Mg2+Transporting P-type ATPases of Salmonella typhimurium Wrong Way, Wrong Place Enzymesa MICHAEL E. MAGUIRE? MARSHALL D. SNAVELY, JONATHAN B. LEIZMAN, SUSAN G U M , DEEPSARAN BAGGA, TAO TAO, AND DEBORAH L. SMITH Department of Pharmacology School of Medicine Case Western Reserve University Cleveland, Ohio 441064965 In both eukaryotes and prokaryotes Mg2+is the most abundant divalent cation within cells.*s2 In contrast to that of the other common cations Na+, K+, and Ca2+, knowledge of the roles of intracellular Mg2+is very limited. This lack of information is primarily centered on two problems. First, technical methods for measuring Mg2+ within cells or Mg2+fluxes across cell membranes are extremely limited and relatively insensitive?-5 As important, however, is the general dogma that intracellular Mg2+ plays no important roles other than those as a membrane-stabilizing agent and as a cofactor with ATP in enzymatic reactions. Nevertheless, recent data show significant changes in intracellular free Mg2+ concentrations after a variety of physiological stimuli, and there is increasing evidence for active hormonal regulation of Mg2+ Consequently, the concept of Mg2+ as a passive cation is undergoing revision, and a regulatory role for Mg2+has been postulated by several a ~ t h o r s . ' ! ~ - l ~ Problem of TransmembraneM , +F l u

To understand the biological roles of intracellular Mg2+,its membrane transport processes must be understood. In addition, there are fundamental chemical reasons for investigating Mg2+.It is the most charge dense of all biologically relevant cations. The volume of the hydrated Mg2+ cation is threefold larger and the volume of its atomic ion is fourfold smaller than that of other common biological cations. Membrane transport systems face the problem that they must initially interact with the hydrated cation but transport the atomic ion.I5 With Na+, K+, and Ca2+,the cation presents as a hydrated sphere 25-30 times the volume of the atomic ion that is transported. Mg2+, however, presents as a hydrated cation over 350 times the volume of the transported atomic ion. Thus, Mg2+transport systems have a unique problem in recognizing, binding, and transporting the ion. It is therefore likely that Mg2+ transport systems will provide interesting, unusual, and possibly distinctive memaThis work was supported by U.S.Public Health Service grants GM39447 and HL18708. bAddress for correspondence: Dr. Michael E. Maguire, Department of Pharmacology, School of Medicine, 10900 Euclid Avenue, Case Western Reserve University, Cleveland, OH

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brane transport systems. Consequently, our laboratory has focused on characterization of transport systems capable of mediating Mg2+flux across membranes.

Experimental System The system of choice for these studies has been the prokaryote Salmonella typhimurium. First, known gene families of transport systems in prokaryotes exhibit significant homology to eukaryotic transport systems.IG2' Second, techniques for genetic and molecular manipulation in prokaryotes are much easier and better developed of those for mammalian cells. The primary route to isolate and study a Mg2+transport system in a mammalian cell would be through conventional biochemical purification, as no mutations are known in eukaryotes and no homology to other transport systems for use in molecular cloning has been determined. However, the technical limitations just noted make even biochemical purification problematic because of the lack of a sensitive and readily available transport assay with which to follow purification. The genetic systems available in prokaryotes obviate most of these problems. As in other cell types, Mg2+ is an essential nutrient for S. typhimurium, and in concentrations below approximately 100 p,M is limiting for growth. Thus, several years ago, this laboratory embarked on studies to isolate and characterize distinct Mg2+transport systems.

IDENTIFICATION AND CLONING OF S. typhimurium Mgz+ TRANSPORT SYSTEMS Previous studies in Escherichia coli had demonstrated that the primary Mg2+ influx system also transported Co2+ and that mutations in this system, designated corA, greatly decreased Mg2+ influx and gave a Co2+-resistant phenotype. Since S. typhimurium is a very closely related species, it seemed likely that the Mg2+transport systems of S. typhimurium were similar, if not identical, to those in E. coli. Thus we showed that Co2+ and Mg2+ uptake in S. typhimurium was mutually competitive; mutations giving Co2+ resistance (corA) mapped to 83.5 minutes on the S. typhimurium chromosome, exhibited decreased Mg2+ uptake, and had lost all Coz+ uptake.z2 Strains carrying mutations in corA exhibited a level of Mg2+ uptake that increased as the level of Mg2+ in the growth medium decreased, suggesting the presence of at least one additional Mg2+uptake system. Mutagenesis of a corA strain with diethylsulfate followed by selection for cells able to grow only at very high extracellular Mgz+ concentrations gave mutant strains that exhibited no detectable Mgz+ uptake and required concentrations of Mgz+ in the extracellular medium of greater than 50 mM for growth. Genetic analysis showed that such strains had acquired two mutations in addition to corA. The additional mutations were termed mgtA and mgtB which mapped at 98 and 80.5 minutes on the S. typhimurium chromosome, r e ~ p e c t i v e l y .All ~ ~ three loci were cloned using a plasmid library constructed from S. typhimurium chromosomal DNA.22.23Appropriate plasmids were identified by their ability to allow the Mg2+-dependentstrain to grow without Mgz+ supplementation of the growth medium. Complementing plasmids were separated into three classes by virtue of independent patterns of their restriction endonuclease digests. Any one of the three classes of plasmids could restore Mg2+ uptake to the Mgz+-dependentstrain with distinct kinetic characteristics. Subsequently, we constructed strains harboring directed mutations in one, two,

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or all three of the l ~ c i .The ~ ~constructed , ~ “triple-mutant” strain, carrying insertion mutations at all three loci, lacked Mg2+transport and required 100 mM Mg2+in the medium for growth. Likewise, strains harboring any two mutant and any one wild-type allele of these three loci could grow without Mg2+supplementation; each such strain had distinct Mg2+transport properties.” Both MgtA and MgtB are much less active transporters than is CorA. Expression of both systems is repressed by extracellular Mg2+,and significant transport can only be measured after growth in micromolar Mg2+concentrations (see below). All three systems mediate the influx of Ni2+ in addition to Mg2+. On the basis of these and additional data, S. typhimurium possesses three independent Mg2+ uptake systems (FIG.1) encoded by different chromosomal l o ~ i . ~ ~ - ~ It is relevant in this context to note that although the uptake of transition metals by MgtA and MgtB is relatively efficient, their uptake is not physiologically relevant. The requirement of S. typhirnuriurn and E. coli (indeed most gram-negative bacteria) for Co2+and Ni2+ is quite low. More importantly, at concentrations sufficiently high to be taken up by MgtA or MgtB, both cations are highly toxic to the cell and eventually cause cell death and lysis. Thus, the three systems described here are physiologically Mg2+transport systems.

FIGURE 1. Model of the transport systems of S. typhimurium. The gram-negative bacteria S. typhimurium and E. coli and likely all gram-negative bacteria possess two distinct classes of Mg2+transport systems. A constitutive system, termed CorA, is the dominant Mg2+transport system upon growth at moderate to high extracellular Mgzt concentrations. CorA mediates only Mg2+influx at moderate Mg2+concentrations, while it mediates Mg2+-Mg2+exchange at high Mg2+ concentrations. At low extracellular MgZf concentrations, uptake via CorA is apparently insufficient for the cells’ needs, and a P-type ATPase, termed Mgt, is expressed that mediates only Mg2+influx. The CorA system is composed of four unlinked genetic loci, corA, corB, corC, and corD. The corA gene product alone can mediate influx but requires interaction with the gene products of the corBCD loci to mediate efflux. There are two distinct Mgt P-type ATPase systems in S. typhimurium, termed MgtA and MgtB. It is not yet known if either requires a p-subunit as some P-type ATPases do (see text).

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MAGUIRE et al.: Mg2+ TRANSPORT SYSTEMS A

ATPase Phosphorylation Site

KdpB

. ... . ..... . ... . ... . .. . . . . . . .. . . . . . ... . ... . . . . . . . . . . . . . . . . . . ... . . . . . . . . ......... ..... PEMLP-MIVSSNLAKGAIAMSRRKVIVKRLNAIQNFGAMDVLCTDKTGTLT ............... .......... ....................................... PEGLP-IIVTVTLALGVLRMAKRKAIVRRLPSVETLGSVNVICSDKTGTLT . . . . . . . . . . ... ... . . . . ... . ... . . . . .. .. .. .. ....... . . .

NC MgtB PMRl

CaST

PTTIGGLLSASAVAGMSRMLGAN-VIATSGRAVEAAGDVDVLLLDKTGTIT

313

PVTLP-AVTTT-MAVGAAYLAKK-AIVQKLSAIESLAGVEILCSDKTGTLT

383

PEGLP-AVITTCLALGTRRMAKKNAIVRSLPSVETIGCTSVICSDKTGTLT

x

385 377 357

B.

“Hinge”Region

KdpB

T P E A K L A L I R Q Y Q A E G R L V A M T G D G T N D A P A L A ~ A D V A V A D L D 552

NC

FPQHKY~EILQQRGYLVAMTGDGVNDAPSLKKADTGIAVEG-SSD~S~DIVFLA 668

MgtB PMRl

CaST

. ... . . . . . ... .. . . ..... .. . . ..... .. . . ... . . . . . . . . ... . . . . . . .. . . . . .. ... . . ... .. . ..... . .. ... . . ... .. . . . . . . . . . . . . . TPLQKTRILQALQKNGHTVGF~DGINDAPALRDADVGISVDS-AADIRKESSDIILLE . ..... . ..... . ..... . ... . . ... . . ... . . ... . . . . . . . . . ... . ... . . TPEHKLNIVRALRKRGDWAMTGDGVNDAPALKLSDIGVSMGRIGTDVAKEASDMVLTD .. .. .. .. . .............. . . . ................................ EPSHKSKIVEFLQSFDEITAMTGDGVNDAPALKKAEIGIAMGS-GTAVAKTASEMVLAD

684 715 736

FIGURE 2. Sequence homology in selected regions of MgtB with other P-type ATPases. Alignments were performed as previously described.26Alignments are shown for two regions highly conserved in all known P-type ATPases. In the regions shown, similarity between MgtB and other P-type ATPases is high regardless of the origin of the ATPase. In other regions, however, MgtB is far less similar to prokaryotic P-type ATPases and is more similar to eukaryotic P-type ATPases, especially the Ca’+-ATPases of mammalian sarcoplasmic and endoplasmic reticulum.

CHARACTERIZATION OF MgtB Genetic analysis and gene expression data indicated that the cloned mgtB locus contained two genes, mgtC and mgtB.26The mgtC gene expresses the 22.5 kD MgtC protein, while the mgtB genes encodes the 102-kD MgtB protein. We previously hypothesized that MgtC is a subunit of the MgtB transport system, and experiments are in progress to determine this. Sequence homology searches indicated that the MgtC protein lacks homology to any known protein. However, the MgtB protein shows clear homology to the ion-motive P-type A T P ~ S ~ S exemplified , ~ ’ , ~ ~ by the mammalian Ca2+-and Na+,K+-ATPases.Although several prokaryotic P-type ATFases have been d e ~ c r i b e d , ~ ~surprisingly, J ~ + * ~ . ~ ~MgtB is only weakly homologous to these enzymes, showing 12-18% identity and an additional 10-20% similarity via conservative substitutions. In great contrast, MgtB is much more similar to sarcoplasmic reticulum Ca2+-ATPasesfrom mammalian skeletal showing about 25% identity and an additional 25% similarity. A partial alignment of MgtB with several other P-type ATPases is shown in FIGURE2 for the area around both the crucial phosphorylated aspartyl residue and the so-called “hinge” r e g i ~ n . * ~The . ~ ’ ~se~~ quence of the cloned mgtA locus indicates that MgtA is also a P-type ATPase, again highly similar to eukaryotic P-type ATPases (M. D. Snavely, S. Gura, and M. E. Maguire, unpublished data). Because of this high homology of MgtA and MgtB to eukaryotic but not prokaryotic P-type ATPases, we have given them the sobriquet of the “Wrong Place” enzymes. When the presumed evolution of the P-type ATPase gene family is analyzed, the resulting dendrogram (evolutionary tree) gives the obviously fallacious result that the prokaryotic S. fyphimun’um enzyme and the sarcoplasmic reticular Ca2+-ATPases

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2.0 eA

.-c

.f 1.8

s

LT

+ m 1.6 I

N

(Y 0

c.

c 0

2

1.4

Q)

n 1.2

c 1

1

I

I

I

0

10

20

30

Minutes FIGURE 3. MgtA and MgtB cannot mediate Mg2+efflux. Cells were loaded with 28Mg2+,and efflux was measured as previously d e s ~ r i b e d ? ~EfRw + ~ ~ . was ~ ~ measured in strains of S. typhimurium in which one of the three Mg2+ transport systems had been inactivated by transposon insertion. Experiments using strains in which two of the three Mgzt transport systems had been inactivated by insertional mutagenesis give identical results. The data show that only the CorA transport system is capable of mediating Mg2+efflux.

evolved at essentially the same time. A more reasonable interpretation of this sequence homology is that the Mg2+-transporting MgtB P-type ATPase begins to define a new, major branch or subfamily among the P-type ATPases. In addition, the great similarity to the Ca2+-ATF'ases allows the MgZ+-ATPasesto be studied not only as Mg2+transporters but also as a model for divalent cation-motive ATPases. This is of importance because genetic and molecular manipulation is significantly easier and more rapid in prokaryotes than in mammalian cells. Given the large electrochemical gradient for Mg2+ in S. typhimurium (-200 mV negative inside and roughly equal free Mg2+ concentrations in the cytosol and extracellular medium), the obvious presumption is that these Mgz+-transporting ATPases mediate Mg2+efflux, using the energy of ATP to provide the energy for the expulsion of Mg2+from the cell. However, the data shown in FIGURE 3 indicate that neither MgtA nor MgtB is capable of mediating Mg2+efflux. When cells are loaded with aMg2+ and the rate of efflux determined, wild-type cells growing aerobically of 10 minutes. Cells carrying deletions with glucose as a carbon source exhibit a of either or both of the mgk4 and mgtB loci show no change whatsoever in rate of efflux. In contrast, when the corA gene is deleted,z~z4~33 efflux of Mg2+is completely abolished regardless of the presence or absence of MgtA or MgB. Cells growing aerobically in glucose have a membrane potential of up to -200 mV negative inside; introduction of an unc mutation renders the FIFOATPase nonfunctional, and thus the energy inherent in ATP and the proton gradient cannot be interconverted. We

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have preliminary data that when such cells are treated with a protonophore to dissipate the proton gradient across the cell membrane, Mg2+ influx via MgtA and MgtB is unaffected, suggesting that the proton motive force is not necessary for their function. These results indicate that MgtA and MgtB are P-type ATPases that mediate only the influx of Mg2+, transporting Mg2+ down its electrochemical gradient. It might be argued that MgtA and MgtB use Mgz+ only as a counter-ion, to balance the efflux of another (unknown) ion against its electrochemical gradient. This seems quite unlikely. First, the cell already possesses numerous transport systems for the monovalent cations and would therefore be unlikely to use Mgz+as a counter-ion for an additional system. Ca2+might be a candidate for the counter-ion except that it not only is not transported by MgtA or MgtB but also is a completely impotent inhibitor of Mgz+influx. The K,,, of Mg2+for MgtB is 6 kM, whereas 30 mM Ca2+,a concentration over three orders of magnitude greater, completely fails to inhibit Mgz+ uptake (FIG. 4). If MgtB were an ATPase mediating Mgzf/Caz+ exchange, Ca2+ would be expected to inhibit Mg2+ influx, albeit perhaps poorly. Furthermore, even if one postulated a counter-ion such as H+ or Ca2+and whether or not Mg2+ influx turns out to be electrogenic or electroneutral, the use of the energy of ATP to transport Mgz+ down its electrochemical gradient is simply unnecessary energetically. Although it seems likely that some environmental growth condition exists for S. typhimunum that would require the expenditure of the energy of ATP for Mg2+influx, these transport systems function extremely well even under growth conditions in which a huge electrochemical gradient for Mg2+influx already exists. Therefore, we have also termed MgtA and MgtB the “Wrong Way” transport systems.

-

100

c

c 0)

2 ao

-a Q)

E

r” *

60

40

n Q)

A

Q

20

c.

n

3

0 0 0.001

0.01

0.1

1 .o

10

[Cation] (mM1 FIGURE 4. Divalent cation inhibition of Mg2+ uptake by MgtB. The uptake of 28Mg2+was measured at an extracellular Mg2+ concentration of 15 p M in the absence or presence of the indicated concentrations of other divalent cations. The K, of Mg2+ for influx is 5-10 pM. The dotted line for Mgz+ shows a calculated curve for inhibition of transport by nonradioactive Mgz+ for comparison.

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PRELIMINARY TOPOLOGY STUDIES OF MgtB

The hydropathy plots of the eukaryotic P-type ATPases and of MgtA and MgtB are all quite similar, suggesting that they share the same membrane topology. Biochemical studies on several different P-type ATPases indicate that both the NH2and COOH-termini are cytosolic. This requires an even number of transmembrane segments to exist. Two distinct models of the membrane topology of P-type ATPases have been suggested based on the hydropathy plots that postulate either 8 or 10 transmembrane sequences. With the 10-segment model as a reference, the cartoon in FIGURE 5 contrasts the two models and indicates where each of the 10 membrane

Models of Membrane Topology

FIGURE 5. Topology models for the P-type ATPases. Two major models have been proposed in the literature for the membrane topology of P-type ATPases in which either 8 or 10 transmembrane sequences or loops are proposed. The cartoon shows the relative position (not to scale) of the membrane loops for the 10-segment model and their relative position in the 8-segment model. Basically, the 8-segment model combines loops 5 and 6 of the 10-segment model and omits the putative loop 10 of that model. One obvious result is that the orientation of the loops labeled 7,8, and 9 is that their relative orientation is inverted in the two models. Using the 10-segment model for reference, epitope tags have been inserted at the COOH-terminus and between loops 8-9 or 9-10 (see text).

segments would be positioned in the 8-segment model. Because of the ease of genetic manipulation in prokaryotes and the high similarity between MgtB and eukaryotic P-type ATPases, we have begun topological studies to decide between these two topological models. With PCR mutagenesis, two different epitope tags have been inserted into the MgtB sequence. The first utilizes the 9 amino acid, very acidic HA-1 epitope from influenza virus. This epitope has been inserted as a COOH-terminal extension and is therefore presumably cytosolic. The second epitope insertion, also acidic, is 11 amino acids in length, derived from the c-myc oncogene, and has been inserted in two

MAGUIRE el al.: Mg2+ TRANSPORT SYSTEMS

25 1

different locations, either between putative membrane loops 8 and 9 or between loops 9 and 10. In addition, two additional mutants have been constructed which carry the HA-1 epitope at the COOH-terminus and a c-myc epitope between loops 8 and 9 or 9 and 10. The mutations were cloned into a pBR-based plasmid and transduced into the “triple mutant” strain in which all three Mg2+transport loci have been inactivated. Introduction of a wild-type allele ofmgtB or of any of the constructs containing one or both epitope tags restored the ability of the “triple mutant” to grow without Mg2+ supplementation of the growth medium and restored cation uptake. Therefore, insertion of one or both epitopes in any combination allows expression of a functional transport protein. This result indicates that the inserted amino acids did not disrupt either membrane insertion or the topology of MgtB, as such disruption would be unlikely to result in a still functional transport system. The corollary to this interpretation is that the locations chosen for these highly charged epitope insertions are not within a membrane segment, but rather between membrane segments. It would be highly unlikely that an insertion carrying a high negative charge could form a membrane loop, much less a functional membrane segment. Therefore, the data suggest that the c-myc epitope insertions define the segments between membrane loops 8-9 and 9-10 if the 10-membrane segment model is correct or the insertions define the segment between loop 7-8 or create a cytosolic insertion if the 8-membrane loop model is correct. Currently, protocols are being developed to determine the exact topology of the epitopes by immunofluorescence. In addition, several unique protease sites can be predicted from the MgtB sequence that will give distinct results depending on which model is correct. REGULATION OF MgtB EXPRESSION Both mgtA and mgtB are highly regulated genes. Under laboratory growth conditions, the dominant Mgz+ transport system is CorA, accounting for 99+% of the total Mg2+ uptake. However, as the Mg2+ concentration in the medium is lowered, CorA becomes less efficient, and expression of both MgtA and MgtB increase^.^^^^^^^^ At concentrations of extracellular Mg2+ below 10 pM, MgtB becomes the dominant Mg2+ transport system.35 This alteration in expression upon change in extracellular Mg2+led us to investigate the regulation of the three Mg2+ transport loci. In-frame protein fusions of the reporter gene IacZ were constructed for c o d , mgvl, and mgtB. The fusions were made within the coding regions of the CorA, MgtA, and MgtB proteins. Thus, changes in transcription at each locus could be measured by determination of P-galactosidase levels. Using such constructs, we showed that mgvl and mgtB are highly regulated genes, whereas corA is apparently expressed constitutively. For example, mgtB expression is increased by the lack of oxygen, by the addition of nonfermentable carbon sources such as glycerol, and by decreased extracellular Mg2+.In addition, millimolar extracellular Ca2+ and Mn2+ concentrations block part of the increase in mgtB transcription caused by low extracellular Mg2+concentrations. The degree of oxygen and carbon source regulation of mgtB is generally three- to sixfold. However, Mg2+regulation of transcription, especially of mgtB, is of much greater magnitude (FIG.6). As extracellular Mg2+ concentration is lowered from 10 mM to 10 pM, c o d transcription remains unchanged, while that of mgtA and mgtB increases in parallel about 10-fold. As the concentration of Mg2+in the growth medium is lowered further, corA transcription remains unaffected and mgtA transcription increases an additional fourfold, for a total increase of about 40-fold. In sharp contrast, transcription of mgtB increases several hundred-fold below 10 pM extracellular Mg2+, for a total increase in

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FIGURE 6. Effect of extracellular MgZCon MgtB transcription. Promoter fusions of the lacZ gene to each of the three Mgz+ transport genes were made using the transposable Mu dJ element.35Transcription of the target gene is then measured by assay of p-galactosidase. In the experiment shown, cells carrying the appropriate fusion construct were grown overnight, washed in medium lacking added Mg2+, and then inoculated into medium containing the indicated Mg2+concentration. Transcription was measured after 4 hours of further incubation. The fold increase in transcription was calculated by dividing the activity obtained after growth in each Mg2+concentration by the activity in cells grown with 10mM Mgz+.The basal activity of p-galactosidase was 0.5-2 units for each of the three fusions.

transcription of about one thousand-fold. Furthermore, the ability of 1mM Ca2+to block this increase in mgtB transcription is evident only at extracellular Mg2+ concentrations greater than 10 kM. Below 10 FM of extracellular Mg2+, Ca2+ is unable to affect mgtB transcription. These results, indicative of a very complex regulation of Mg2+transport, suggest that Mg2+homeostasis is of great importance to the cell. It might be argued that the transcriptional results do not completely reflect the physiological situation as they measure only transcription and further measure transcription from a gene carried on a multicopy plasmid. However, the amount of Mg2+ uptake, a reflection of functional protein inserted in the membrane, is also greatly increased. We measured the induction of Mg2+uptake using strains carrying either the chromosomal wild-type mgtB gene, a plasmid carrying a wild-type mgtB allele, or a plasmid carrying a mutant mgtB allele containing either or both of the epitope tags just discussed. In all cases, expression of a functional MgtB transport system is greatly increased by growth of the strain in medium containing a low Mg2+ concentration. Furthermore, the ability of Mgz+ to regulate expression is retained both for plasmid-borne mgtB and for mgtB alleles containing the epitope tags. Surprisingly, the magnitude of the expression is extremely high. Overexpression of proteins in general and membrane proteins in particular often results in formation of inclusion bodies and degraded proteins and may be toxic to the cells. Functional expression of MgtB, however, can clearly be increased over 1,000-fold (FIG.7). When a strain carrying mgtE as the only functional Mgz+transporter is grown in millimolar

MAGUIRE et af.: Mgz+ TRANSPORT SYSTEMS

253

Mg2+,the amount of MgtB expressed is barely detectable by our transport assay. We have seen increases in expression of over 3,000-fold upon incubation in medium lacking added Mg2+.After resuspension of cells in medium lacking added Mg2+,the expression of mgtB exhibits a lag period before increasing and, under some conditions, is still increasing at 24 hours, even though the cells are essentially vegetative and not growing because of the lack of Mg2+.That this huge increase in expression may have been of physiological importance is shown by a similarly large increase in mgtB expression about 4 hours after a virulent strain of S. typhimunum invades a mammalian epithelial In addition, it is obvious that this great increase in expression of a functional protein coupled with the epitope tag will be of use for eventual purification of these Mg2+-transportingP-type ATPases. Thus, in summary, S.typhimurium and likely most gram-negative bacteria possess two Mgz+-transportingP-type ATPases, MgtA and MgtB. These ATPases are highly homologous to eukaryotic P-type ATPases but much less similar to other currently known prokaryotic ATPases. Both MgtA and MgtB mediate the influx of Mg2+with rather than against the Mg2+electrochemical gradient. Both systems, but especially MgtB, are highly regulated. Growth of cells in a low extracellular Mg2+concentration induces at least a thousand-fold increase in the expression of a functional MgtB transporter. These results indicate that the S. typhimun’um Mg2+ transport systems are a powerful model to study not only Mg2+ transport, but also the structurefunction of P-type ATPases in general.

0 P) Y

no added Mg2+

m

CI

3

1000

.-c Q v)

m

2

-0c -0 0

500

U

0 0

3

6

9

12

15

Hours in Growth Medium FIGURE 7. Time course of the increase in Mgz+ influx at various extracellular Mg2+ concentrations. The MgtB gene, cloned on a pBR-based plasmid, was introduced into the “triple mutant” S. typhimun‘um strain (see text). The resulting strain regained the ability to grow o n medium containing n o added Mg2+. The strain was grown in N-minimal medium overnight, washed in medium without Mg2+, and inoculated at a density of approximately 0.7 x lo8 cells/ml in medium containing the indicated Mg2+concentrations. 6RNi2+was used as a Mgz+ surrogate to measure uptake via MgtB, as previously d e s ~ r i b e d . Uptake ~ ~ . ~ ~ was measured in N-minimal medium for 30 minutes over which period uptake is completely linear. T h e fold increase on the ordinate is probably an underestimate, as uptake at t = 0 is extremely low, e.g., 5&100 cpm over a background of 500 cpm. In various experiments, uptakes of as much a s 300,000 cpm of 63Ni2+ can be measured in medium without added Mgz+. Under these conditions, the cells remain viable but do not grow because of the lack of Mgz+.

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ACKNOWLEDGMENT We thank Dr. Ronald Smith for critical discussions of the data and for his generous assistance with the figures.

REFERENCES

1. REINHART,R. A. 1988. Magnesium metabolism. A review with special reference to the relationship between intracellular content and serum levels. Arch. Intern. Med. 1 4 8 2415-2420. 2. ROTEVATN, S., H. SARHEIM & E. MURPHY. 1991. Intracellular free magnesium concentration: Relevance to cardiovascular medicine. Acta Physiol. Scand. 142 (Suppl. 599): 125133. 3. LONDON,R. E. 1991. Methods for measurement of intracellular magnesium: NMR and fluorescence. Annu. Rev. Physiol. 53: 241-258. 4. ROTEVATN, S., E. MURPHY, L. A. LEVY,B. RAJU,M. LIEBERMAN & R. E. LONDON.1989. Cytosolic free magnesium concentration in cultured chick heart cells. Am. J. Physiol. 257: C1414146. 5. MURPHY,E., C. C. FREUDENRICH & M. LIEBERMAN. 1991. Cellular magnesium and Na/Mg exchange in heart cells. Annu. Rev. Physiol. 53: 273-287. 6. GRUBBS,R. D. 1991. Effect of epidermal growth factor on magnesium homeostasis in BC3H1 myocytes. Am. J. Physiol. Cell Physiol. 260 C115841164. 7. ERDOS,J. J. & M. E. MAGUIRE. 1983. Hormone-sensitive magnesium transport in murine S49 lymphoma cells: Characterization and specificityfor magnesium. J. Physiol. 337: 351371. 8. QUAMME, G. A. 1989. Control of magnesium transport in the thick ascending limb. Am. J. Physiol. 256 F197-F210. 9. WALKER,G. M. & J. H. DUFFUS.1983. Magnesium as the fundamental regulator of the cell cycle. Magnesium 2: 1-16. 10. MAGUIRE,M. E. 1984. Hormone-sensitive magnesium transport and magnesium modulation of hormone response. Trends Pharmacol. Sci. 5: 73-77. 11. MAGUIRE,M. E. 1990. Magnesium: A regulated and regulatory cation. Metal Ions Biol. 2 6 135-153. 12. GRUBBS, R. D. 1990. Hormonal regulation of magnesium homeostasis in cultured mammalian cells. Metal Ions Biol. 2 6 177-192. 13. SEELIG,M. 1989. Cardiovascular consequences of magnesium deficiency and loss: Pathogenesis, prevalence and manifestations-Magnesium and chloride loss in refractory potassium repletion. Am. J. Cardiol. gj: 4G-21G. 14. KASS,E. H. 1989. Magnesium and the pathogenesis of toxic shock syndrome. Rev. Infect. Dis. 11(Suppl): S167-S175. 15. HILLE,B. 1992. Ionic Channels of Excitable Membranes, 2nd Ed.: 292-314, 355-361. Sinauer. Sunderland, MA. 16. HESSE,J. E., L. WIECZOREK, K. ALTENDORF, A. S. REICIN,E. DORUS& W. EPSTEIN. 1984. Sequence homology between two membrane transport ATPases, the Kdp-ATPase of Escherichia coli and the CazC-ATPaseof sarcoplasmic reticulum. Proc. Natl. Acad. Sci. USA 81: 47464750. W. 1990. Bacterial transport ATPases. In The Bacteria, Vol. XII: Bacterial 17. EPSTEIN, Energetics. T. A. Krulwich, ed.: 87-110. Academic Press. New York. 18. HENDERSON, P. J. F. & M. C. J. MAIDEN.1990. Homologous sugar transport proteins in Escherichia coli and their relatives in both prokaryotes and eukaryotes. Philos. Trans. R. SOC.Lond. [Biol.] 3 2 6 391-410. 19. BLIGHT, M. A. & I. B. HOLLAND.1990. Structure and function of haemolysin B, P-glycoprotein and other members of a novel family of membrane translocators. Mol. Microbiol. 4: 873-880. 20. AMES,G. F.-L., C. S. MIMURA& V. SHYAMALA.1990. Bacterial periplasmic permeases

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21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.

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belong to a family of transport proteins operating from Escherichia coli to human: Tratfic ATPases. FEMS Microbiol. Rev. 75: 429446. MALONEY, P. C. 1990. Microbes and membrane biology. FEMS Microbiol. Rev. 87: 91102. C. G. MILLER& M. E. MAGUIRE.1986. Magnesium HMIEL,S. P., M. D. SNAVELY, transport in Salmonella typhimun'um: Characterization of magnesium influx and cloning of a transport gene. J. Bacteriol. 168: 14441450. J. B. FLORER, M. E. MAGUIRE & C. G. MILLER.1989. HMIEL,S. P., M. D. SNAVELY, Magnesium transport in Salmonella typhimun'um: Genetic characterization and cloning of three magnesium transport loci. J. Bacteriol. 171: 47424751. 1989. Magnesium SNAVELY, M. D., J. B. FLORER,C. G. MILLER& M. E. MAGUIRE. transport in Salmonella typhimun'um: ZXMgZ+ transport by the CorA, MgtA, and MgtB systems. J. Bacteriol. 171: 4761-4766. SNAVELY, M. D., J. B. FLORER,C. G. MILLER& M. E. MAGUIRE.1989. Magnesium transport in Salmonella fyphimun'um: Expression of cloned genes for three distinct Mg2+transport systems. J. Bacteriol. 171: 47524760, M. D., C. G . MILLER & M. E. MAGUIRE. 1991.The mgtB Mgz+transport locus of SNAVELY, Salmonella fyphimun'um encodes a P-type ATPase. J. Biol. Chem. 2 6 6 815-823. 1987. Ion motive ATPases. 1. Ubiquity, properties, and PEDERSON, P. L. & E. CARAFOLI. significance to cell function. TIBS 1 2 146-150. P. L. & E. CARAFOLI. 1987. Ion motive ATPases. 11. Energy coupling and work PELIERSON, output. TIBS 12: 186-189. E. D. LITWACK & W. EPSTEIN.1989. Wide distribution of homoloes WALDERHAUG, M. 0.. of Escherichia coli Kdp K+-ATPase among gram-negative bacteria. J. Bacteriol. 171: 1192-1195. & P. FORST.1987. Cloning of the K+-ATPase of Streptococcus SOLIOZ, M., S. MATHEWS faecalis. Structural and evolutionary implications of its homology to the KdpB protein of Escherichia coli. J. Biol. Chem. 262: 7358-7362. & D. H. MACLENNAN. 1986. Two Ca" ATPase BRANDL, C. J., N. M. GREEN,B. KORCZAK genes: Homologies and mechanistic implications of deduced amino acid sequences. Cell 44.597-607. S ~ R R A NR. O . 1988. Structure and function of proton translocating ATPase in plasma membranes of plants and fungi. Biochim. Biophys. Acta 947: 1-28. GIBSON,M. M., D. A. BAGGA,C. G. MILLER& M. E. MAGUIRE.1991. Magnesium transport in Salmonella typhimunurn: The influence of new mutations conferring Co2+ resistance on the CorA Mg2+transport system. Mol. Microbiol. 5: 2753-2762. F. G., J. W. FOSTER,M. E. MAGUIRE & B. B. FINLAY. 1992. Characterization of PORTILLO, the microenvironment of Salmonella typhimun'um-containing vacuoles within MDCK epithelial cells. Molec. Microbiol. 6 in press. T. T. CHEUNG, C. G. MILLER & M. E. MAGUIRE. 1991. SNAVELY. M. D., S. A. GRAVINA, Magnesium transport in Salmonella typhimurium: Regulation of mgtA and mgtB expression. J. Biol. Chem. 266: 824-829. S. P. HMIEL& M. E. MAGUIRE.1989. Magnesium GRUBBS,R. D.. M. D. SNAVELY, transport in eukaryotic and prokaryotic cells using magnesium-28 ion. Methods Enzymol. 173: 546-563.

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DISCUSSION JACK KAPLAN(University of Pennsylvania, Philadelphia, PA): A m I right in thinking that the only evidence that your system is a P-type ATPase (or ion pump) is based on sequence homology? Phosphorylation and stoichiometric transport/ ATPase determinations have not yet been made. MICHAELMAGUIRE:Correct. However, the extremely high sequence homology

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ANNALS NEW YORK ACADEMY OF SCIENCES

( 90% at the phosphotylation and ATP binding sites) makes it highly unlikely that this system does not function as an ATPase. The high level of functional expression that I showed now makes such experiments possible. PETERPEDERSEN(Johns Hopkins University, Baltimore, MD): Perhaps I missed something, but if your ATPase catalyzes the influx of Mg2+in the “wrong” direction against the membrane potential, does this not indicate that another positive charge must be transported out? MICHAELMAGUIRE:The level of transport that we induce suggests that the transport is not electrogenic and therefore further suggests that there is a counterion transported out of the cell. Regardless, our data clearly show that Mg2+influx is the system’s physiological function. Furthermore, the complete lack of Ca2+inhibition strongly suggests that CA2+is not the postulated counter-ion. N. GREEN(NIMR, London, UK): The putative proton pump of Leishmania has a polar insert in the normally short M5 M6junction. If you insert one of your epitopes in this position, it could provide a crucial distinction between the 8 and 10 helix models. Have you tried this? MICHAELMAGUIRE:No we haven’t, but it is an obvious experiment in which to distinguish the 10 from the 8 membrane segment model. N. GREEN:By analogy with almost all the other pumps, is it not Mgz+ that is the counter-ion? What is missing is the principal ion that normally activates phosphorylation by ATP. This is a purely formal distinction, and it does not imply that the Mgz is not the physiologically important ion. In this respect, MgtB resembles Escherichia coli KdpB, for which K+ also behaves as a counter-ion. Formally, you are correct, and it is certainly possible that MICHAELMAGUIRE: the cell has simply “appropriated” a P-type ATPase that mediated efflux of an ion and now uses it to mediate Mg2+influx. Physiologically, as you note, MgtB is clearly functioning as a Mg2+influx system. ERNESTO CARAFOLI(ETH, Zurich, Switzerland):As you know, it has been very difficult to produce Mgz+ ligands, that is, ligands that would prefer Mg2+over Ca2+. This may be a premature question, but do you have any results on mutants that would tell you something about the Mg2+binding site? MICHAELMAGUIRE:No we haven’t, although the ability to do this (as has been done with the Kdp system) is one of the beauties of a good genetic system. We do plan to ask this question.

Mg2+ transporting P-type ATPases of Salmonella typhimurium. Wrong way, wrong place enzymes.

Mg2+Transporting P-type ATPases of Salmonella typhimurium Wrong Way, Wrong Place Enzymesa MICHAEL E. MAGUIRE? MARSHALL D. SNAVELY, JONATHAN B. LEIZMAN...
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