The Yeast Plasma Membrane Protein Alr1 Controls Mg2+ Homeostasis and Is Subject to Mg2+-dependent Control of Its Synthesis and Degradation*

Anton GraschopfDagger §, Jochen A. StadlerDagger §, Maria K. HoellererDagger , Sandra Eder, Monika Sieghardt||, Sepp D. Kohlwein, and Rudolf J. SchweyenDagger **

From the Dagger  Vienna Biocenter, Institute of Microbiology and Genetics, University of Vienna, A-1030 Vienna, the   Biomembrane Research Center (SFB), Department of Biochemistry, Technical University of Graz, A-8010 Graz, and the || Institute of Forest Ecology, University of Agricultural Sciences, Vienna, A-1180 Vienna, Austria

Received for publication, February 16, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 characterized npi1 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 (Delta alr1) 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, whereas Delta alr1 mutant cells did not. Expression of the bacterial Mg2+ transporter CorA in the yeast Delta alr1 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+.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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. 1 and 2).

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 (1, 3). 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 (4).

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. 1, 2, and 5-9). Further transport systems are to be expected in membranes of intracellular compartments that are likely to sequester and release Mg2+ (10). 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 (11-13).

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. 14). 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. 14).

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 (12, 13).

Alr1p is essential for growth of yeast cells, except in media with high Mg2+ concentrations, and its overexpression confers resistance to aluminum (15). 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 Delta alr1 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 (15).

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+. In Delta alr1 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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Strains, Plasmids, and Media-- Escherichia coli strains were cultivated at 37 °C in LB medium supplemented with 100 µg/ml ampicillin when appropriate. Yeast strains were grown at 28 °C in YPD medium (yeast extract/peptone/dextrose), standard SD medium (0.67% yeast nitrogen base, 2% glucose, and amino acids), or synthetic SD medium (16) supplemented with MgCl2 where indicated. E. coli strain DH5alpha (Stratagene) and the following plasmids were used for subcloning: YEp351HA and YIp211HA (12) and pUG34 and pUG23 (17). Yeast strains FY1679 (18), GA74D (12), and 23344c (NPI1) and 27038a (npi1) (19) were described previously.

Delta alr1 Disruption-- For disruption of the ALR1 gene, a disruption cassette was PCR1-amplified using the pJJ244-URA3 cassette (20) and oligonucleotide primers of sequences flanking the ALR1 (YOL130w) gene: ALR1-S1, 5'-AAGATCATCGAATATTGCTAGAAAGCGTAAAAGCATTTTACCATGCTGGCGAAAGGGGGATGTGC-3'; and ALR1-S2, 5'-TCTGTGACTTAAATCTTCTATCTTTATCACATCACTAATCAGTCGCTGGCACGACAGGTTTCCCG-3'. The PCR product was transformed into the yeast strain GA74D, and Ura+ colonies were selected. The resulting strain, JS74 (ALR1/Delta alr1 (allele alr1-1)), was sporulated, and tetrads were dissected on YPD medium supplemented with 200 mM MgCl2 at 28 °C, generating JS74A (ALR1) and JS74B (alr1-1).

Delta end3 Disruption-- To disrupt the END3 gene, the loxP-kanMX-loxP disruption cassette from the vector pUG6 (21) was PCR-amplified using the oligonucleotide primers end3-KAN5 (5'-AGTGGGTATTGGAAAGGCCGGTAAAGATAACAGGGATCTCTGAAAGCTGAAGCTTCGTACGCTGC-3') and end3-KAN3 (5'-ACAAACAGTAAATATTACACATTCATGTACATAAAATTAATTATCCATAGGCCACTAGTGGATCTG-3'). The resulting PCR product was transformed into the yeast strain FY1679, followed by selection for G418-resistant colonies. The resulting strain, JS331 (END3/Delta end3), was sporulated, and tetrads were dissected on YPD medium at 28 °C, generating JS034C (END3) and JS034B (Delta end3).

Delta pep4 Disruption-- To disrupt the PEP4 gene, the loxP-kanMX-loxP disruption cassette was PCR-amplified from the vector pUG6 (21) using the oligonucleotide primers PEP4-KANF (5'-AAGCCTAGTGACCTAGTATTTAATCCAAATAAAATTCAAGCTGAAGCTTCGTACGCTGC-3') and PEP4-KANR (5'-CAGAAAAGGATAGGGCGGAGAAGTAAGAAAAGTTTACATAGGCCACTAGTGGATCTG-3'). The PCR product was transformed into the yeast strain FY1679, and G418-resistant colonies were selected. The resulting strain, AG1679 (PEP4/Delta pep4), was sporulated, and tetrads were dissected on YPD medium at 28 °C, generating AG1679C (PEP4) and AG1679A (Delta pep4). Correct replacements of the ALR1, END3, and PEP4 open reading frames by the disruption constructs were verified using analytical PCR (data not shown).

Plasmid Constructions-- Plasmid pUG34-CorAGFP was generated by subcloning the Salmonella typhimurium corA gene PstI fragment from the vector YEpCorA (12) into the plasmid pSK+ (Stratagene), from where it was further subcloned as an EcoRI/BamHI fragment into pUG34 (17). To generate C-terminally HA-tagged Alr1p, the ALR1 gene was PCR-amplified from GA74D chromosomal DNA using the mutagenic oligonucleotide primers ALR1-SalI (5'-AAAGTCGACTGTCGTAGCGGCTATATC-3') and ALR1-SacI (5'-AAAGTCGACGAGCTCATTTAATTGCCG-3'), starting at positions -502 and +2578 relative to the ALR1 start codon, respectively, introducing therewith SacI and SalI cleavage sites (underlined) for cloning the gene into the vector YEp351HA. The PCR product was digested with SacI and SalI and then ligated to the SacI- and SalI-cleaved vector YEp351HA to obtain the plasmid YEpALR1HA or to the SacI- and SalI-cleaved vector YIp211HA to obtain the plasmid YIpALR1HA. Correct in-frame fusion of ALR1 to the HA tag was verified by DNA sequencing (data not shown). For chromosomal integration of a HA-tagged ALR1 allele, the plasmid YIpALR1HA was linearized by ApaI digestion and transformed into yeast, followed by selection for Ura+ transformants. To generate the centromeric plasmid pUG23-ALR1GFP, expressing C-terminally GFP-tagged Alr1p under the control of the pMet-25 promoter, the ALR1 coding sequence was PCR-amplified from YEpALR1HA DNA using the mutagenic oligonucleotide primers pALR1-pUG23direct (5'-CGCGCGGATCCATGTCATCATCCTCAAGTTCATCAGAG-3') and pALR1- pUG23reverse (5'-GCCACGCGTCGACGTCGTAGCGGCTATATCTACTAG-3'), introducing a BamHI and a SalI restriction site, respectively (underlined). The PCR product was digested with BamHI and SalI and then ligated to the BamHI- and SalI-digested vector pUG23. To express GFP-tagged Alr1p via its endogenous promoter, a SacI/StuI fragment of pUG23-ALR1GFP consisting of the pMet-25 promoter and 120 base pairs of the ALR1 5'-part was replaced by the 863-base pair SacI/StuI fragment from YEpALR1HA containing the same 120 base pairs of ALR1 as well as the ALR1 5'-untranslated region, giving rise to the plasmid pUG123-ALR1GFP. To generate pUG135-ALR1HA, the SacI/SalI fragment from pUG123-ALR1GFP containing the endogenous ALR1 promoter as well as the ALR1 coding region was subcloned into the SacI/SalI-digested vector pUG35 (17). All ALR1-bearing plasmids described above were able to complement the Delta alr1 phenotype (data not shown).

Membrane Fractionation-- FY1679 YIpALR1HA cells were grown in synthetic SD medium supplemented with 5 µM MgCl2 overnight to A600 = 1.2. Spheroplasts were prepared and homogenized as described (22). Unbroken cells and cell debris were pelleted at 1500 × g, and the supernatant was centrifuged for 15 min at 15,000 × g. The pellet, enriched in membranes, was resuspended in 10 mM Tris-Cl (pH 7.5), 0.2 mM Na2EDTA, 0.2 mM dithiothreitol, and 10% glycerol; loaded on a 12-60% continuous sucrose gradient (12-60% (w/v) sucrose in 20 mM Tris-Cl (pH 7.4)); and ultracentrifuged at 100,000 × g for 2 h. 1-ml fractions were collected from the bottom to the top using an Isco Retriever 500 sample collector. Protein concentrations were determined using Bio-Rad protein assay reagent according to the manufacturer's instructions. Equal amounts of protein from the various fractions were separated on a 10% SDS-polyacrylamide gel, followed by immunoblotting.

Analysis of Ion Composition-- Cells were grown to A600 = 1.2 in SD medium supplemented with 200 mM MgCl2, washed twice in HPLC-grade water, and incubated in the respective media containing different levels of MgCl2. Samples were drawn at the indicated time points, and the cells were washed three times in HPLC-grade water and dried at 105 °C. To digest the cells, 5 ml of 65% HNO3 and 2 ml of 60% HClO4 were added to 300 mg (dry weight), and the samples were heated in steps: 50 °C, 45 min; 75 °C, 15 min; 100 °C, 15 min; 125 °C, 15 min; 150 °C, 15 min; and 200 °C, 60 min. The extracts were diluted with 75 ml of ultrapure water (18 megaohms), filtered (No. 589, Schleicher & Schüll), and stored at 4 °C until analysis. Ions were quantified using a PerkinElmer Life Sciences OPTIMA3000XL-ICP-OES.

Yeast Whole Cell Extracts-- Cells were harvested and washed twice in ice-cold 1 mM EDTA to complex cations and once in HPLC-grade water. For lysis, cells were incubated in 2 N NaOH and 1.25% beta -mercaptoethanol for 10 min on ice, and then proteins were precipitated with trichloroacetic acid (25% final concentration) for at least 60 min. Subsequent washings of the pelleted precipitate were performed using acetone and 1 M Tris base. The precipitated protein extracts were dissolved by boiling in 5% SDS. Protein concentrations were determined using the Bio-Rad dye reagent concentrate protein assay according to the manufacturer's protocols.

Microscopy-- GFP fluorescence was analyzed with a Leica TCS 4D confocal microscope (argon/krypton laser; lambda ex = 488 nm and lambda em = 500-500; and 100×/1.4 oil objective). Cells were prepared for microscopy as described (23). For FM4-64 staining of the yeast vacuole, cells were pulse-labeled for 1 h in synthetic SD medium containing 30 µg/µl FM4-64 (T-3166, Molecular Probes, Inc.), and FM4-64 fluorescence was visualized after a chase of 1 h.

Antibodies-- The antibodies used in this study were mouse anti-HA (12); rabbit anti-Hxk1p, rabbit anti-Pma1p, rabbit anti-Sec61p, and rabbit anti-Aac2p (kindly provided by G. Schatz); mouse anti-Alp2p (A-6458, Molecular Probes, Inc.); and horseradish peroxidase-conjugated goat anti-mouse IgG (W4021) and horseradish peroxidase-conjugated goat anti-rabbit IgG (W4011, Promega).

mRNA Quantification-- Yeast mRNA was isolated from JS74A and JS74B cells using the Promega SV total RNA isolation system and the QIAGEN Oligotex mRNA mini-kit according to the manufacturers' protocols. Reverse transcription-PCR analysis (Access RT-PCR system, Promega) was performed as described by the manufacturer using oligonucleotide primers Alr1-rtp (5'-CAGGGTATGGATGAAACGGTTGC-3'), Alr1-rtm (5'-TGATCCCGAAGTGGAAGTAGAGC-3'), ACT1_plus (5'-ACCAAGAGAGGTATCTTGACTTTACG-3'), and ACT1_minus (5'-GACATCGACATCACACTTCATGATGG-3'). For each reaction, 30 ng RNA were used.

Miscellaneous-- Sequencing of DNA was performed by the Automated DNA Sequencing Service OLIGO-OCOM at the Vienna Biocenter. Immunodetection (Pierce SuperSignal West Pico chemiluminescent substrate) was performed according to the manufacturer's protocols.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mg2+-dependent Phenotype of Delta alr1 Mutant Yeast Cells-- To investigate the phenotype of the Delta alr1 deletion mutant, the ALR1 gene was disrupted in the diploid yeast strain GA74D by one-step gene disruption, replacing the entire ALR1 open reading frame with the URA3 gene. Tetrads were dissected and grown on YPD medium supplemented with 200 mM Mg2+. As reported previously for another yeast strain (15), growth of the Delta alr1 mutant was found to depend on high Mg2+ concentrations in the medium. Wild-type cells of the yeast strain used here (JS74A) grew well when provided with Mg2+ concentrations as low as 30 µM in synthetic SD medium (Fig. 1). Delta alr1 mutant cells (JS74B) stopped growing upon transfer to medium with 30 µM Mg2+. Growth rates increased proportionally to the Mg2+ concentrations in the medium, reaching wild-type rates at 50 mM Mg2+ in synthetic SD medium.


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Fig. 1.   Mg2+-dependent growth phenotype of the Delta alr1 mutant. JS74A wild-type (WT; left panel) and JS74B Delta alr1 (right panel) cells were cultured in synthetic SD medium containing 200 mM Mg2+ to A600 = 1; washed three times in synthetic SD medium without Mg2+; and then inoculated (A600 = 0.05) in synthetic SD medium containing 30 µM or 1, 10, 50, 100, or 200 mM Mg2+. Cells were incubated at 28 °C with shaking, and growth was followed by measuring the A600. black-triangle, 200 mM Mg2+; black-square, 100 mM Mg2+; triangle , 50 mM Mg2+; , 10 mM Mg2+; +, 1 mM Mg2+; , 30 µM Mg2+.

The Bacterial Mg2+ Transporter CorA Partially Suppresses the Delta alr1 Phenotype-- To investigate whether the S. typhimurium Mg2+ transporter CorA can substitute for its putative homolog Alr1p in yeast and thus overcome the Mg2+-dependent Delta alr1 phenotype, N-terminally GFP-tagged CorA protein was expressed in the Delta alr1 mutant from the strong pMet-25 promoter. Serial dilutions of the wild-type and Delta alr1 strains either carrying the empty plasmid pUG34 or expressing the corA-GFP gene fusion via pMet-25 were spotted on synthetic SD plates containing 10 µM, 100 µM, 1 mM, or 100 mM Mg2+. As shown in Fig. 2, the bacterial CorA protein partially suppressed the Delta alr1 phenotype at low Mg2+ concentrations. Although we failed to allocate the CorA-GFP signal to a particular cellular compartment, we assume that a minor amount of the highly expressed fusion protein is inserted into the plasma membrane, where it can promote the observed suppression.


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Fig. 2.   The bacterial Mg2+ transporter CorA partially suppresses the Delta alr1 phenotype. The Delta alr1 disrupted strain JS74B and the wild-type (wt) strain JS74A were transformed with the plasmid pUG34 either without insert or with the S. typhimurium corA gene. Serial dilutions of cells were spotted onto synthetic SD medium containing 10 µM (A), 100 µM (B), 1 mM (C), and 100 mM (D) Mg2+ and grown at 28 °C for 5 days.

Altered Ion Concentrations in Delta alr1 Cells-- To investigate the possible effects of Alr1p on cellular metal ion homeostasis, we determined the total intracellular concentrations of certain ions in the wild-type strain JS74A and in the Delta alr1 mutant strain JS74B. Cells were incubated either in standard SD medium or in standard SD medium supplemented with 200 mM Mg2+ for 6 h. The total cellular amounts of Mg2+, Ca2+, Na+, K+, phosphorus, and sulfur were quantified using inductively coupled plasma/optical emission spectrophotometry (Table I). When incubated in standard SD medium, the total cellular Mg2+ level of Delta alr1 was reduced to 40% of the wild-type level. Grown in the same medium supplemented with 200 mM Mg2+, Delta alr1 cells came up to 75% of the Mg2+ measured in wild-type cells. In the latter case, the non-physiologically high Mg2+ concentration in the medium seems to compensate for the absence of Alr1 protein. Other uptake systems, which still have to be found, might replenish internal pools to an extent that allows cells to grow under these conditions. The cellular Ca2+ concentration was found to be increased when the cellular Mg2+ level was decreased. This correlates with previous observations (4, 24). Cellular Na+ levels were similarly increased. This is consistent with previous work reporting an increased Na+/H+ exchange in Mg2+-depleted yeast cells (25). The levels of K+, phosphorus, and sulfur were just slightly influenced by the Mg2+ concentrations in the medium or the Delta alr1 mutation.

                              
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Table I
Total ion levels in wild-type and Delta alr1 cells
Values are expressed in milligrams/g (dry weight) ± S.E. WT, wild-type.

Time-dependent Change in the Cellular Mg2+ Concentration-- To shed more light on the relevance of Alr1p for Mg2+ uptake, both wild-type and Delta alr1 mutant strains were pre-grown in synthetic SD medium supplemented with 200 mM Mg2+ and then shifted to synthetic SD medium lacking Mg2+ for 24 h at 28 °C. Under these conditions, both strains stopped growing, but stayed viable; and addition of 200 mM Mg2+ immediately induced growth of both the wild-type and Delta alr1 cells (data not shown). The cellular level of Mg2+ decreased by a factor of 3-4 within a few hours of Mg2+ starvation (Fig. 3A). When provided with 1 mM Mg2+, Mg2+-starved wild-type cells increased their Mg2+ content by 100% within 1 h from 1 to 2 mg/g (dry weight) and, after 4 h, finally reached 2.4 mg/g (dry weight). In contrast, Delta alr1 disrupted cells showed only a 20% increase in the Mg2+ level from 1 to 1.2 mg/g (dry weight) (Fig. 3B). This inability of the Delta alr1 cells to accumulate Mg2+ indicates that Alr1p is essential for Mg2+ uptake.


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Fig. 3.   Time-dependent changes in total cellular Mg2+. JS74A wild-type cells (black-triangle) and JS74B Delta alr1 cells (triangle ) were starved of Mg2+ in synthetic SD medium without Mg2+ for 24 h. Aliquots were taken after 0, 3, 6, and 24 h, and the total cellular amount of Mg2+ was determined by inductively coupled plasma/optical emission spectrophotometry (A). 1 mM MgCl2 was added to cells starved of Mg2+ for 24 h, and the total cellular amount of Mg2+ (milligrams/g (dry weight)) was determined by inductively coupled plasma/optical emission spectrophotometry after 0, 1, 2, and 4 h (B).

Localization of Alr1p-- To determine the subcellular localization of Alr1p, FY1679 cells carrying chromosomally integrated YIpALR1HA (expressing Alr1p C-terminally tagged with a triple copy of the Hemophilus influenzae hemagglutinin epitope) were grown in synthetic SD medium containing 5 µM Mg2+. Extracts were prepared and subfractionated by velocity sedimentation through a sucrose gradient (see "Experimental Procedures"), and equal amounts of protein from each fraction were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted. The Alr1p-HA fusion protein was found to cofractionate with the plasma membrane marker Pma1p, whereas the endoplasmic reticulum (Sec61p) and vacuolar (Alp2p) marker proteins were found in other fractions (Fig. 4). Although signals for mitochondrial Aac2 protein and for Alr1p-HA partially overlapped, results obtained by fluorescence microscopy excluded a mitochondrial localization of Alr1p (Fig. 5).


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Fig. 4.   Subcellular localization of Alr1p by cell fractionation. Spheroplasts of FY1679 cells expressing C-terminally HA-tagged Alr1p from integrated YIpALR1HA were homogenized, and membranes were enriched by centrifugation at 15,000 × g and separated by ultracentrifugation at 100,000 × g on a 12-60% sucrose gradient. Fractions were collected, and equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted for HA (Alr1p-HA), plasma membrane ATPase (Pma1p), the endoplasmic reticulum marker Sec61p, the vacuolar alkaline phosphatase (Alp2p), and the mitochondrial ADP-ATP carrier (Aac2p).


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Fig. 5.   Subcellular localization of Alr1p by fluorescence microscopy. JS74A cells expressing C-terminally GFP-tagged Alr1p from the centromeric vector pUG123-ALR1GFP were grown in synthetic SD medium containing 5 µM (A), 1 mM (B), or 200 mM (C) Mg2+ at 28 °C for 12 h and then examined by confocal microscopy. GFP fluorescence (left panels) and corresponding differential interference contrast images (right panels) are shown. Scale bars = 10 µm.

To determine the cellular location of Alr1p by a different method, C-terminally GFP-tagged Alr1 protein was expressed in JS74A wild-type cells from the centromeric plasmid pUG123-ALR1GFP (see "Experimental Procedures"). Cells were grown in synthetic SD medium containing various concentrations of Mg2+ and examined by confocal microscopy. The Alr1p-GFP signal was highest in cells incubated at low (5 µM) Mg2+ (Fig. 5A). Fluorescence was preferentially detected as a rim structure at the cell surface, consistent with a location of Alr1p in the plasma membrane. Addition of either 1 or 200 mM Mg2+ to the growth medium led to a dramatic reduction of this signal (Fig. 5, B and C). Upon elevation of the Mg2+ concentrations, the Alr1p-specific GFP signal also appeared in small vesicular structures, which are likely to be endocytic vesicles internalizing Alr1p for subsequent degradation in response to the Mg2+ concentration in the growth medium.

Mg2+-dependent Expression of Alr1p-- To address the question whether the ALR1 mRNA level is modulated according to the Mg2+ concentration in the media, wild-type and Delta alr1 cells were grown in synthetic SD medium containing different Mg2+ concentrations. mRNA was isolated and analyzed by semiquantitative reverse transcription-PCR. As shown in Fig. 6, the steady-state level of ALR1 mRNA was considerably higher in cells grown in medium containing 5 µM Mg2+ than in medium containing a standard (1 mM) or high (200 mM) Mg2+ concentration. Consequently, the ALR1 mRNA level is highly dependent on the Mg2+ concentration provided to the cells. This indicates that ALR1 expression is regulated at the mRNA level.


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Fig. 6.   Regulation of the ALR1 mRNA levels by external Mg2+. Shown are the results from the semiquantitative reverse transcription-PCR analysis of ALR1 and ACT1 mRNAs isolated from JS74A wild-type (WT) and JS74B Delta alr1 cells grown in synthetic SD medium containing 5 µM, 1 mM, or 200 mM Mg2+ for 12 h. Aliquots were taken after 25, 30, 35, and 40 cycles of PCR amplification and visualized on a 2% agarose gel.

Mg2+-dependent Alr1 Protein Level-- To determine the steady-state level of cellular Alr1p at different Mg2+ concentrations, FY1679 cells carrying the chromosomally integrated HA-tagged ALR1 gene with its endogenous promoter were grown in synthetic SD medium supplemented with 5 µM, 1 mM, or 200 mM Mg2+. Total cell extracts were analyzed by immunoblotting. As shown in Fig. 7A, the Alr1p level was much higher when the cells were grown in medium containing a low (5 µM) Mg2+ concentration than when they were grown in medium containing either a standard (1 mM) or very high (200 mM) Mg2+ concentration.


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Fig. 7.   Alr1p level and stability are dependent on external Mg2+. A, FY1679 cells expressing C-terminally HA-tagged Alr1p from integrated YIpALR1HA were grown in synthetic SD medium containing 5 µM, 1 mM, or 200 mM Mg2+ for 12 h; total cell extracts were prepared; and equal amounts of protein were immunoblotted for HA-tagged Alr1p as well as hexokinase (Hxk1p). B, FY1679 YIpALR1HA cells were grown in synthetic SD medium containing 5 µM Mg2+ for 12 h and then simultaneously exposed to 100 µg/ml cycloheximide and 5 µM, 1 mM, or 10 mM Mg2+. Aliquots were taken at 0, 30, 90, and 180 min and immunoblotted for HA-tagged Alr1p and hexokinase (Hxk1p).

We further investigated whether Mg2+ concentrations in the growth medium directly affect the turnover of Alr1p. FY1679 cells carrying the triple HA-tagged ALR1 gene on the chromosome were incubated in synthetic SD medium containing 5 µM Mg2+ for 12 h. Protein synthesis was then inhibited by addition of 100 µg/ml cycloheximide, and 1 mM or 10 mM Mg2+ was simultaneously added. Samples were taken after 0, 30, 90, and 180 min, and total cell extracts were prepared and separated on an SDS-polyacrylamide gel, followed by immunoblotting. As shown in Fig. 7B, the Alr1 protein was stable within 180 min at very low (5 µM) Mg2+ concentrations. When the cells were exposed to 1 or 10 mM Mg2+, an additional band with slightly lower mobility appeared within 30 min, suggesting that Alr1p was modified, and total Alr1p was severely decreased (Figs. 7B and 8A). This shows that Mg2+ exceeding a certain concentration induces a rapid modification/degradation process of Alr1p, indicating a post-translational regulatory mechanism.


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Fig. 8.   Ubiquitination and endocytosis are involved in Alr1p degradation. Strains 23344c (NPI1) (A), 27038a (npi1) (B), and JS034B (end3) (C) were grown in synthetic SD medium containing 5 µM Mg2+ for 12 h and then simultaneously exposed to 100 µg/ml cycloheximide and 10 mM Mg2+. Aliquots were taken at 0, 30, 90, and 180 min and immunoblotted for HA-tagged Alr1p and hexokinase (Hxk1p).

Pathway of Alr1p Degradation-- Several plasma membrane proteins have been shown to undergo ubiquitin-dependent endocytosis, including the pheromone receptors Ste2p and Ste3p, the ABC transporters Ste6p and Pdr5p, the uracil permease Fur4p, sugar permeases, and the Zn2+ transporter Zrt1p (reviewed in Ref. 26). To examine whether the Mg2+-induced Alr1p degradation is dependent on the endocytic pathway, we followed Alr1p turnover in the genetic backgrounds of npi1, Delta end3, and Delta pep4 mutants.

NPI1 encodes an E3 ubiquitin-protein ligase that has been shown to be involved in attaching ubiquitin to the amino group of lysine residues in the substrate proteins (27). Since npi1 null mutants are lethal, we used an npi1 promoter mutation that expresses <10% of the wild-type protein level (28). 23344c (NPI1) and 27038a (npi1) cells expressing chromosomally integrated HA-tagged Alr1p were grown in synthetic SD medium containing 5 µM Mg2+ for 12 h. Cycloheximide (100 µg/ml) and Mg2+ (10 mM) were simultaneously added, and samples were drawn after 0, 30, 90, and 180 min and analyzed by immunoblotting. Upon addition of 10 mM Mg2+ to the medium, degradation of Alr1p was impaired in the ubiquitination-insufficient npi1 mutant cells in contrast to the wild-type cells (Fig. 8, A and B), implying that in the npi1 mutant, Alr1p is not sufficiently ubiquitinated to be targeted for degradation.

The End3 protein has been shown to be required for the internalization step of endocytosis and presumably functions in a multiprotein complex that coordinates the early stages of endocytosis (29, 30). Several plasma membrane proteins like Ste2p, Ste6p, Mal61p, Gal2p, Fur4p, and Zrt1p undergo End3p-dependent endocytosis (29, 31-35).

We generated an Delta end3 null mutant (see "Experimental Procedures") and followed Alr1p stability upon addition of Mg2+ in this genetic background. Delta end3 cells grown at 5 µM Mg2+ were exposed to 10 mM Mg2+ and 100 µg/ml cycloheximide simultaneously. Unlike the isogenic wild-type cells (Fig. 7B), Alr1p stayed stable upon addition of Mg2+ to the Delta end3 cells (Fig. 8C), implying that endocytosis is crucial for Alr1p degradation. It is worth noting that in both npi1 and Delta end3, Alr1p was enriched in a modified form even at low Mg2+ concentrations (Fig. 8, B and C, 0-min lanes), indicating that its modification precedes End3p- and Npi1p-controlled endocytosis.

The vacuolar protease Pep4p plays a major role in vacuolar protein degradation and maturation of several vacuolar proteases (36, 37). To determine whether Alr1p is degraded in the vacuole in response to Mg2+, AG1679A (Delta pep4) and AG1679C (wild-type) cells expressing C-terminally GFP-tagged Alr1p from the plasmid pUG135-ALR1GFP were grown in 200 mM Mg2+ at 28 °C for 12 h and then examined by confocal microscopy. This high Mg2+ concentration resulted in a weak Alr1p signal in the plasma membrane of both strains. Although Alr1p appeared to be degraded in wild-type cells (Fig. 9, a-d), consistent with the protein stability studies described above, the protein was accumulated in the vacuoles of cells lacking the vacuolar protease Pep4p (Delta pep4) (Fig. 9. e-h). Also at low Mg2+ concentrations, slight accumulation of the protein in the vacuole was observed in Delta pep4 cells, which is consistent with the notion of vacuolar Alr1p degradation (data not shown). Taken together, the results obtained with the npi1, Delta end3, and Delta pep4 mutants indicate that Mg2+ induces substrate-specific endocytosis and vacuolar degradation of Alr1p.


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Fig. 9.   Alr1p degradation is dependent on the vacuolar protease Pep4p. AG1679C (wild-type) pUG135-ALR1GFP (a-d) and AG1679A (Delta pep4) pUG135-ALR1GFP (e-h) cells were grown in synthetic SD medium containing 200 mM Mg2+ at 28 °C for 12 h and then examined by confocal microscopy. GFP fluorescence (a and e), FM4-64 staining of vacuolar membranes (b and f), overlay of GFP and FM4-64 fluorescence (c and g), and corresponding differential interference contrast images (d and h) are shown.

We also investigated whether other divalent cations are able to trigger rapid degradation of Alr1p. Cells were incubated in synthetic SD medium with 5 µM MgCl2, and then various amounts of MgCl2, CaCl2, CoCl2, MnCl2, ZnCl2, NiSO4, or CuCl2 were added together with 100 µg/ml cycloheximide. After 90 min of incubation at 28 °C, cells were collected, and total protein extracts were prepared. Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted (Fig. 10). As expected, Alr1p degradation proved to be highly sensitive to Mg2+. Addition of as little as 100 µM Mg2+, which is just 5-10% of the level in standard SD or YPD medium, resulted in a significant reduction of Alr1p. Co2+ and Mn2+ also influenced Alr1p degradation, although only at concentrations exceeding those in standard media by 20-100-fold. In contrast, Ca2+, Zn2+, Ni2+, and Cu2+ had no significant effect on Alr1p stability. These results indicate that rapid Alr1 protein turnover is selectively triggered by its substrate Mg2+. This process is effective even at concentrations of Mg2+ far below those of standard synthetic and complete yeast media. Co2+ and Mn2+ are unlikely to contribute to the regulation of the Alr1p level in yeast cells because they affect Alr1p stability only when added at exceptionally high concentrations.


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Fig. 10.   Dependence of Alr1p turnover on various metal ions. FY1679 YIpALR1HA cells were grown in synthetic SD medium containing 5 µM Mg2+ for 12 h and then exposed for 90 min to cycloheximide (100 µg/ml) and various concentrations of MgCl2, CaCl2, CoCl2, MnCl2, ZnCl2, NiSO4, or CuCl2 as indicated. Cells were collected, and total cell extracts were prepared, followed by immunoblotting for HA-tagged Alr1p and hexokinase (Hxk1p). Standard ion concentrations in synthetic SD medium are 2 mM Mg2+, 1 mM Ca2+, <0.2 µM Co2+, 2.5 µM Mn2+, 2.9 µM Zn2+, <0.2 µM Ni2+, and 0.3 µM Cu2+.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The yeast protein Alr1 was the first candidate for a transporter of Mg2+ in eukaryotic cells (15). 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 by Delta alr1, Delta mrs2, or Delta lpe10 mutations can be partially suppressed by expression of the bacterial CorA protein in yeast cells (Refs. 12 and 13 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 (35, 38, 39). Data obtained here with the npi1, end3, and pep4 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 (26, 29, 31-35). 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 (14, 15), 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 (40, 41), or by mRNA turnover.

Mg2+ appears to be the only ion whose intracellular concentration becomes growth-limiting in Delta alr1 cells. 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 (15). 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; Delta alr1 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 (4), 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 when Delta alr1 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 yeast Schizosaccharomyces pombe (42), 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 Delta alr1 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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* This work was supported by Austrian Science Fund (FWF) Project F706, Austrian National Bank Project P7273, European Union Grant BIO4-CT97-2294 (EUROFAN II), the Austrian Ministry of Education, Science, and Culture EUROFAN II Supplement Project, and AUSTROFAN Grant GZ 200.042/2-Pr/4/2000 (to S. D. K. and R. J. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Both authors contributed equally to this work.

** To whom correspondence should be addressed. Tel.: 43-1-4277-54604; Fax: 43-1-4277-9546; E-mail: schweyen@gem.univie.ac.at.

Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M101504200

    ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; HA, hemagglutinin; GFP, green fluorescent protein; HPLC, high pressure liquid chromatography.

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