant at ∼3 mg/g (dry weight) with extracellular Mg2+ concentrations ranging from 1 to 200 mm. Only further studies will reveal if reduced influx or increased efflux (or both) accounts for the lack of cellular Mg2+accumulation in the presence of very high external concentrations. Low intracellular Mg2+ concentrations of ∼1 mg/g (dry weight) were detected both when wild-type cells were grown in essentially Mg2+-free medium (data not shown) and whenΔalr1 mutant cells were grown in medium containing ≤1 mm Mg2+. It remains to be shown whether this arrest is due to specific signals resulting from low intracellular Mg2+, leading to a defined state of cellular differentiation, as previously described for the fission yeastSchizosaccharomyces pombe (42Walker G.M. Magnesium. 1986; 5: 9-23PubMed Google Scholar), or whether the demand of a certain essential enzymatic function for Mg2+ can no longer be met, leading to growth arrest.Taken together, the data presented here consistently show that Alr1p expression is essential to maintain cellular Mg2+concentrations at levels suitable for growth of yeast cells. The suppression of the Δalr1 mutant phenotype by high Mg2+ (but not by other ions) and the control of expression and stability of Alr1p by Mg2+ suggest a specificity for Mg2+. Changes in steady-state levels of other ions appear to be secondary, reflecting charge compensation. The location of Alr1p in the plasma membrane of yeast cells and its apparent functional and structural homology to the bacterial Mg2+ transporter CorA protein tentatively classify Alr1p as a Mg2+ transporter of the yeast plasma membrane. It remains to be shown by which mechanisms Alr1p mediates uptake of Mg2+ into yeast cells and whether homologs of Alr1p exist in plasma membranes of higher eukaryotes. The yeast protein Alr1 was the first candidate for a transporter of Mg2+ in eukaryotic cells (15MacDiarmid C.W. Gardner R.C. J. Biol. Chem. 1998; 273: 1727-1732Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The results presented here show its location in the plasma membrane and elucidate the specific role of Alr1p in cellular Mg2+ homeostasis as well as in the ion-specific expression and turnover of this protein. With two predicted transmembrane domains in its carboxyl-terminal part, the first of which is followed by the conserved motif (Y/F)GMN, Alr1p appears to be distantly related to the bacterial CorA proteins. Additional members of this family of putative Mg2+transporters are Mrs2p and Lpe10p in yeast mitochondria. This relationship is confirmed by the finding that growth defects caused byΔalr1, Δmrs2, orΔlpe10 mutations can be partially suppressed by expression of the bacterial CorA protein in yeast cells (Refs. 12Bui D.M. Gregan J. Jarosch E. Ragnini A. Schweyen R.J. J. Biol. Chem. 1999; 274: 20438-20443Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar and13Gregan J. Bui M.D. Pillich R. Fink M. Zsurka G. Schweyen R.J. Mol. Gen. Genet. 2001; 264: 773-781Crossref PubMed Scopus (54) Google Scholar and this work). Cell fractionation and fluorescence microscopy data reveal that Alr1p is located in the plasma membrane. This location is predominant in cells grown in limiting concentrations of Mg2+, whereas cells grown in medium containing standard or high Mg2+ concentrations show reduced total amounts of Alr1p, and the residual protein is found partly in the plasma membrane and partly in intracellular vesicles. Mg2+ plays a crucial role in Alr1 protein stability. Exposure of cells to even standard Mg2+ concentrations leads to a dramatic decrease in the stability of this protein. This is reminiscent of regulation of the plasma membrane manganese transporter Smf1p and contrary to the copper transporter Ctr1p and the zinc transporter Zrt1p, where turnover of these proteins is induced only by relatively high copper and zinc concentrations (35Gitan R.S. Luo H. Rodgers J. Broderius M. Eide D. J. Biol. Chem. 1998; 273: 28617-28624Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 38Liu X.F. Culotta V.C. J. Biol. Chem. 1999; 274: 4863-4868Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 39Ooi C.E. Rabinovich E. Dancis A. Bonifacino J.S. Klausner R.D. EMBO J. 1996; 15: 3515-3523Crossref PubMed Scopus (178) Google Scholar). Data obtained here with the npi1, end3, andpep4 mutants affecting ubiquitination, endocytosis, and vacuolar degradation, respectively, reveal that Alr1p is internalized via the endocytic pathway and delivered to the vacuole for degradation. The data do not exclude additional routes of degradation of Alr1p. Interestingly, these mutants also accumulate a modified form of Alr1p, which constitutes a minor fraction of Alr1p in wild-type cells. This modification apparently precedes ubiquitination and endocytosis of Alr1p. It still remains to be shown how Mg2+ triggers the initial steps of Alr1 protein turnover and in which form Alr1p enters the endocytic pathway. The mechanism of Alr1p turnover is reminiscent of substrate-triggered degradation of plasma membrane proteins such as Ste2p, Ste3p, Pdr5p, Fur4p, sugar permeases, and the Zn2+transporter Zrt1p, which are removed from the plasma membrane by endocytosis for vacuolar degradation (26Rotin D. Staub O. Haguenauer-Tsapis R. J. Membr. Biol. 2000; 176: 1-17Crossref PubMed Google Scholar, 29Benedetti H. Raths S. Crausaz F. Riezman H. Mol. Biol. Cell. 1994; 5: 1023-1037Crossref PubMed Scopus (237) Google Scholar, 31Berkower C. Loayza D. Michaelis S. Mol. Biol. Cell. 1994; 5: 1185-1198Crossref PubMed Scopus (88) Google Scholar, 32Medintz I. Jiang H. Han E.K. Cui W. Michels C.A. J. Bacteriol. 1996; 178: 2245-2254Crossref PubMed Google Scholar, 33Chiang H.L. Schekman R. Hamamoto S. J. Biol. Chem. 1996; 271: 9934-9941Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 34Volland C. Urban-Grimal D. Geraud G. Haguenauer-Tsapis R. J. Biol. Chem. 1994; 269: 9833-9841Abstract Full Text PDF PubMed Google Scholar, 35Gitan R.S. Luo H. Rodgers J. Broderius M. Eide D. J. Biol. Chem. 1998; 273: 28617-28624Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Rapid decay of these plasma membrane receptors and transporters is induced by their physiological substrates. Similarly, Alr1p degradation appears to be triggered by Mg2+, with high selectivity over other divalent metal ions. Only cobalt, which has been shown to be taken up by the Mg2+ transport systems (14Smith R.L. Maguire M.E. Mol. Microbiol. 1998; 28: 217-226Crossref PubMed Scopus (148) Google Scholar, 15MacDiarmid C.W. Gardner R.C. J. Biol. Chem. 1998; 273: 1727-1732Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), and manganese affect Alr1p stability, but only when present at non-physiologically high concentrations. In addition to this post-translational control,ALR1 mRNA steady-state levels are down-regulated in medium containing standard or high Mg2+ concentrations compared with Mg2+ limiting growth conditions. It still remains to be shown whether this regulation is exerted by metal ion-sensitive transcription factors, as in the case of the zinc transporters Zrt1p, Zrt2p, and Zrt3p (40Zhao H. Butler E. Rodgers J. Spizzo T. Duesterhoeft S. Eide D. J. Biol. Chem. 1998; 273: 28713-28720Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 41MacDiarmid C.W. Gaither L.A. Eide D. EMBO J. 2000; 19: 2845-2855Crossref PubMed Scopus (301) Google Scholar), or by mRNA turnover. Mg2+ appears to be the only ion whose intracellular concentration becomes growth-limiting in Δalr1cells. First, the total intracellular concentration of Mg2+(but not of other ions) is significantly reduced in this mutant. Second, out of many metal ions tested, only Mg2+ at non-physiologically high concentrations can efficiently restore growth of the deletion mutant (15MacDiarmid C.W. Gardner R.C. J. Biol. Chem. 1998; 273: 1727-1732Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Mg2+ uptake or homeostasis therefore appears to be the essential function of Alr1p when low or standard Mg2+ concentrations are provided. In growth media containing non-physiologically high Mg2+ concentrations, Alr1p is dispensable; Δalr1 cells regain growth; and wild-type cells severely reduce the amount of Alr1 protein in the plasma membrane. Uptake of Mg2+ under these conditions apparently is mediated by other uptake mechanisms. Consistent with previous studies on wild-type yeast cells (4Beeler T. Bruce K. Dunn T. Biochim. Biophys. Acta. 1997; 1323: 310-318Crossref PubMed Scopus (39) Google Scholar), the data reported here confirm a very tight control of total intracellular Mg2+ concentrations, allowing a 3–4-fold decrease only, when the external concentrations change by 4 orders of magnitude (from 100 mm to 10 μm). They were found to be kept rather constant at ∼3 mg/g (dry weight) with extracellular Mg2+ concentrations ranging from 1 to 200 mm. Only further studies will reveal if reduced influx or increased efflux (or both) accounts for the lack of cellular Mg2+accumulation in the presence of very high external concentrations. Low intracellular Mg2+ concentrations of ∼1 mg/g (dry weight) were detected both when wild-type cells were grown in essentially Mg2+-free medium (data not shown) and whenΔalr1 mutant cells were grown in medium containing ≤1 mm Mg2+. It remains to be shown whether this arrest is due to specific signals resulting from low intracellular Mg2+, leading to a defined state of cellular differentiation, as previously described for the fission yeastSchizosaccharomyces pombe (42Walker G.M. Magnesium. 1986; 5: 9-23PubMed Google Scholar), or whether the demand of a certain essential enzymatic function for Mg2+ can no longer be met, leading to growth arrest. Taken together, the data presented here consistently show that Alr1p expression is essential to maintain cellular Mg2+concentrations at levels suitable for growth of yeast cells. The suppression of the Δalr1 mutant phenotype by high Mg2+ (but not by other ions) and the control of expression and stability of Alr1p by Mg2+ suggest a specificity for Mg2+. Changes in steady-state levels of other ions appear to be secondary, reflecting charge compensation. The location of Alr1p in the plasma membrane of yeast cells and its apparent functional and structural homology to the bacterial Mg2+ transporter CorA protein tentatively classify Alr1p as a Mg2+ transporter of the yeast plasma membrane. It remains to be shown by which mechanisms Alr1p mediates uptake of Mg2+ into yeast cells and whether homologs of Alr1p exist in plasma membranes of higher eukaryotes. We thank Mirjana Iliev for technical assistance and Gerlinde Wiesenberger and Gábor Zsurka for critical and helpful suggestions. Special thanks go to G. Schatz for providing antibodies.
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