From the 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
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ABSTRACT |
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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 ( 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 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
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 DH5 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 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% Microscopy--
GFP fluorescence was analyzed with a Leica TCS
4D confocal microscope (argon/krypton laser; 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.
Mg2+-dependent Phenotype of The Bacterial Mg2+ Transporter CorA Partially
Suppresses the Altered Ion Concentrations in Time-dependent Change in the Cellular Mg2+
Concentration--
To shed more light on the relevance of Alr1p for
Mg2+ uptake, both wild-type and
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).
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 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.
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.
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,
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
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 (
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.
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
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 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
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 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
alr1 mutant cells did not. Expression of the
bacterial Mg2+ transporter CorA in the yeast
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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).
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(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.
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/
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).
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/
end3), was sporulated, and
tetrads were dissected on YPD medium at 28 °C, generating JS034C
(END3) and JS034B (
end3).
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/
pep4), was sporulated, and
tetrads were dissected on YPD medium at 28 °C, generating AG1679C
(PEP4) and AG1679A (
pep4). Correct
replacements of the ALR1, END3, and
PEP4 open reading frames by the disruption constructs were
verified using analytical PCR (data not shown).
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
alr1 phenotype (data not shown).
-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.
ex = 488 nm
and
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.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
alr1 Mutant
Yeast Cells--
To investigate the phenotype of the
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
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).
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.
View larger version (13K):
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Fig. 1.
Mg2+-dependent growth
phenotype of the alr1 mutant. JS74A
wild-type (WT; left panel) and JS74B
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.
,
200 mM Mg2+;
, 100 mM
Mg2+;
, 50 mM Mg2+;
, 10 mM Mg2+; +, 1 mM Mg2+;
, 30 µM Mg2+.
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
alr1 phenotype, N-terminally GFP-tagged CorA
protein was expressed in the
alr1 mutant from
the strong pMet-25 promoter. Serial dilutions of
the wild-type and
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
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 alr1
phenotype. The
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.
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
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
alr1 was reduced to 40% of the wild-type
level. Grown in the same medium supplemented with 200 mM
Mg2+,
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
alr1 mutation.
Total ion levels in wild-type and alr1 cells
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
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,
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
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 ( ) and JS74B
alr1 cells (
) 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).
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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.
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 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.
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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).
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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).
end3, and
pep4 mutants.
end3 null mutant (see
"Experimental Procedures") and followed Alr1p stability upon
addition of Mg2+ in this genetic background.
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
end3 cells (Fig.
8C), implying that endocytosis is crucial for Alr1p
degradation. It is worth noting that in both npi1 and
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.
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
(
pep4) (Fig. 9. e-h). Also at low
Mg2+ concentrations, slight accumulation of the protein in
the vacuole was observed in
pep4 cells, which
is consistent with the notion of vacuolar Alr1p degradation (data not
shown). Taken together, the results obtained with the npi1,
end3, and
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 ( 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.
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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
alr1,
mrs2, or
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.
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;
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.
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.
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.
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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.
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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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