MNR2 Regulates Intracellular Magnesium Storage in Saccharomyces Cerevisiae

8/14/2019 MNR2 Regulates Intracellular Magnesium Storage in Saccharomyces Cerevisiae

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MNR2 regulates intracellular magnesium storage inSaccharomyces cerevisiae

Nilambari P. Pisat, Abhinav Pandey1, and Colin W. MacDiarmid

Department of Biology, University of Missouri-St Louis, St Louis, MO 63121-4400

1Present address: Ranbaxy Research Laboratories, Gurgaon, Haryana-122001, India


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Running title:MNR2 regulates magnesium storage

Keywords: CorA, Mnr2, Alr1, magnesium homeostasis, vacuole

Corresponding author: Colin MacDiarmid

Department of Biology,

University of Missouri-St Louis,

St Louis, MO 63121-4400.

Phone: 314-516-7013

Fax: 314-516-6233

e-mail: [email protected]


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ABSTRACT

Magnesium (Mg) is an essential enzyme cofactor and a key structural c

biological molecules, but relatively little is known about the molecular component

Mg homeostasis in eukaryotic cells. The yeast genome encodes four characterized

the CorA Mg transporter superfamily located in the plasma membrane (Alr1 and

mitochondrial inner membrane (Mrs2 and Lpe10). We describe a fifth yeast C

(Mnr2) required for Mg homeostasis. MNR2 gene inactivation was associated with

both the Mg requirement and Mgcontent of yeast cells. In Mg-replete conditions, w

accumulated an intracellular store of Mg that supported growth under deficient c

mnr2 mutant was unable to access this store, suggesting that Mg was trapped in an

compartment. Mnr2 was localized to the vacuole membrane, implicating this org

storage. The mnr2 mutant growth and Mg-content phenotypes were dependent

proton-ATPase activity, but were unaffected by the loss of mitochondrial Mg upta

a specific dependence on vacuole function. Overexpression of Mnr2 suppresse

defect of an alr1 alr2 mutant, indicating that Mnr2 could function independentl

genes. Together, our results implicate a novel eukaryotic CorA homolog in the

intracellular Mg storage.


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C-terminus, and a triad of conserved residues [glycine-methionine-asparagine] tha

for Mg transport (KNOOP et al. 2005). The low number of transmembrane domain

be present in MIT proteins suggested that oligomerization was required for

(KOLISEKet al. 2003; WARREN et al. 2004), and independent crystallographic stud

homolog from Thermotoga maritima support this model (ESHAGHI et al. 2006;

2006; PAYANDEH and PAI 2006). T. Maritima CorA formed a homopentamer o

which the C-terminal transmembrane domains clustered together to form a membr

pore. The N-terminal regions of the subunits formed a cytosolic "funnel"

incorporated several apparent Mg-binding sites, suggesting a regulatory role for

Genetic studies provided evidence that the binding of Mg ions to these site

conformation of the complex and decreased channel activity (PAYANDEH et al. 200

and PAI 2006). The activity of the Mrs2 protein was also shown to be depen

concentration (SCHINDL et al. 2007), suggesting that both prokaryotic and euk

proteins can respond directly to the cytosolic or matrix Mg concentration in ord

homeostasis.

A fifth CorA homolog (YKL064w) is present in the yeast genome, but h

characterized (MACDIARMID and GARDNER 1998). The Ykl064w protein sh

predicted transmembrane domains and conserved GMN motif characteristic of M

members of the MIT family (KNOOP et al. 2005; MACDIARMID and GAR

Phylogenetic analysis revealed that Ykl064w belongs to a subgroup of fungal MI


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CorA proteins suggested that the Ykl064w-related proteins perform a novel func

reason, we decided to investigate the role of Ykl064w in ion homeostasis. Here w

Ykl064w is a vacuolar membrane protein that is required for Mg homeostasis

evidence implicating this protein in the regulation of intracellular Mg storage.

MATERIALS AND METHODS

Growth media and general methods: Yeast were routinely grown in YPD or

with 2% glucose and required auxotrophic supplements. Low magnesium medium

prepared using a commercial YNB mix lacking divalent cations (Q-Biogene). Di

were added to give final concentrations of 1 mM CaCl2, 0.15 M CuCl2, 0.75 M

M MnCl2, and 2.5 M ZnCl2. The Mg concentration of LMM was below levels

atomic absorption spectroscopy (< 10 nM). For some experiments, LMM was suppl

MgCl2 to give the concentration required, or with 1 mM Na2-EDTA to chelate trac

Mg. Cell number/ml of yeast suspensions was determined by measuring the opti

595 nm (A595) and comparing with a standard curve ofA595 vs viable cells.Yeast tra

were performed using standard methods (GIETZ et al. 1992).

Plasmid construction:The plasmids pFL38, pFL44-S, pFL45-S (BONNEAUD

and YCpALR1 (MACDIARMID and GARDNER 1998) were described previously.

palr2TRP1, a 5 kbKpnI genomic clone ofALR2 in pBCSK-(Stratagene) was cut w


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transformed with the mnr2::KANR

PCR product and the inserts of palr1HIS3 an

The resulting diploid was sporulated to obtain clones of the appropriate genot

NP180, NP193 and NP201 were generated by sequential transformation of DY1

insert of pmnr2SpHIS5 and a tfp1::KANR

PCR product, followed by sporulation. N

and NP112 were derived by transformation of DBY747, lpe10!-1 and mrs2!-2 (G

2001a) with the mnr2::KANR PCR product.

Atomic absorption spectroscopy (AAS) and inductively coupled

spectrometry (ICP-MS): Measurement of total cell-associated Mg was routine

using atomic absorption spectroscopy. Yeast cultures (5 ml) were harvested durin

growth, collected by centrifugation, and washed twice with 1 mM Na2-EDTA (

twice with deionized water. Cells were resuspended in 1 ml of water and the A595 o

recorded. One ml of each suspension was added to 1 ml of concentrated nitric ac

glass tube, and digested by incubation for 16 h at 95. After addition of 2 ml 1x LaC

mM LaCl3, 240 mM HCl), each digest was adjusted to a total volume of 4 ml

water. Digests were diluted 5-fold in 0.5x LaCl3 buffer before measurement with a

instrument. ICP-MS analysis was performed essentially as previously described

2004). Aliquots of yeast cultures (2-5 ml) were filtered through 0.45 M nitroce

(Millipore), washed twice with 5 ml of wash buffer (20 mM sodium citrate, pH 4, 1

and once with 5 ml of Millipore water. The filters were digested with concentrat

before analysis The contribution of the filters was subtracted from the final value


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using standard techniques (HARLOW and LANE 1988) and immunoblots develope

Pico chemiluminescent substrate (Pierce Chemical Co.). Antibodies for immuno

obtained from Sigma-Aldrich, Molecular Probes, and Pierce Chemical

immunofluorescence microscopy was performed as previously described (MACD

2000), using a mouse "-myc primary (Abcam) and a goat anti-mouse secondary ant

with Alexa Fluor 488 (Molecular Probes). FM4-64 staining of the vacuole membra

EMR1995) was performed by incubation of yeast cultures in medium containing 2

for 15 min. The cells were washed once with in dye-free medium, then resuspen

medium and incubated at 30 for 30 minutes to allow complete internalization

Fluorescence images were overlaid using Photoshop CS software (Adobe), which

to filter image noise and optimize contrast.

RESULTS

Effect ofYKL064won elemental content: Initial phenotypic characterization

deletion mutant revealed that this mutation was associated with a substantia

sensitivity to manganese ions, and a less severe sensitivity to calcium, zinc, an

(Figure 1A). For this reason, the gene was designated MNR2 (for Manganese res

potential role of Mnr2 in divalent cation homeostasis was further investigated by c

total elemental content of wild-type and mnr2 strains after growth in a stand


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Somewhat surprisingly, the mnr2 mutant showed a growth defect in Mg-deficie

(


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ATPase). Mutations that inactivated essential subunits of the V-ATPase reduc

content (EIDE et al. 2005), which is consistent with a dependence of the vacuola

system on this enzyme. If Mnr2 regulates the release of Mg from vacuolar stores, m

prevent vacuolar sequestration of Mg should suppress phenotypes associated w

mutation. To test this prediction, we determined the effect of combining the tf

mutations on Mg accumulation under Mg-replete and deficient conditions (Figur

encodes subunit A of the V1 domain of the V-ATPase, which is essential for the a

enzyme (LIU et al. 1997). Consistent with the above model, a tfp1 mutant had a mu

content than wild-type after growth in Mg-replete conditions. The Mg content of th

was similar to that of Mg-deficient wild-type cells (approximately 20 nmol/106 cell

4A), which as previously noted, represents the minimum Mg content of viable wild

addition, there was little difference in Mg content between tfp1 cells grown under

deficient conditions, indicating that this mutant was unable to accumulate exces

mnr2 and tfp1 mutations were combined, the tfp1 mutation completely suppres

phenotype of elevated Mg content in deficient conditions. TFP1 thus acts upstream

genetic pathway controlling intracellular Mg accumulation.

To investigate the effect of the tfp1 mutation on Mg storage, we compared t

mutant strains in Mg-free medium, conditions in which all growth is dependent on

of intracellular Mg (Figure 4B). Consistent with its initial low Mg content, th

displayed a severe growth defect under these conditions (Figure 4A) Growth of


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To investigate the specificity of this genetic interaction, we also determined

combining the mnr2 mutation with deletions of genes encoding other organelle M

(Figure 4A). Previous reports indicated that both the Mrs2 and Lpe10 proteins were

Mg uptake by mitochondria, as lpe10 and mrs2 mutants displayed a similar

mitochondrial Mg content (GREGAN et al. 2001a; KOLISEKet al. 2003). To determ

of these mutations on whole cell Mg content, we grew strains carrying combinat

lpe10 and mrs2 mutations in Mg-replete and deficient conditions. Unlike the tfp1

lpe10 and mrs2 mutations did not reduce total cellular Mg content, suggesting that

Mg is a relatively minor component of total cellular Mg. In addition, neither the l

mutations affected the severity of the mnr2 Mg content phenotype, indicating that M

regulate the Mg content of the mitochondrial compartment. In addition, th

reproducible effect of the lpe10 ormrs2 mutations on growth in Mg-deficient con

alone or in combination with the mnr2 mutation (Figure 4B), indicating that the m

compartment does not make a significant contribution to the intracellular storage of

Interaction of the ALR and MNR2 genes: To determine the relative contri

ALR1, ALR2, and MNR2 genes to Mg homeostasis, we determined their combin

growth and Mg content (Figure 5). Several studies have suggested thatALR2 does

to Mg homeostasis in S288C-derived strains (DA COSTA et al. 2007; MACDIARMID a

1998; WACHEKet al. 2006). However,ALR1 inactivation had a less severe effect on

W303-derived strain (TRAN et al 2000) suggesting that ALR2 was functional in


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had no effect. However, combining the two mutations enhanced the alr1-assoc

defect, supporting a function for Alr2. In addition, whereas growth of the alr1 sing

enhanced by supplementation with excess Mg, supplementation had no effect on g

double mutant, consistent with a more severe block of Mg uptake in this strai

content of the strains was determined under the same conditions, both the a

mutations reduced Mg content (Figure 5B), but the double mutant displayed the lo

again indicating that both Alr proteins contribute to Mg uptake. In summary, these

that in the W303 genetic background, inactivation of both Alr proteins is requ

effectively block Mg uptake.

We then determined the effect of combining the alr1, alr2 and mnr2 muta

content. As previously observed (Table II), the mnr2 mutation increased Mg conte

replete conditions (Figure 5C). However, when the mnr2 mutation was combined

and alr2 mutations, its effect on Mg content was eliminated. Complete suppression

phenotype by the alr1/2 mutations was also observed when the effect of the mnr2

accentuated by Mg starvation (Figure 5D). The observation that theALR genes a

MNR2 is consistent with the dependence of intracellular Mg storage on access to

supply of Mg. Our data indicate that, even when supplied with excess Mg, alr1

accumulated approximately 20 nmol Mg/106

cells (Figure 5C) which as prev

represents the minimum Mg content of viable cells. For this reason, alr1 alr2 mut

unlikely to sequester significant quantities of Mg within intracellular stores which


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membrane might increase Mg influx, which could suppress phenotypes associated

of Alr1/2 protein activity. To determine if the overexpression of Mnr2 would m

protein, the myc-Mnr2 ORF was expressed from a galactose-inducible prom

subcellular location of the protein was determined using indirect immunofluoresc

6A). Although myc-Mnr2 was primarily associated with the vacuole membrane, a

was also detected at the periphery of the cells (white arrows), suggesting accum

plasma membrane. Although this peripheral staining might also indicate an ER

absence of a distinct signal at the nuclear membrane meant that the overall patte

consistent with a plasma membrane location.

To determine the phenotypic effect of this change, we assayed growth o

overexpressing alr1 alr2 mutant and control strains under non-permissive (stand

medium with 2% galactose), and permissive conditions (the same medium suppl

excess Mg) (Figure 6B). Either normal expression of Alr1 (using a low copy A

clone) or overexpression of myc-Mnr2 (GAL1-MNR2) restored growth of the mutan

non-permissive conditions. Suppression by Mnr2 was dependent on a high level of

the introduction of a low copy MNR2 genomic clone had no effect (Figure 6B, low

To quantify this effect, growth curves were performed by culturing the same set

standard synthetic medium with 4 mM Mg (Figure 6C). After 16 h incubation, the

complemented strains completed approximately 2.5 doublings, while the non-c

strain grew very little (0 25 doublings) In comparison the cell number of the sup


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We also examined the effect of Mnr2 overexpression on the Mg content of

mutant (Figure 6D). The low Mg phenotype of this mutant was fully comp

expression of Alr1. However, overexpressing myc-Mnr2 did not increase Mg cont

level seen in a non-complemented control strain. The implications of this observ

examined further in the discussion, but in general, the experiments shown

demonstrate that Mnr2 overexpression partially compensated for the loss of Alr prot


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DISCUSSION

Model for Mnr2 function in yeast: The data reported here add signific

understanding of fundamental processes of Mg homeostasis in eukaryotic cells, as

Figure 7. In part, this model proposes that in Mg-replete conditions, an

compartment (most probably the vacuole) provides a storage site for excess Mg. Co

this idea, the vacuolar Mg concentration is approximately four-fold higher than th

concentration in Mg-replete yeast cells (SIMM et al. 2007). The accumulation of exc

vacuole would require active transport to overcome the charge gradient generated b

activity. Although the transporter responsible has not been identified, several

identified Mg2+/H+ antiport activities in the vacuole membrane (BORRELLY

OKOROKOV et al. 1985). Mutations that inactivate V-ATPase activity (e.g. tfp1),

reduced total Mg content of yeast (Figure 4A) (EIDE et al. 2005), as would be exp

mutations inhibit proton-coupled Mg transport. However, it should be noted that th

phenotype of reduced Mg content may not represent a direct effect of a reductio

exchanger activity, as this phenotype could result from the inhibition of other proce

influence Mg accumulation in this compartment, such as polyphosphate synthesis (

1997; SIMM et al. 2007).

The model also proposes that vacuolar stores of Mg can be utilized to offset

deficiency. Previous work (BEELERet al. 1997) as well as our observations (Figure

h i i i l M f ild ll ff h f h f


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maintain the required concentration in other critical cellular compartments. The effe

mutation on Mg content, and the severe growth defect displayed by tfp1 cells trans

deficient conditions (Figure 4B), provides support for this view. In addition, se

evidence indicate that Mnr2 is required for the release of vacuolar Mg. Although an

strain accumulated more Mg than a wild-type strain during growth in Mg-replete co

increased content had no effect on the subsequent growth of the mutant un

conditions (Figure 3). The growth defect exhibited by the mnr2 mutant under the

suggests that an essential Mg-dependent compartment (such as the cytosol) was

The opposite effect of the mnr2 and alr1/alr2 mutations on Mg content (Figur

location of the Mnr2 protein (Figure 2), is consistent with Mnr2 supplying

compartment with Mg from vacuolar stores, rather than the external environm

evidence for a direct role of Mnr2 in Mg transport came from the observatio

overexpression appeared to relocate a fraction of the protein to the cell surfac

partially suppressing the growth defect of an alr1 alr2 mutant (Figure 6B, C).

experiment did not provide direct evidence for Mg transport by Mnr2, it did dem

Mnr2 could still function in the absence of the Alr proteins, suggesting that M

simply act as a regulatory or structural component of these proteins. This abili

distinguish Mnr2 from a class of yeast transporter orthologs that do not mediate sol

functioning instead as cell surface receptors. For example, the Rgt2 and Snf3

glucose receptors required for a signal transduction pathway that regulates gluco


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replete and deficient cells, and YFP-tagged Mnr2 accumulated in the vacuole

deficient cells (Figure 2, Figure 1S), indicating that Mnr2 is present in the co

under the appropriate conditions to directly regulate the Mg content of the vacuole.

Several other lines of evidence support general aspects of this model. First, the

proton-coupled secondary transport systems in the vacuole by the inactiva

suppressed the mnr2 phenotype of increased Mg accumulation (Figure 4A),

expected if Tfp1 is required for the initial accumulation of Mg in the vacuol

determines Mg content of the vacuole by regulating its release from this o

dependence of Mg storage on V-ATPase activity was also supported by the severe

tfp1 mutation on growth in Mg-free medium (Figure 4B), which indicates the ab

stores in this strain. Second, eliminating Mg uptake into mitochondria had no

severity of the mnr2 Mg-accumulation phenotype, indicating that the mnr2 mut

cause a general overaccumulation of Mg within all intracellular compartments.

function was dependent on the efficient accumulation of excess Mg; when a severe

uptake was imposed by the inactivation of both the Alr proteins (Figure 5), the hig

phenotype of the mnr2 mutation was suppressed. Together, these observations a

with Mnr2 functioning to directly mediate the release of Mg from a vacuolar

responsible for the storage of excess Mg.

Effect of Mnr2 overexpression on alr1 alr2 phenotypes: One somewhat surp

f hi k h l h h M 2 i i d h h f


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requirement of yeast cells (i.e., without improving the supply or redistribution of M

model is that Mnr2 might have increased the rate of Mg release from the vacuole,

Mg through this compartment, thus improving Mg supply to the cytosol. In su

model, we note that Mnr2 overexpression significantly increased its accumulation i

membrane (Figure 6A). Finally, it is possible that (as previously suggested), the

Mnr2 to the plasma membrane did enable a small increase in Mg uptake, thus

directly for the loss of the Alr proteins. This minor increase in Mg uptake may h

sufficient Mg to improve growth, but not enough to completely refill intracellul

restore normal cellular Mg content. In support of this model, we note that Mnr2 o

did not completely restore wild-type growth (Figure 6C), which together with

content, suggests that the suppressed strain was still Mg-deficient. However, the d

here do not yet allow us to definitively distinguish between these three models, and

required to determine if the Mnr2 protein can directly mediate Mg transport.

Mg homeostasis and elemental content of yeast: In addition to the effect

mutation on Mg, we also observed other changes in elemental content related to Mg

(Table II). In both wild-type and mnr2 strains, variation in Mg content was as

altered phosphate content. Previous reports have also noted a close relationship

supply and phosphate accumulation by yeast (BEELERet al. 1997; SIMM et al. 200

1975). The basis for this correlation is still unclear however, and further work i

understand the relationship In addition to this effect Mg supply also altered the ac


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cations via a Mg-inhibited, low affinity uptake system. The existence of such a syst

supported by several previous reports. For example, restriction of Mg supply

sensitivity of S. cerevisiae and S. pombe to manganese, nickel and cobalt (BLAC

1997; BLACKWELL et al. 1998; EITINGER et al. 2000; JOHO et al. 1991), consiste

cations competing with Mg for the same low-affinity transport system. Several

linked this low-affinity uptake system to the Alr1 and Alr2 proteins and their h

overexpression of either Alr1 or Alr2 conferred sensitivity to calcium, manganese

and nickel ions, and enhanced cobalt uptake (MACDIARMID and GARDNER1998), w

of an Alr1 homolog from S. pombe increased nickel and zinc tolerance (EITINGE

SARIKAYA et al. 2006). If fungal Alr homologs are capable of accumulating div

other than Mg, the availability of Mg may also influence divalent cation accu

changes in gene expression. Both the level ofALR1 mRNAand the stability of the

were reported to increase under Mg-deficient conditions (GRASCHOPF et al. 2

deficient cells, elevated Alr1 expression might further enhance divalent cation a

perhaps accounting for the enhanced sensitivity of Mg-starved yeast to ma

(BLACKWELL et al. 1997; BLACKWELL et al. 1998). Mg deficiency might also

regulate the activity of the Alr proteins. Although little is known about Alr1 r

activity of bacterial and yeast CorA proteins is coupled to Mg2+ availability via the

Mg-sensing domain located in the cytosol or mitochondrial matrix (PAYANDEH

SCHINDL et al. 2007), and a similar mechanism may operate for the Alr proteins.


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expression might be increased in mnr2 strains. A mechanism for this effect is sug

observation that Alr1 and Alr2 expression may be regulated by Mg supply (GRA

2001; WACHEK et al. 2006), which may in turn be determined by Mnr2 acti

regulation of the Alr systems might also explain the divalent cation sensitivity of

(Table II and Figure 1A). To test this model, we are currently studying the effect

and the mnr2 mutation on Alr1 expression and activity.

Vacuolar Mg storage in higher eukaryotes: One important implication of thi

homologs of Mnr2 in higher eukaryotes may also play a role in regulating intr

storage. Differentiated plant cells contain large vacuoles that may play an importa

storage of inorganic nutrients [reviewed in (MARTINOIA et al. 2007)], and tonopla

can mediate active transport of Mg (AMALOU et al. 1992; AMALOU et al. 1994), su

Mg could be sequestered in these organelles. Most plant genomes encode multiple

(LI et al. 2001; SCHOCKet al. 2000). The Arabidopsis plasma membrane protein A

al. 2001), chloroplast inner membrane protein AtMrs2-11 (DRUMMOND et al

mitochondrial protein AtMgt5 (CHEN et al. 2009) have all been shown to play impo

Mg homeostasis, but the function of many other members of this large gene fa

unknown. The identification of a functional homolog of Mnr2 in plants would fu

our understanding of Mg homeostasis in higher eukaryotes, as well as providing

useful tool to manipulate the nutrient content of economically important species.


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ACKNOWLEDGMENTS

This work was supported by a Research Award from the University of Misso

The authors wish to thank Amanda Bird, David Eide, Richard Gardner, and Anton G

supplying yeast strains and plasmids; David Eide, Bethany Zolman, Wendy

Christopher Wolin for comments on the MS; James O'Brien for assistance with AA

Salt for assistance with ICP-MS.

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ABBREVIATIONS

The abbreviations used are: Mg, Magnesium; MIT, metal ion transporter;

membrane; AAS, atomic absorption spectroscopy; ICP-MS, inductively-coupled

spectroscopy; DIC, differential interference contrast.


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and the indicated number of cells were applied to SC-ura plates with no metal ad

SC-ura plates with metal ions added as chloride salts. Plates were incubated for thre

photography. (B) Mg requirement of wild-type (DY1457) and mnr2(NP4) strain

LMM containing the indicated concentration of Mg were inoculated to an A595

incubated for 16 h. Values for final cell density are shown (means of three rep

SEM). Inset graph shows growth curve of DY1457 and NP4 in LMM + 3 M M

cellular Mg content of wild-type (DY1457) and mnr2(NP4) yeast strains. Yeast

grown in synthetic LMM containing the indicated concentration of Mg ions, and c

Mg assayed using AAS. Data points are the means of four independent replicates

and six independent replicates for NP4, +/- 1 SEM.

FIGURE 2. Mnr2 is a vacuolar membrane protein. BY4743 cells expressing YFP

low copy vector (YCpCit-MNR2) or transformed with an empty vector (pFL38,

grown for 6 h in LMM + 1 mM Mg (high Mg) or LMM + 1 M Mg (low Mg

treated with FM4-64 to visualize the vacuole membrane and images captu

appropriate filter set (FM4-64 or YFP). Signal overlap is shown (Merge). DIC

interference contrast.

FIGURE 3. The mnr2 mutation prevents access to intracellular Mg stores. Cult


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determination of cell density (A) and Mg content (B). Error bars indicate +

independent experiments).

FIGURE 4. Interaction of the MNR2, TFP1,LPE10 and MRS2 genes. (A) Effect

mrs2 and lpe10 mutations on Mg content. Wild-type (WT, DY1457), mnr2 (NP4),

and mnr2tfp1 (NP201) strains isogenic to DY1514 (DY1514 BG), or wild-type (W

lpe10 (lpe10!-1), mrs2 (mrs2-!2), mnr2lpe10 (NP107) and mnr2mrs2 (NP112) st

to DBY747 (DBY747 BG) were cultured in LMM + 1 mM or 1 M Mg for 1

content determined by AAS. Error bars indicate +/- 1 SEM (three replicates for DY

strains, five replicates for DBY747-derived strains). (B) Effect of the mnr2,tfp1, m

mutations on growth in Mg-free conditions. Strains described in A were cultured in

washed free of Mg, and used to inoculate aliquots of Mg-free LMM to an initia

After 16 h incubation, cell density was recorded. Error bars indicate +/- 1 SEM (thre

FIGURE 5. Interaction of the MNR2, ALR1 and ALR2 genes. (A, B) Cultures

(WT, DY1457), alr1 (NP10), alr2 (NP27), and alr1 alr2 (NP14) strains were grown

in YPD + 250 mM Mg, washed, and used to inoculate aliquots of LMM with the

concentration to an initial A595 of 0.01. After 16 h incubation, cell density of the

determined (A), and cell-associated Mg content measured via AAS (B). (C, D) Cu


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FIGURE 6. MNR2 overexpression in an alr1 alr2 mutant. (A) Subcellular d

overexpressed myc-Mnr2. A wild-type strain (DY1514) was transformed with YEp

(myc-Mnr2) or pFL38 (control). Cultures were grown to exponential phase in

galactose medium and processed for indirect immunofluorescence. White arrows

Mnr2 signal at the cell periphery. (B). Wild-type (WT, DY1457) andalr1 alr2 (

were transformed with vector control (pFL38), low copy ALR1 (YCpALR1), low

(YCpmycMNR2), orGAL1 promoter-driven MNR2 (YEpGmycMNR2) constructs.

grown in SD-ura medium with 100 mM Mg, and the indicated number of cells appl

plates + 2% galactose with a standard (4 mM) or high (50 mM) Mg concentration

incubated for 2 days before photography. (C, D) Strains described in panel A w

saturation in SC-ura supplemented with 100 mM Mg, washed twice with water

inoculate aliquots of SC-ura with 2% galactose (4 mM Mg) to an initial A595 of 0

growth, finalA595 was recorded (C), and Mg content was determined using AAS (D

D, error bars indicate +/- 1 SEM (four independent experiments).

FIGURE 7. Model for Mg homeostasis in yeast. Under Mg-replete conditions, Mg

cell via the Alr1 and Alr2 proteins to maintain the concentration of an essential cyto

([Mg2+

]). Excess cytosolic Mg is sequestered in the vacuole via an as yet unidentifie


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inhibits growth under Mg-deficient conditions by restricting the supply of Mg to the

compartment.


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FIGURE 1


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FIGURE 2


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FIGURE 3


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FIGURE 4


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FIGURE 5


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FIGURE 6


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FIGURE 7


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TABLE I. YEAST STRAINS

Strain Relevant genotype Full genotype

DY1514 Wild-type MATa/" ade2/+ ade6/+ can1-100oc/- his3-11,15/- le

1/- ura3-52/-

NP4 mnr2! MAT" ade6 can1-100oc his3-11,15 leu2-3,112

mnr2::KANR

NP10 alr1! MATa ade2 can1-100oc his3-11,15 leu2-3,112

alr1::HIS3

NP27 alr2! MATa can1-100ochis3-11,15 leu2-3,112 trp1-1 ura3

NP14 alr1! alr2! MATa ade6 can1-100oc his3-11,15 leu2-3,112

alr1::HIS3 alr2::TRP1

NP20 alr1! alr2! mnr2! MATa ade2 can1-100oc his3-11,15 leu2-3,112

alr1::HIS3 alr2::TRP1 mnr2::KANR

NP174 Wild-type MAT"ade2 can1-100ochis3-11,15 leu2-3,112 trp1-1

NP180 mnr2! MAT" ade2 can1-100oc his3-11,15 leu2-3,112

mnr2::SpHIS5

NP193 tfp1! MAT" ade2 can1-100oc his3-11,15 leu2-3,112

tfp1::KANR

NP201 mnr2! tfp1! MAT" ade2 can1-100oc his3-11,15 leu2-3,112

mnr2::SpHIS5 tfp1::KANR

DBY747 Wild-type MATa his3-!1 leu2-3,112 ura3-52 trp1-289

lpe10!-1 lpe10! MATa his3-!1 leu2-3,112 ura3-52 trp1-289 lpe10::U


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NP112 mnr2! mrs2! MATa his3-!1 leu2-3,112 ura3-52 trp1-289

mnr2::KANR

All strains were generated during this study except for DY1514 (obtained from Dr

DBY747, mrs2!-2, and lpe10!-1 (obtained from Dr. Anton Graschopf). All strains are isoge

except for those isogenic to DBY747 (lpe10!-1, mrs2!-2, NP103, NP107, and NP112).

Ele

Mg

PSKCa

Mn

Fe

Co

Zn

det

Un


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44

TABLEII.EL

EMENTALCONTENTOFWILD-TYPEANDmnr2STRAINS.

ement

WT(+Mg)

mnr2(+M

g)

Foldchange(+Mg)*

WT(-Mg)

mn

r2(-Mg)

Foldchange(-M

g)*

g

5

0.11+/-2.52

84.16+/-2.91

1.7

9.88+/-0.79

53.1

4+/-1.53

5.4

299.28+/-7.84

423.41+/-13.62

1.4

236.08+/-9.59

464.9

6+/-16.28

2.0

3

1.09+/-5.17

46.31+/-6.04

1.5

35.72+/-2.66

35.9

6+/-5.10

1.0

169.75+/-4.90

184.81+/-

5.08

1.1

73.40+/-3.75

169.44+/-5.47

2.3

a

5.35+/-0.37

6.58+/-0

.99

1.2

22.60+/-0.58

43.4

3+/-0.41

1.9

n

0.038+/-0.004

0.047+/-0

.001

1.2

0.107+/-0.006

0.189+/-0.019

1.8

0.071+/-0.009

0.068+/-0

.016

1.0

0.169+/-0.037

0.242+/-0.012

1.4

o

0.387+/-0.013

0.415+/-0

.015

1.1

0.499+/-0.082

0.991+/-0.060

2.0

1.46+/-0.08

1.89+/-0

.18

1.3

2.23+/-0.08

4.70+/-0.17

2.1

Wild-type(W

T,NP174)andmnr2(NP180)

strainsweregrownfor12hin

LMM

+1mM

Mg(+Mg)or1M

Mg(-Mg),andelementalcon

tent

terminedbyIC

P-MSanalysis,asdescribedinMaterialsandMethods.Data

arethemeanoffivereplicates

fromtwoindependentexperime

nts.

itsarenmol/1

06yeastcells+/-1SEM,except

cobalt(pmol/106yeastcells).O

nlyelementsforwhichreproduc

ibledatawereobtainedaresho

wn.

*Ratioofelementalcontent(mnr2/WTstrain

).

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MNR2 Regulates Intracellular Magnesium Storage in Saccharomyces Cerevisiae