Bacterial homologs of eukaryotic membrane proteins: the 2-TM-GxN family of Mg²⁺ transporters (Review)

Abstract

Magnesium is essential for all forms of life. It is the cofactor for many enzymes and plays a key role in many biological processes. Thus, the acquisition of Mg²⁺ is crucial for cell survival. The best characterized Mg²⁺ transporters to date belong to the 2-TM-GxN type family of transporters. The name indicates the two C-terminal transmembrane (TM) domains and a conserved GxN motif present in all members of this family towards the C-terminal end of TM1. In most members of the family, this conserved motif is generally YGMNF. The prototypical member of this family is CorA. Other characterized members of this family include Mrs2p, Alr, Mnr, AtMGT and ZntB. CorA is widely distributed throughout the prokaryotic world. It is the primary Mg²⁺ uptake system in most bacteria and many Archaea. A homolog, Mrs2p, is a eukaryotic mitochondrial Mg²⁺ channel. The Mrs2p related AtMGT transporters are found in plants and other eukaryotes. Alr1p and Mnr are Mg²⁺ transporters found in the plasma membrane of many fungi. ZntB is a bacterial member of the 2-TM-GxN family but mediates efflux of Zn²⁺ instead of influx of Mg²⁺. The recent crystal structure of a bacterial CorA shows that the structure of this family is unlike that of any other class of transporter or channel currently known.

• Magnesium
• magnesium transport
• CorA
• zinc
• protein structure

Mg^2 + transport via the 2-TM-GxN family

Prokaryotes

Mg^2 + transport by a 2-TM-GxN family member has been best studied in bacteria. Webb first showed Mg^2 + accumulation in gram negative bacteria during growth [1]. Mg^2 + transport was further characterized by the laboratories of Simon Silver and Eugene Kennedy in the 1970s. In Escherichia coli, ²⁸Mg^2 + uptake was shown to be inhibited by Co^2 +. The same system apparently also transported Co^2 +[2], [3]. Because Co^2 + accumulation is toxic, Co^2 + resistant mutants could be selected, thus allowing for the selection of mutants defective for Mg^2 + uptake. The corA (cobalt resistance) gene was first identified via complementation of a resistant mutant with a recombinant plasmid carrying the corA gene from Salmonella enterica serovar Typhimurium [4]. In the late 1980s, the kinetic properties of Mg^2 + transport in E. coli and Salmonella enterica serovar Typhimurium were characterized in Michael Maguire's laboratory [5], [6]. CorA was shown to mediate influx of Mg^2 +, Ni^2 +, and Co^2 +. Cation hexaammines are potent and selective inhibitors of CorA transport [7]. Smith et al. [8] showed that CorA is widely distributed in gram negative bacteria. Subsequent genomic sequencing has indicated that CorA is present in over half of all Bacteria and Archaea [9], [10]. The remainder appear to express another Mg^2 + transporter, MgtE, homologous to the SLC41A family in eukaryotes [11–14].

CorA family members are essentially two-domain proteins, with a large soluble cytoplasmic N-terminal domain and a small C-terminal membrane domain [15], [16]. The functional transporter is a homo-oligomer [16]. Crystal structures of Thermatoga maritima CorA (see below) were recently described and show that CorA is a homopentamer [17–19]. These are the first structures not only of a member of the 2-TM GxN family but of any divalent cation channel.

A CorA homolog, ZntB was first characterized in S. Typhimurium [20]. The ZntB family typically shares 20–30% sequence identity with CorA. Unlike resistance to Co^2 + in corA mutants, mutations in zntB confer Zn^2 + and Cd^2 + resistance. Rather than mediating cation influx, ZntB mediates Zn^2 + efflux.

Eukaryotes

Work on eukaryotic members of the 2-TM GxN family has also developed over the last 20 years. The importance of Mrs2p in Group II RNA splicing was identified before its function as a Mg^2 + transporter in the mitochondria was discovered [21]. Mrs2p is required for the excision of Group II introns from certain yeast mitochondrial transcripts in vivo. Mutation of the Mrs2 gene also leads to a respiratory deficiency even in yeast strains that do not have introns. Several suppressor mutants were isolated which rescued either the splicing defect or respiratory deficiency of the Mrs2 mutant [22]. Subsequently, Rim2/Mrs12, a nuclear gene, was found to rescue the respiratory deficiency [23]. This protein is also a member of the mitochondrial carrier family; thus suggesting a link between transport and suppression of the respiratory deficiency. CorA was found to functionally substitute for Mrs2p in yeast implying that Mrs2p could mediate Mg^2 + flux [24]. Subsequent work has clearly demonstrated that Mrs2p is a Mg^2 + transporter active in the inner membrane of the mitochondrion in most if not all eukaryotes [25]. Weghuber et al. directly correlated mitochondrial Group II intron splicing to Mg^2 + levels and identified several Mrs2p domains necessary for function [26]. As with CorA, Mg^2 + flux via Mrs2p is inhibited by cation hexaammines [25].

An additional putative homolog in yeast mitochondria (Lpe10) has also been reported [27], [28]. Lpe10p is 32% identical to Mrs2p and contains the YGMNF signature motif of the CorA family; nonetheless, its function as a Mg^2 + transporter is unclear since it cannot readily substitute for Mrs2p [27].

In 1999, while studying Al^3 + toxicity, MacDiarmid and Gardner showed that Saccharomyces cerevisiae Alr1p and Alr2p (aluminum resistance) proteins are homologous to CorA [29] and that Alr1p is required for Mg^2 + homeostasis [30]. Inhibition of Alr1p by Al^3 + leads to Al^3 + toxicity in yeast apparently from Mg^2 + deprivation. Kern et al. also reported that Alr1p helps prevent Cd^2 + toxicity [31]. Alr proteins are found in the plasma membrane of yeast. Two forms of Alr exist, Alr1p and Alr2p [32]. Alr2p does not significantly transport Mg^2 + but a single amino acid substitution in the loop between TM1 and TM2 allows it to do so. Several residues critical for Alr1p Mg^2 + transport have been determined; interestingly, large sections of Alr1p protein were found to be unimportant for Mg^2 + transport [33].

Plants also have homologs of the 2-TM-GxN family. Arabidopsis has at least 10 homologs, AtMGT1-AtMGT10, some of which are localized to distinct tissues. Not all AtMGT homologs can transport Mg^2 +, although clearly AtMGT1 and AtMGT10 can do so [34], [35]. Both of these proteins are sensitive to Al^3 +. While they are clearly important for Mg^2 + influx, the plant members of this family may be more broad spectrum divalent cation transporters than the CorA and Mrs2p homologs. The functional significance of this is currently unknown.

The presence of a short signature motif, YGMN(F), in the Mrs2p, Alr1p, AtMGT and both Eubacterial and Archaeal CorA proteins presumably indicates an evolutionary as well as a functional relationship [9], [24], [36]. Extensive phylogenetic analyses of several hundred homologs have confirmed the relationship of the 2-TM-GxN family proteins and their apparent role in Mg^2 + homeostasis in Eubacteria, Archaea and Eukaryota [9], [10]. In addition, a subfamily appears to mediate Zn^2 + efflux while an additional substantial number of 2-TM-GxN family members have not yet been functionally classified.

Crystal structure of T. maritima CorA

The crystal structure of Thermatoga maritima CorA has been solved at a resolution of 3.9 Å for the whole protein and 1.85 Å for the soluble domain [17]. Subsequently, 3.7 Å [18] and 2.9 Å [19] structures were published. All three crystal structures are essentially identical (Figure 1). S. Typhimurium CorA shares 20% sequence identity with T. maritima CorA but is virtually an exact match in terms of secondary structure predictions. Similarly, secondary structure predictions for other CorA's, Mrs2p, AtMGT and even ZntB proteins are virtually identical and compatible with the T. maritima crystal structure [37]. CorA is a funnel-shaped homopentamer [16], [17]. It has a large, soluble, cytoplasmic amino terminus followed by two C-terminal TM segments. A Mg^2 + appears bound between each of the 5 monomers. The cytoplasmic domain consists of seven β sheets sandwiched between 2 sets of three α helices. An additional and very long α helix of about 100 Å extends from the cytoplasmic domain into the membrane becoming the first TM segment; TM1 contains the YGMNF signature sequence of the CorA family at its C-terminal end. Mutation of the YGMNF sequence abolishes transport through CorA, Mrs2p, and Alr1p [38], [25], [33]. TM1 is followed by a short, negatively charged loop residing in the periplasm, TM2 and a short 6 a.a. C-terminal cytoplasmic domain. The structure of the loop connecting TM1 and TM2 is unresolved in all three crystal structures. We have suggested that this loop is the portion of CorA that first interacts with the Mg^2 + ion prior to transport [7], [17]. In contrast, Payandeh and Pai [18] have suggested that this loop lies relatively flat against the outer surface of the membrane well away from the pore and any possible cation interaction. Further work will be needed to define the role of the periplasmic loop in CorA function.

Figure 1.  Panel A shows the CorA homopentamer oriented with the membrane domain at the top. Each monomer is colored independently, and both TM segments are indicated. Panel B shows the homopentamer from the cytosol facing out through the pore (center). The red dots represent the 5 Mg^2 + bound between the α₃-helix of one monomer and the long α₇-helix of an adjacent monomer. The α₇ helices form most of the inner face of the funnel in the cytosol. See ref. [37] for additional details.

Several amino acids (N314, M302, and L294 & M291) in the crystal structure appear to obstruct the pore indicating that the three current structures are of a closed form of CorA. Consistent with this, mutation of residues near L294/M291 alters the transport properties of CorA [38]. An electrostatic view of CorA indicates a ring of positive charges exists at the membrane interface comprised of 20 Lys from the C-terminal segment and 10 Lys from the very long α-helix. Next to this ring are α helices in the cytoplasm on the outside of the funnel containing a plethora of negatively charged Asp and Glu residues. Interactions between these highly charged regions are potentially critical to the mechanism of transport of CorA.

Hydropathy profiles indicate that Mrs2p's topology is similar to that of CorA and Alr. Each has 2 C-terminal TM segments, a large soluble N-terminus with both the N- and C- termini in the cytoplasm (or mitochondrial matrix for Mrs2p) [24], [32]. Crosslinking data reveal that Mrs2p is also a pentamer [25] while Alr1p is at least a tetramer [32]. Alr1p and Alr2p have been shown to form both homo-oligomers and hetero-oligomers when overexpressed. However since Alr2p does mediate Mg^2 + transport, its oligomerization to Alr1p results in a dominant negative action on the protein [32]. Together, the highly similar secondary structures of the 2-TM-GxN family and the existence of homo-oligomers in several members strongly suggest that the T. maritima crystal structure is representative of the entire CorA family.

Kinetics and expression of the 2-TM-GxN type family

Kinetics of Mg^2 + transport by a 2-TM-GxN type family have been best characterized for S. Typhimurium CorA. CorA is non-repressible, constitutively expressed, and the major Mg^2 + transporter for S. Typhimurium [39]. At 20°C, the K_m of CorA for Mg^2 +is 15 µM with a V_max of 0.25 nmol of Mg^2 + min⁻¹ 10⁸ cells⁻¹[4], [6]. The K_m of the E. coli CorA for Mg^2 + is identical. Cation selectivity for inhibition of CorA has the rank order Mg^2 +>Mn^2 +>Co^2 +>Ni^2 +> > Ca^2 +. Of these, only Co^2 + and Ni^2 + are transported via CorA, with a K_m of 20 µM and 200 µM, respectively. However, transport of Ni^2 + and Co^2 + is unlikely to be physiologically relevant as their accumulation is toxic, and typically they are not encountered under most biological conditions in the concentrations needed for efficient uptake by CorA. corA mutants do not appear to have any obvious Mg^2 + deficiency and intracellular content of Mg^2 + is unchanged (Papp-Wallace and Maguire, unpublished observations). This is presumably because S. Typhimurium carries two other Mg^2 + transporters, MgtA and MgtB [6], [40], [41].

Kinetic properties of Mg^2 + transport by Mrs2p, the yeast mitochondrial Mg^2 + transporter, are less well understood. Mag-Fura2, a Mg^2 + sensitive fluorescent dye, was used to measure intramitochondrial Mg^2 + levels and rates of Mg^2 + influx [25]. Basal intracellular Mg^2 + concentration in Mg^2 + free medium is 0.69 mM in yeast mitochondria. When Mg^2 + concentration in the medium is increased, a corresponding increase in intracellular Mg^2 + is observed. These increases occurred rapidly, within 50 seconds, and a maximum of 5 mM free Mg^2 + could be achieved in the mitochondrial matrix space when extracellular Mg^2 + was 10 mM. A rate of ≈160 micromole sec⁻¹ was measured when the external Mg^2 + was 1 mM. This corresponds to a 25% increase in internal Mg^2 + per second. Curiously, unlike most other transporters or enzymes, incubation temperature had no effect on the rate of Mg^2 + uptake via Mrs2p [25]. Mg^2 + uptake was shown to be dependent on the electrochemical gradient. Interestingly, marked overexpression of Mrs2p only slightly increased the concentration of Mg^2 + in the mitochondria, suggesting that Mrs2p may be functionally regulated [25]. Again in contrast to corA, a mutation in Mrs2p decreased intramitochondrial Mg^2 + by 38%. The rate of Mg^2 + uptake in the Mrs2 mutant decreased from 160 to 27 micromole sec⁻¹. Mg^2 + is required for group II RNA splicing because mutation of the YGMNF motif in Mrs2p results in defective RNA splicing [25], [42]. Moreover, gain of function Mrs2 mutants have better RNA splicing, whereas loss of function mutants are deficient in splicing [26]. Other cations such as Mn^2 +, Ni^2 +, Ca^2 +, Cu^2 +, and Zn^2 + have no effect on splicing if present up to 10 mM in the extra-mitochondrial medium [42].

Less kinetic data is available for the yeast Alr homologs. Total Mg^2 + content between wild type and Alr1p mutant differs by 50–75% depending on the growth media, and Alr1p mutants fail to accumulate Mg^2 +[30]. Expression of Alr1p is regulated at the mRNA level with expression increasing in medium containing low Mg^2 + concentrations. The increase in mRNA correlates with an increase in Alr1p protein. Conversely, when yeast are grown in a medium containing a high Mg^2 + concentration, Alr1p protein is rapidly degraded by a ubiquitin dependent pathway. While Mg^2 + had the most effect on degradation, Co^2 + and Mn^2 + affected degradation at 20–100 times their normal medium concentration. Ca^2 +, Zn^2 +, Ni^2 +, and Cu^2 + had no effect on Alr1p degradation [30].

Efflux of Mg^2 +

In S. Typhimurium, CorA is also essential for Mg^2 + efflux. In cells preloaded with ²⁸Mg^2 +, efflux of ²⁸Mg^2 + can be demonstrated when extracellular Mg^2 + is raised into the mM range. No efflux is detectable at extracellular Mg^2 + concentrations below about 0.5 mM. Three other Co^2 + resistant genes, corB, corC, and corD, affect efflux markedly but have only a modest effect on influx [43]. While mutation of corA completely abolishes Mg^2 + efflux, mutation of corB, corC, and corD individually or in combination markedly reduces the ability of extracellular Mg^2 + to elicit efflux via CorA. Mrs2p is also capable of efflux when the electrochemical gradient is abolished. Mg^2 + loaded mitochondria that were depolarized in a Mg^2 + free medium lost 27% of their intracellular Mg^2 + in 30 min whereas mitochondria lacking Mrs2p showed no significant decrease under these conditions [25]. No efflux studies have been completed on ALR or AtMGT homologs. It is not clear that efflux via CorA is physiologically relevant however, and further studies are needed with purified protein.

Inhibition of Mg^2 + transport

Cation hexaammines are selective inhibitors of CorA transport [7]. Cobalt (III) hexaammine, ruthenium (II) hexaammine and ruthenium (III) hexaammine potently and competitively inhibit ⁶³Ni^2 + uptake through both the Bacterial S. Typhimurium CorA and the Archaeal Methanococcus jannaschii CorA but do not enter the cell. The MgtA and MgtB Mg^2 + transporters are not inhibited by the cation hexaammines. In a mgtA mgtCB mutant strain, dependent on CorA for Mg^2 + uptake, growth is completely inhibited by the cation hexaammines. Cobalt hexaammine also inhibits both Mg^2 + influx and efflux via Mrs2p [25]. It is not known if cation hexaammines can inhibit transport via Alr1p or AtMGT1.

Other properties of the 2-TM-GxN family

In S. Typhimurium, mutation of corA abrogates the Fe^2 + hypersensitivity phenotypes of mgtA mgtCB and phoP mutants [44]. Addition of a cation hexaammine decreases the Fe^2 + hypersensitivity phenotype of the phoP mutant. The V_max for ⁶³Ni^2 + uptake by CorA is increased 15-fold in a phoP mutant and 5-fold in a mgtA mgtCB mutant without change in the K_m of CorA for Mg^2 +[44]. These results could imply that CorA mediates the uptake of Fe^2 +, but direct measurement of transport indicates that CorA does not mediate Fe^2 + uptake nor can Fe^2 + inhibit CorA transport of ⁶³Ni^2 +[45]. Since neither corA transcription nor CorA protein level are increased in either the phoP or mgtA mgtCB mutants, the most parsimonious explanation of these data is that CorA is regulated in some unknown manner.

Lactoperoxidase oxidizes different substrates using hydrogen peroxide; thiocyanate is its major substrate. Addition of lactoperoxidase to cultures of E. coli or S. Typhimurium result in induction of corA expression. A corA mutant is killed after just 2 hours of exposure to this system, whereas wild type cells are not sensitive to hydrogen peroxide alone. Cobalt (III) hexaammine was able to rescue the toxic effects of lactoperoxidase seen in the corA mutant [46], [47].

Alr1p was first identified through a screen inn yeast selecting for increased resistance to Al^3 + and Ga^3 +[29]. Overexpression of Alr1p increases tolerance to both of these trivalent cations but decreases tolerance to Ni^2 +, Mn^2 +, Co^2 +, and Zn^2 +. Al^3 + is apparently toxic because it inhibits Mg^2 + uptake via Alr1p. Overexpression provides more Mg^2 +, overcoming the Al^3 + inhibition. In contrast, the increased toxicity to other divalent cations following Alr1p overexpression suggests that Alr1p mediates the uptake of these cations, an indication that Alr proteins are likely more broad spectrum divalent cation transporters than CorA or Mrs2p. The AtMGT transporters may also be involved in Al^3 + toxicity, presumably through the same mechanism as with the yeast proteins [34], [35].

Conclusions

The CorA or 2-TM-GxN family of proteins plays an important role in Mg^2 + uptake in most domains of life. Conversely, some of these same proteins can mediate uptake of toxic metals such as Co^2 +. The crystal structure of the T. maritima CorA is the first structure of an apparent divalent cation channel to be determined. The structure is quite unlike that of monovalent cation channels. The overall CorA structure appears to be retained in bacterial, archaeal and eukaryotic homologs. Although determination of the structure of CorA has greatly advanced our knowledge of this family of proteins, their role in Mg^2 + homeostasis is still poorly defined.