From the School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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EXPERIMENTAL PROCEDURES |
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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 (MAT
alr1::HIS3), CM46 (MATa
alr1::HIS3
alr2::URA3), CM48 (MATa
alr2::URA3), and CM52
(MAT
ALR1 ALR2). The last four strains are
isogenic derivatives of the FY series (13) and share the additional
markers his3-
200, ura3-52, leu2-
1, lys2-
202, and
trp1-
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).
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Cloning, DNA Manipulation, and Sequence Analysis--
Most
constructs were generated by restriction fragment subcloning (see Fig.
1) (15). pSHA203, pA8
4, and pA8
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 pA8
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.
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RESULTS |
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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).
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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).
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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+.
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DISCUSSION |
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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 |
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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.
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FOOTNOTES |
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* 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.
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.
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REFERENCES |
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