Overexpression of the Saccharomyces cerevisiae Magnesium Transport System Confers Resistance to Aluminum Ion*

Colin W. MacDiarmidDagger and Richard C. Gardner§

From the School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Ionic aluminum (Al3+) is toxic to plants, microbes, fish, and animals, but the mechanism of its toxicity is unknown. We describe the isolation of two yeast genes (ALR1 and ALR2) which confer increased tolerance to Al3+ and Ga3+ ions when overexpressed while increasing strain sensitivity to Zn2+, Mn2+, Ni2+, Cu2+, Ca2+, and La3+ ions. The Alr proteins are homologous to the Salmonella typhimurium CorA protein, a bacterial Mg2+ and Co2+ transport system located in the periplasmic membrane. Yeast strains lacking ALR gene activity required additional Mg2+ for growth, and expression of either ALR1 or ALR2 corrected the Mg2+-requiring phenotype. The results suggest that the ALR genes encode the yeast uptake system for Mg2+ and other divalent cations. This hypothesis was supported by evidence that 57Co2+ accumulation was elevated in ALR-overexpressing strains and reduced in strains lacking ALR expression. ALR overexpression also overcame the inhibition of Co2+ uptake by Al3+ ions. The results indicate that aluminum toxicity to yeast occurs as a consequence of reduced Mg2+ influx via the Alr proteins. The molecular identification of the yeast Mg2+ transport system should lead to a better understanding of the regulation of Mg2+ homeostasis in eukaryote cells.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Aluminum is the most abundant metallic element, constituting about 8% by weight of the outer crust of the Earth (1). In neutral to slightly acidic conditions (pH > 6) aluminum is locked in silicate and oxide minerals and is consequently nontoxic. In more acidic conditions, however, aluminum is leached from insoluble forms, and the bioavailability and toxicity of the element are increased. The pH-dependent chemistry of aluminum may be one reason why, despite its abundance in the environment, the Al3+ ion does not appear to be utilized for any biological purpose and is generally recognized to be nonessential for life (2). Ionic aluminum is toxic to many organisms including microbes (3), plants (4), fish (5), and mammals (6). Despite a plethora of hypotheses regarding the mechanism of toxicity, no clear consensus has emerged (4).

The economic impact of aluminum toxicity to agriculture has focused research interest on the effects of ionic aluminum in plant systems. Aluminum-tolerant cultivars have been obtained by conventional breeding (7), but this approach is limited by the availability of suitable aluminum-tolerant germplasm. Very recently, a gene transfer strategy was used successfully to increase the aluminum tolerance of tobacco and papaya (8). Expression of a bacterial citrate synthase gene resulted in increased citrate secretion from roots. The aluminum tolerance of the transgenic plants presumably occurred as a consequence of citrate binding and detoxifying aluminum outside the plant cell. This result is very promising from a biotechnological standpoint, but it is likely that a range of genes will be required to confer high levels of aluminum tolerance. Moreover, the result reveals little about the mechanism of aluminum toxicity to plant cells. A better understanding of aluminum toxicity will allow more directed genetic improvements to be made, leading to increased levels of tolerance.

The genetic analysis of metal ion toxicity has been accelerated by the use of microbial model systems to clone and analyze genes contributing to metal tolerance. To this end, we have described conditions that enable the use of bakers' yeast (Saccharomyces cerevisiae) for the genetic analysis of aluminum toxicity (9). During this work, aluminum toxicity was found to be ameliorated by Mg2+ ions, and Al3+ ion was found to inhibit the Mg2+ and Co2+ uptake system. The results suggested that Al3+ induced Mg2+ deficiency in yeast. In this paper we describe the isolation and characterization of two yeast genes (ALR1 and ALR2) which increase aluminum resistance when overexpressed. The ALR genes encode redundant systems mediating the influx of several divalent cations, including Mg2+. Our results provide genetic confirmation that the inhibition of Mg2+ uptake is the primary cause of aluminum toxicity to yeast.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Yeast Strains and Growth Conditions-- Yeast were grown in standard synthetic or complete culture media (SC and YPD, respectively; Ref. 10), with the required auxotrophic supplements and 2% glucose, galactose or raffinose. Methods for yeast transformation and plasmid rescue were as described (11, 12). Yeast strains used were: SH2332 (MATa pho3-1 pho4::HIS3 his3-532 leu2-3,112 ura3-1,2 trp1-289 ade2), CG379 (MATa ade5 can1 leu2-3,112 trp1-289aura3-52 gal2 [Kil-0]), CM45 (MATalpha alr1::HIS3), CM46 (MATa alr1::HIS3 alr2::URA3), CM48 (MATa alr2::URA3), and CM52 (MATalpha ALR1 ALR2). The last four strains are isogenic derivatives of the FY series (13) and share the additional markers his3-Delta 200, ura3-52, leu2-Delta 1, lys2-Delta 202, and trp1-Delta 63. ALR1 and ALR2 deletion mutants were generated by one-step gene disruption (11) using the alr1::HIS3 polymerase chain reaction product or the palr2::URA3 plasmid (Fig. 1). Correct disruption was verified by Southern hybridization (data not shown).


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 1.   Identification and disruption of the ALR genes in aluminum resistance plasmids pSHA20 (panel A) and pCGA8 (panel B). The locations of restriction sites used to construct derivatives of the two plasmids (B, BamHI; Bg, BglII; K, KpnI; p, PstI; X, XhoI) and significant open reading frames within the plasmid inserts (gray arrows) are indicated. The extent of the inserts in the derivative clones (solid lines) and the Al3+ resistance conferred by each are shown. Oligonucleotide primers (arrows 1-4) were used to amplify the ALR reading frames for cloning behind the GAL1 promoter (GAL1pr). Constructs used for ALR gene disruption are also shown.

To assay cation tolerance, cultures were grown in SC-uracil medium (2% glucose), and dilutions were applied to synthetic media with 2% galactose. The toxicity of trivalent metals was tested using a synthetic medium with a low magnesium content (LPM medium with 100 µM Mg2+, pH 3.8) (12). Trivalent metals were added to concentrations of 100 µM Al3+, 150 µM Ga3+, 500 µM La3+, or 10 µM In3+. The toxicity of divalent cations was tested using LPM medium with 2 mM Mg2+ and toxic divalent cations added to give concentrations of 1 mM Co2+, 5 mM Zn2+, 250 µM Ni2+, 3 mM Mn2+, 500 µM Cu2+, or 20 µM Cd2+. Ca2+ sensitivity was tested using low sulfate SC-uracil medium with 500 mM CaCl2 (14).

Cloning, DNA Manipulation, and Sequence Analysis-- Most constructs were generated by restriction fragment subcloning (see Fig. 1) (15). pSHA20Delta 3, pA8Delta 4, and pA8Delta 6 are deletion derivatives of pSHA20 and pCGA8. pCMA20-1 and pCMA81 were derived from pFL46-S (2 µ LEU2) (16) containing subcloned restriction fragments of the pSHA20 and pCGA8 inserts, respectively. To construct YCpALR1 and YEpALR1, a fragment of pSHA20 containing the ALR1 gene was subcloned into the pFL38 (ARS/CEN, URA3) or pFL44-S (2 µ origin, URA3) shuttle vectors (16). YCpALR2 and YEpALR2 contain pCGA8 insert DNA subcloned into pFL38 or pFL44-S. For construction of GAL1 promoter gene fusions (YEpGALR1 and YEpGALR2), the ALR1 and ALR2 coding sequences were amplified using the High Fidelity polymerase chain reaction kit (Boehringer Mannheim) and the following oligonucleotides (Fig. 1), 5'-GGCCTCGAGCGAATATTGCTAGAAAGCGT-3' (primer 1), 5'-CGGCGGCCGCCACATCACTAATCAGTCGT-3' (primer 2), 5'-GGCCTCGAGCTTCGTAATGTCGTCCTTATC-3' (primer 3), and 5'-CGGCGGCCGCAGATCTGCCGACCTACCATA-3' (primer 4). The polymerase chain reaction products were cleaved and ligated into the NotI and SalI sites of the pYES3 expression vector (2 µ URA3 GAL1p) (17). The alr1::HIS3 polymerase chain reaction product was amplified from the HIS3 gene using oligonucleotides with flanking ALR1 homology (18). The oligonucleotides used were as follows. His31A was 5'-CCATCCAATGACCCGGCGTATTGCTCTTACCAGGGTACAGACTTTGGCCTCCTCTAGTACACTC-3'. His32B was 5'-TTTGGCTCCACTTTCAGCGGCCTCGTTAAGTGTTGCAGGAGGGTCCTTGCCACCTATCACCACA-3'. Plasmid palr2::URA3 was constructed as follows (Fig. 1). A XhoI fragment encompassing ALR2 was subcloned into pBC (Stratagene) to create pBC5. The central BglII fragment of the pBC5 insert was then replaced with the URA3 gene from pFL38. This deletion inactivated ALR2, as determined by the lack of aluminum resistance conferred by plasmid pA8Delta 6 (Fig. 1). The palr2::URA3 plasmid was digested with XhoI prior to yeast transformation. To predict membrane-spanning regions within proteins, related protein sequences were aligned using Pileup (19) and the alignment interpreted using PHDhtm (20). Conventional hydropathy plots were generated using Top-PredII (21).

Co2+ Uptake Assay-- Co2+ uptake by cell suspensions was assayed as described (9), using 200 µM total Co2+. To measure uptake in overexpressing strains, yeast were grown in SC-uracil with 2% raffinose, and the GAL1 promoter was induced by the addition of galactose (2%) to cultures at early log phase. After a 3-h induction, cells were harvested, washed, and pretreated with 1% galactose and 1% raffinose at 25 °C for 20 min before addition of the tracer.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Identification of the ALR Genes-- A genomic library of strain S288C in a high copy episomal vector (22) was introduced into the SH2332 and CG379 yeast strains by transformation. A modified synthetic medium with 200 µM Mg2+ and 200 µM ionic Al3+ (LPM) (9) was used to select aluminum-resistant transformants. Plasmids were rescued from tolerant strains, and those that reconferred Al3+ resistance to the original strain were selected. After restriction mapping and Southern analysis (not shown), two plasmids (pSHA20 and pCGA8; Fig. 1) were representative of the six isolated. Deletion mapping of the clones, partial sequence analysis, and comparison with the yeast genome sequence identified two previously uncharacterized open reading frames on chromosomes XV and VI (YOL130w and YFL050c) as responsible for the aluminum resistance (Fig. 1). These two genes were designated ALR1 and ALR2, respectively.

The ALR Genes Encode Homologs of the CorA Mg2+ Transport System-- ALR1 and ALR2 encode closely related proteins (70% identity) with similar molecular masses (95.9 and 96.7 kDa, respectively) and isoelectric points (6.24 and 6.28, respectively). Data base searches revealed that the Alr proteins shared 34% identity with the product of the yeast YKL064w gene on chromosome XI (Fig. 2, A and B) (23). Searches also revealed that the Alr proteins show a low level of similarity to Salmonella typhimurium CorA, a periplasmic membrane protein known to transport divalent cations including Mg2+ and Co2+ (24, 25). S. typhimurium CorA is the best characterized member of a large bacterial gene family. The ALR1, ALR2, and YKL064w proteins share several structural features with CorA, including a highly charged N-terminal domain (greatly expanded in the yeast proteins; Fig. 2B) and two C-terminal hydrophobic regions. Hydropathy analysis indicates that these regions are likely to form transmembrane domains (Fig. 2B, black bars). A third region of the CorA protein known to be membrane-spanning (Fig. 2B, gray bar) was not predicted by the algorithms used here (data not shown).


View larger version (85K):
[in this window]
[in a new window]
 
Fig. 2.   Yeast ALR1 and ALR2 genes encode homologs of the CorA protein. Panel A, sequence alignment of the C-terminal region of the CorA-related proteins with homology to the Alr1p sequence highlighted (gray boxes). Transmembrane domains predicted for the ALR1 and ALR2 sequences (TM2, TM3) and the cryptic transmembrane domain in the S. typhimurium CorA sequence (TM1) are shown. Abbreviations and data base accession numbers for each protein sequence are: ALR1, ALR2, and YKL, S. cerevisiae YOL130w, YFL050c, and YKL064w predicted proteins (PID g1209711, Swissprot P43553 and P35724, respectively); S. ty., S. typhimurium (PIR A64109); H. in., Hemophilus influenzae (PIR A64109); Syn. 1 and Syn. 2, Synechocystis sp. (DDBJ D64006 and D64005, respectively); M. ja., Methanococcus jannaschii (PID g1499876); M. le., Mycobacterium leprae (PID g699153); B. su., Bacillus subtilis (PID g732250). An insertion in the Syn. 1 sequence was removed to clarify the figure (residues 329-334, open triangle). Panel B, schematic comparison of the domain structure of the yeast Alr and YKL064w proteins with the S. typhimurium CorA protein, based on sequence alignment.

The similarity of the CorA and ALR genes suggested that the latter are involved in divalent cation transport. For this reason we characterized the effect of increased ALR gene expression on resistance to various divalent and trivalent metal ions. To maximize ALR gene expression, the coding sequences of both genes were fused to the strong GAL1 promoter in a multicopy plasmid. Fig. 3 shows that the ALR genes conferred increased resistance to Al3+ and Ga3+ (but not In3+) and increased sensitivity to Co2+, Zn2+, Ni2+, Mn2+, Ca2+, La3+, and Cu2+ (but not Cd2+). Several of these divalent cations have been implicated as substrates for the same yeast cation transport system, either via uptake studies (26-28) or by a genetic correlation (29). The enhanced sensitivity to divalent cations seen in strains overexpressing either ALR gene is consistent with the ALR genes encoding such an influx system. The YEpGALR1 construct was generally less effective than YEpGALR2 at altering metal tolerance (evident in the results for Cu2+ and Ca2+, Fig. 3); the reason for this difference is unclear.


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 3.   Overexpression of the ALR1 and ALR2 genes alters cation tolerance. The FY73 strain was transformed with pYES3 (control) or the YEpGALR1 (+ALR1) or YEpGALR2 (+ALR2) overexpression construct. Cation tolerance was recorded after 4 days growth on synthetic media containing the indicated metal salts.

The ALR1 Gene Is Required for Growth at Low Mg2+ Concentrations-- To define further the role of the ALR genes, mutant strains were obtained by gene disruption (Fig. 1; see also "Experimental Procedures") using yeast strains derived from S288C (13). Haploid strains carrying the expected alr2::URA3 allele were viable, and no novel phenotype was observed to be associated with this marker in subsequent crosses. These results indicate that the ALR2 gene is not essential for growth. In contrast, ALR1 could not be disrupted in a haploid strain. Segregation analysis of an alr1::HIS3/+ heterozygous diploid strain confirmed that ALR1 inactivation was lethal (not shown). In an attempt to rescue haploid alr1 mutants, spores were dissected to a variety of growth conditions. The growth of alr1 mutants was increased substantially by supplementation with additional Mg2+ (50-500 mM MgCl2 or MgSO4; Fig. 4). However, no growth was observed on media with high salt (1 M NaCl), high calcium (100-500 mM CaCl2), or osmotic stabilizers (1 M sorbitol, 1 M glucose), or after incubation at a lower temperature (25 °C) (data not shown).


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 4.   Complementation of an alr1 mutant. CM45 (alr1) was transformed with three control plasmids (pFL38, pFL44-S, and pYES3), the ALR genes in low copy (YCpALR1 and YCpALR2) and high copy (YEpALR1 and YEpALR2) vectors, or fused to the GAL1 promoter in a high copy vector (YEpGALR1 and YEpGALR2). The strains were grown to saturation in SCM-uracil (SC-uracil + 500 mM MgCl2) and 2 × 104 cells applied to SCM-uracil plates (left) or standard SC-uracil (right, 4 mM Mg2+). Growth is shown after 3 days at 30 °C.

The phenotype of the alr1 mutation suggested a defect in Mg2+ uptake was present in alr1 strains. In contrast, no phenotype was observed to be associated with the alr2 mutation, and alr1 alr2 double mutant strains had the same Mg2+-dependent phenotype as alr1 single mutants (data not shown). The similarity of the overexpression phenotypes conferred by the ALR genes (Fig. 3) had suggested that the two genes might perform a similar function. To test this hypothesis, we introduced plasmids carrying ALR2 into an alr1 mutant strain. Increased copy number of an ALR2 genomic clone, or transcription of the ALR2 coding sequence from a heterologous promoter, eliminated the Mg2+-dependent phenotype of the alr1 mutant (Fig. 4). Only a small increase in ALR2 expression (that conferred by an extra one or two copies/cell) was required to correct the alr1 defect. Together with the similar phenotypes shown in Fig. 3, this result suggested that the ALR genes were functionally redundant but that ALR2 was normally not expressed in the S288C genetic background. Poor expression of ALR2 in some strains is consistent with later observations that increased copy number of an ALR2 genomic clone did not confer Al3+ tolerance to FY73 (a strain derived from S288C), although tolerance of the SH2332 and CG379 strains was increased (data not shown).

To measure transcription of the ALR genes, Northern hybridization was performed using total RNA extracted from FY73. A 3-kilobase pair transcript hybridizing to an ALR1 probe was detected in these experiments, but no mRNA species was observed to hybridize to an ALR2-specific probe (data not shown). The sequence of the ALR2 gene and surrounding regions was examined for features that could potentially affect ALR2 expression. Two autonomous replication sequences were found close to the 3'-end of ALR2 (ARS601 and 602) (30). In yeast, ARS elements are thought to contribute to the silencing of nearby genes (31, 32). In addition to this potential for transcriptional silencing, the ALR2 coding sequence is directly preceded by a small (27 nucleotides) reading frame of unknown function. If this reading frame is included in the ALR2 mRNA, it may negatively affect translation from the ALR2 start codon (33). No equivalent reading frame is present in the 5'-region of the ALR1 gene. The ALR2 5'-untranslated region was not included in YEpGALR2, so it would not be expected to interfere with ALR2 expression from this construct.

The ALR Genes Mediate Co2+ Uptake by Yeast-- We determined the effect of altered ALR gene expression on divalent cation accumulation, using 57Co2+ as a tracer. Co2+ has been reported to be accumulated by the yeast Mg2+ transport system (26, 29) and was used as a substitute for the unavailable 28Mg2+ isotope. As described previously (27), Co2+ uptake was energy-dependent: the addition of a fermentative carbon source was required, and tracer accumulation was inhibited by low temperature (0 °C) or the presence of the metabolic uncoupler 2,4-dinitrophenol (250 µM, data not shown). The rate of uptake was dependent on the Co2+ concentration, and the uptake system was saturable (data not shown). Together these observations indicated that Co2+ uptake was mediated by an enzyme-like transport system in yeast.

The alr1 mutation was associated with a lower rate of Co2+ uptake than found for the wild type, whereas deletion of ALR2 had no effect (Fig. 5A). As measurement of Co2+ uptake required growth of alr1 mutants in medium with 500 mM Mg2+, wild-type (ALR1) strains were grown with normal and high Mg2+ concentrations prior to the assay. Transport by ALR1 wild-type strains was decreased substantially by growth in 500 mM Mg2+ (Fig. 5A), but alr1 mutants still exhibited significantly less uptake than ALR1 wild-type strains under equivalent conditions. Strains overexpressing either ALR gene showed greatly increased Co2+ uptake (Fig. 5B). Because overexpression of the ALR genes altered Al3+ tolerance, the effect of Al3+ on Co2+ uptake in strains overexpressing the ALR genes was also tested. Al3+ strongly inhibited Co2+ uptake in the control strain (Fig. 5B), but strains overexpressing either ALR gene maintained a robust uptake capacity that was equal to, or greater than, the control strain without Al3+.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 5.   The ALR genes enhance Co2+ uptake. Panel A, time course of 57Co2+ uptake compared in a wild-type strain (solid squares, solid triangles), alr1 (open squares), and alr2 (solid circles, open triangles) single mutants, and an alr1 alr2 double mutant (open circles). Growth of the yeast strains in YPDM prior to the assay is indicated (+Mg). Panel B, rate of 57Co2+ uptake in a control strain (carrying pYES3) compared with strains overexpressing the ALR genes (+ALR1 and +ALR2). The effect of simultaneous addition of Al3+ (100 µM) with the Co2+ tracer is shown (+Al3+). Strains used were identical to the three previously described (Fig. 3). All values represent the average of four experiments (error bars = ± S.E.).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The Alr Proteins Mediate Transport of Mg2+ and Other Divalent Cations-- Previous work suggests that Mg2+ enters the yeast cell via a low affinity transport system, which can also accumulate Co2+, Mn2+, Zn2+, and Ni2+ ions (26-29). Our data are consistent with the ALR genes encoding this transport system. Clear evidence that both the Alr proteins transport Mg2+ came from the Mg2+-dependent growth of alr1 mutants, which could be corrected by increased ALR2 expression. The increased sensitivity of overexpressing strains to Co2+, Mn2+ Ni2+, and Zn2+ is consistent with the Alr proteins directing the influx of these divalent cations. Use of a 57Co2+ tracer revealed increased Co2+ accumulation in overexpressing strains and reduced uptake in alr1 mutant strains. The observed differences between the effects of the alr1 and alr2 mutations can be explained simply, by poor expression of ALR2 in the S288C genetic background.

Despite the broad substrate specificity of the Alr proteins, two lines of evidence suggest that the main physiological role of these systems is to allow Mg2+ influx. First, an increased requirement for divalent cations other than Mg2+ was not observed in alr1 strains. Second, specific uptake systems for Mn2+ (34), Zn2+ (35), and Cu2+ (36) have been identified previously in yeast. These systems typically display a high affinity for their substrates, with reported Km values of 1-4 µM (35, 36). In contrast, the Km determined for Co2+ uptake via the Alr proteins ranges from 77 µM (27) to 105 µM.1 Given the low requirement of yeast for divalent cations other than Mg2+ (37), the Alr proteins are unlikely to contribute to the accumulation of such cations at physiologically significant concentrations.

The ALR Genes Are Likely to Encode Plasma Membrane Proteins-- The location of the Alr proteins in the cell has not been determined directly. However, the results reported here support their assignment to the plasma membrane. First, the related CorA protein is known to mediate Mg2+ influx over the bacterial periplasmic membrane (25). Second, inactivation of ALR1 increased the yeast requirement for Mg2+. Yeast mutants lacking plasma membrane-localized systems required for nutrient uptake commonly display an increased requirement for particular nutrients, for example SO42-, K+, or NH4+ ions (17, 38, 39). Third, the results of tracer uptake assays are consistent with the Alr proteins mediating the uptake of Co2+ over the plasma membrane. Overexpression of the ALR genes increased Co2+ uptake, and loss of ALR gene activity reduced uptake. Parallel changes in uptake rates have been observed in yeast with altered expression of other plasma membrane cation transport systems (34-36). Although increased expression of transporters located in organelle membranes can also enhance ion uptake (for example, the Zrc1 and Cot1 proteins; Ref. 40), it is unusual for the inactivation of such systems to result in an increased requirement for the transporter substrate. Hence, the phenotypes associated with altered ALR gene expression strongly suggest that these proteins mediate cation influx over the yeast plasma membrane.

Mechanism and Importance of Mg2+ Transport in Eukaryotes-- The energy dependence of Co2+ uptake by yeast and its sensitivity to 2,4-dinitrophenol (an uncoupler of transmembrane proton gradients) suggested that the Alr proteins mediate active transport, possibly via a proton-coupled symport mechanism. However, passive cation influx via a channel-like system may also be dependent on metabolic energy and the proton gradient, since both these factors would affect the electrical potential generated by the activity of the plasma membrane H+-ATPase. It has been proposed that this electrical potential difference alone could drive sufficient Mg2+ uptake by bacterial and eukaryote cells (41, 42). For these reasons it is also possible that the CorA protein family (including the Alr proteins) could represent a new class of divalent cation channel. Apparent Mg2+-specific channel activities have been described in other eukaryotes. For example, in the yeasts Schizosaccharomyces pombe and Kluyveromyces fragilis, rapid and short lived influx of Mg2+ occurs just prior to cell division, suggesting that a channel-like activity is transiently activated (43). In Paramecium tetraurelia, a novel Mg2+-specific channel has been characterized using electrophysiological methods (44); and in mammalian kidney cells, a tightly regulated transport system is responsible for Mg2+ influx after depletion of internal Mg2+ stores (45). The use of newly available methods to investigate yeast electrophysiology, combined with the mutant strains described in this work, may enable the definition of the mechanism of cation influx via the Alr proteins.

Recently, there has been renewed interest in the role of Mg2+ in the control of cell growth and development. Although most intracellular Mg2+ is bound, the concentration of free ionized Mg2+ can alter rapidly in response to environmental signals (46). In S. pombe and K. fragilis, the level of intracellular Mg2+ regulates the timing of cell cycle progression (43, 47). Mg2+ is also required for germ tube formation in Candida albicans vegetative cells and consequently regulates the morphogenesis and pathogenicity of this species (48). Free ionized Mg2+ has been proposed to regulate cell growth and division in some mammalian cells (46), possibly via an effect on the activity of enzymes involved in signal transduction (49). Study of the regulation of free ionized Mg2+ in the cytoplasm is hampered by a lack of molecular information on Mg2+ transport by eukaryote cells. The conditional lethal phenotype of alr1 mutants may allow the molecular cloning of other Mg2+ transporters via functional complementation, as has been achieved successfully for other nutrient transporters (50).

Al3+ Toxicity to Yeast and Plants-- Although previous work in our laboratory characterized mutations that conferred increased aluminum sensitivity to yeast (51), this is the first report of the identification and molecular characterization of a gene capable of increasing yeast aluminum resistance. The identification of the ALR genes provides genetic support for the proposal that, under conditions of low Mg2+ availability, Al3+ prevents Mg2+ uptake by yeast and consequently induces Mg2+ deficiency (9). The results described here show that increased activity of the yeast Mg2+ transport system confers aluminum resistance (Fig. 3) and also overcomes the inhibition of cation uptake by aluminum (Fig. 5). Our results also suggest that Ga3+ and Al3+ ions may be similar in their mechanism of action; these two cations have similar effects on growth and Co2+ uptake (9), and ALR gene overexpression confers tolerance to both (Fig. 3). In contrast, La3+ is an ineffective inhibitor of the Mg2+ uptake system (9), and ALR overexpression increased La3+ toxicity, suggesting that this trivalent cation effectively permeates the Mg2+ transporter.

Several parallels exist between our observations in yeast and Al3+ toxicity to plants. Mg2+ ameliorates Al3+ toxicity (52, 53), and Mg2+ deficiency is observed in aluminum-intoxicated cereals and grasses (53-55). In addition, Al3+ has inhibitory effects on Mg2+ uptake by root cells (56-58). However, there are also key differences between the two systems. The most immediate symptom of Al3+ toxicity to plants is the inhibition of root elongation (4), an effect that is not duplicated by the reduced availability of Mg2+ (52). Nevertheless, an Al3+-induced reduction in Mg2+ uptake could contribute to the low yields observed in species such as the Graminae, where Mg2+ deficiency is a notable feature of long term exposure to Al3+ (53-55). Expression of the ALR genes in aluminum-sensitive plant species may be one way to investigate the interaction between Mg2+ utilization and aluminum toxicity to plants.

    ACKNOWLEDGEMENTS

We thank A. Goldstein for the YEp24 library and HIS3 clone; E. Schott, J. Putterill, and D. Christie for discussions during the research; S. Harashima for providing sequence information before publication; and R. Bellamy, G. Cooper, D. Christie, and J. Putterill for reviewing the manuscript.

    FOOTNOTES

* This work was supported in part by Foundation for Research Science and Technology Grant AGR 06-052 to the New Zealand Pastoral Agriculture Research Institute.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.

Dagger Supported by a University of Auckland Doctoral Scholarship during this work.

§ To whom correspondence should be addressed: Center for Gene Technology, School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand. Tel.: 64-9-373-7599; Fax: 64-9-373-7416; E-mail: r.gardner{at}auckland.ac.nz.

1 C. W. MacDiarmid and R. C. Gardner, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Moeller, T., Bailar, J. C., Kleinberg, J., Guss, C. O., Castellion, M. E., Metz, C. (1984) Chemistry with Inorganic Qualitative Analysis, p. 929, Academic Press, Orlando
  2. Exley, C., and Birchall, J. D. (1992) J. Theor. Biol. 159, 83-98[Medline] [Order article via Infotrieve]
  3. Piña, R. G., and Cervantes, C. (1996) Biometals 9, 311-316[Medline] [Order article via Infotrieve]
  4. Kochian, L. V. (1995) Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 237-260 [CrossRef]
  5. Driscoll, C. T., Baker, J. P., Bisogni, J. J., Schofield, C. L. (1980) Nature 284, 161-164
  6. Wisniewski, H. M., Moretz, R. C., Sturman, J. A., Wen, G. Y., Shek, J. W. (1990) Env. Geochem. Health 12, 115-120
  7. Aniol, A. (1991) in Plant-Soil Interactions at Low pH (Wright, R. J., Baligar, V. C., and Murrmann, R. P., eds), pp. 1007-1017, Kluwer Academic, Dordrecht, The Netherlands
  8. de la Fuente, J. M., Ramírez-Rodríguez, V., Cabrera-Ponce, J. L., Herrera-Estrella, L. (1997) Science 276, 1566-1568[Abstract/Free Full Text]
  9. MacDiarmid, C. W., and Gardner, R. C. (1996) Plant Physiol. 112, 1101-1109[Abstract/Free Full Text]
  10. Sherman, F. (1991) Methods Enzymol. 194, 3-21[Medline] [Order article via Infotrieve]
  11. Kaiser, C., Michaelis, S., and Mitchell, A. (1994) Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, pp. 141-143, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  12. Gietz, R. D., Schiestl, R. H., Willems, A. R., Woods, R. A. (1995) Yeast 11, 355-360[Medline] [Order article via Infotrieve]
  13. Winston, F., Dollard, C., and Ricupero-Hovasse, S. L. (1995) Yeast 11, 53-55[Medline] [Order article via Infotrieve]
  14. Pozos, T. C., Sekler, I., and Cyert, M. S. (1996) Mol. Cell. Biol. 16, 3730-3741[Abstract]
  15. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., pp. 1.1-1.110, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  16. Bonneaud, N., Ozier-Kalogeropoulos, O., Li, G., Labouesse, M., Minvielle-Sebastia, L., and Lacroute, F. (1991) Yeast 7, 609-615[Medline] [Order article via Infotrieve]
  17. Smith, F. W., Hawkesford, M. J., Prosser, I. M., Clarkson, D. T. (1995) Mol. Gen. Genet. 247, 709-715[Medline] [Order article via Infotrieve]
  18. Baudin, A., Ozier-Kalogeropoulos, O., Denouel, A., Lacroute, F., and Cullin, C. (1993) Nucleic Acids Res. 21, 3329-3330[Medline] [Order article via Infotrieve]
  19. Devereux, J., Haeberli, P., and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395[Abstract]
  20. Rost, B., Casadio, R., Fariselli, P., and Sander, C. (1995) Protein Sci. 4, 521-533[Abstract/Free Full Text]
  21. Claros, M. G., and von Heijne, G. (1994) Cabios Application Notes 10, 685-686
  22. Carlson, M., and Botstein, D. (1982) Cell 28, 145-154[Medline] [Order article via Infotrieve]
  23. Rasmussen, S. W. (1994) Yeast 10, S63-S68[Medline] [Order article via Infotrieve]
  24. Hmiel, S. P., Snavely, M. D., Miller, C. G., Maguire, M. E. (1986) J. Bacteriol. 168, 1444-1450[Medline] [Order article via Infotrieve]
  25. Smith, R. L., Banks, J. L., Snavely, M. D., Maguire, M. E. (1993) J. Biol. Chem. 268, 14071-14080[Abstract/Free Full Text]
  26. Fuhrmann, G.-F., and Rothstein, A. (1968) Biochim. Biophys. Acta 163, 325-330[Medline] [Order article via Infotrieve]
  27. Norris, P. R., and Kelly, D. P. (1977) J. Gen. Microbiol. 99, 317-324
  28. Okorokov, L. A., Lichko, L. P., Kadomtseva, V. M., Kholodenko, V. P., Titovsky, V. T., Kulaev, I. S. (1977) Eur. J. Biochem. 75, 373-377[Abstract]
  29. Conklin, D. S., Kung, C., and Culbertson, M. R. (1993) Mol. Cell. Biol. 13, 2041-2049[Abstract]
  30. Shirahige, K., Iwasaki, T., Rashid, M. B., Ogasawara, N., Yoshikawa, H. (1993) Mol. Cell. Biol. 13, 5043-5056[Abstract]
  31. Diffley, J. F., and Stillman, B. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2120-2124[Abstract]
  32. Triolo, T., and Sternglanz, R. (1996) Nature 381, 251-253[CrossRef][Medline] [Order article via Infotrieve]
  33. Schneider, J. C., and Guarente, L. (1991) Methods Enzymol. 194, 373-388[Medline] [Order article via Infotrieve]
  34. Supek, F., Supekova, L., Nelson, H., and Nelson, N. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5105-5110[Abstract/Free Full Text]
  35. Zhao, H., and Eide, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2454-2458[Abstract/Free Full Text]
  36. Dancis, A., Yuan, D. S., Haile, D., Askwith, C., Eide, D., Moehle, C., Kaplan, J., and Klausner, R. D. (1994) Cell 76, 393-402[Medline] [Order article via Infotrieve]
  37. Loukin, S., and Kung, C. (1995) J. Cell Biol. 131, 1025-1037[Abstract]
  38. Gaber, R. F., Styles, C. A., and Fink, G. R. (1988) Mol. Cell. Biol. 8, 2848-2859[Medline] [Order article via Infotrieve]
  39. Marini, A. M., Vissers, S., Urrestarazu, A., and André, B. (1994) EMBO J. 13, 3456-3463[Abstract]
  40. Conklin, D. S., Culbertson, M. R., and Kung, C. (1994) Mol. Gen. Genet. 244, 303-311[Medline] [Order article via Infotrieve]
  41. Flatman, P. W. (1991) Annu. Rev. Physiol. 53, 259-271[CrossRef][Medline] [Order article via Infotrieve]
  42. Maguire, M. E., Snavely, M. D., Leizman, J. B., Gura, S., Bagga, D., Tao, T., Smith, D. L. (1992) Ann. N. Y. Acad. Sci. 671, 244-256[Medline] [Order article via Infotrieve]
  43. Walker, G. M., and Duffus, J. H. (1980) J. Cell Sci. 42, 329-356[Abstract]
  44. Preston, R. R. (1990) Science 250, 285-288[Medline] [Order article via Infotrieve]
  45. Dai, L.-J., and Quamme, G. A. (1991) J. Clin. Invest. 88, 1255-1264[Medline] [Order article via Infotrieve]
  46. Maguire, M. E. (1988) Ann. N. Y. Acad. Sci. 551, 201-217[Abstract]
  47. Walker, G. M. (1986) Magnesium 5, 9-23[Medline] [Order article via Infotrieve]
  48. Walker, G. M., Sullivan, P. A., and Shepherd, M. G. (1984) J. Gen. Microbiol. 130, 1941-1945[Medline] [Order article via Infotrieve]
  49. Chien, M. M., and Cambier, J. C. (1990) J. Biol. Chem. 265, 9201-9207[Abstract/Free Full Text]
  50. Frommer, W. B., and Ninnemann, O. (1995) Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 419-444 [CrossRef]
  51. Schott, E. J., and Gardner, R. C. (1997) Mol. Gen. Genet. 254, 63-72[CrossRef][Medline] [Order article via Infotrieve]
  52. Kinraide, T. B., and Parker, D. R. (1987) Plant Physiol. 83, 546-551
  53. Tan, K., Keltjens, W. G., and Findenegg, G. R. (1991) Plant Soil 136, 65-71
  54. Wheeler, D. M., Edmeades, D. C., and Christie, R. A. (1992) J. Plant Nutr. 15, 387-402
  55. Tan, K., and Keltjens, W. G. (1995) Plant Soil 171, 147-150
  56. Rengel, Z., and Robinson, D. L. (1989) Plant Physiol. 91, 1407-1413
  57. Robinson, D. L., and Rengel, Z. (1991) Curr. Top. Plant Biochem. Physiol. 10, 107-116
  58. Schimansky, C. (1991) Z. Pflanzenernähr. Bodenk. 154, 195-198


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.