Магний тасымалдаушылары: эволюция және түрлердің арасындағы айырмашылықтар

Magnesium transporters are proteins that transport magnesium across the cell membrane. All forms of life require magnesium, yet the molecular mechanisms of Mg2+ uptake from the environment and the distribution of this vital element within the organism are only slowly being elucidated. The ATPase function of MgtA is highly cardiolipin dependent and has been shown to detect free magnesium in the μM range

In bacteria, Mg2+ is probably mainly supplied by the CorA protein and, where the CorA protein is absent, by the MgtE protein. In yeast the initial uptake is via the Alr1p and Alr2p proteins, but at this stage the only internal Mg2+ distributing protein identified is Mrs2p. Within the protozoa only one Mg2+ transporter (XntAp) has been identified. In metazoa, Mrs2p and MgtE homologues have been identified, along with two novel Mg2+ transport systems TRPM6/TRPM7 and PCLN 1. Finally, in plants, a family of Mrs2p homologues has been identified along with another novel protein, AtMHX.

The evolution of Mg2+ transport appears to have been rather complicated. Proteins apparently based on MgtE are present in bacteria and metazoa, but are missing in fungi and plants, whilst proteins apparently related to CorA are present in all of these groups. The two active transport transporters present in bacteria, MgtA and MgtB, do not appear to have any homologies in higher organisms. There are also Mg2+ transport systems that are found only in the higher organisms.

There are a large number of proteins yet to be identified that transport Mg2+. Even in the best studied eukaryote, yeast, Borrelly has reported a Mg2+/H+ exchanger without an associated protein, which is probably localised to the Golgi. At least one other major Mg2+ transporter in yeast is still unaccounted for, the one affecting Mg2+ transport in and out of the yeast vacuole. In higher, multicellular organisms, it seems that many Mg2+ transporting proteins await discovery. The CorA domain containing Mg2+ transporters (CorA, Alr like and Mrs2 like) have a similar but not identical array of affinities for divalent cations. In fact, this observation can be extended to all of the Mg2+ transporters identified so far. This similarity suggests that the basic properties of Mg2+ strongly influence the possible mechanisms of recognition and transport. However, this observation also suggests that using other metal ions as tracers for Mg2+ uptake will not necessarily produce results comparable to the transporter's ability to transport Mg2+. Ideally, Mg2+ should be measured directly. Since 28Mg2+ is practically unobtainable, much of the old data will need to be reinterpreted with new tools for measuring Mg2+ transport, if different transporters are to be compared directly. The pioneering work of Kolisek and Froschauer using mag fura 2 has shown that free Mg2+ can be reliably measured in vivo in some systems. By returning to the analysis of CorA with this new tool, we have gained an important baseline for the analysis of new Mg2+ transport systems as they are discovered. However, it is important that the amount of transporter present in the membrane is accurately determined if comparisons of transport capability are to be made. This bacterial system might also be able to provide some utility for the analysis of eukaryotic Mg2+ transport proteins, but differences in biological systems of prokaryotes and eukaryotes will have to be considered in any experiment.

Comparing the functions of the characterised Mg2+ transport proteins is currently almost impossible, even though the proteins have been investigated in different biological systems using different methodologies and technologies. Finding a system where all the proteins can be compared directly would be a major advance. If the proteins could be shown to be functional in bacteria (S. typhimurium), then a combination of the techniques of mag fura 2, quantification of protein in the envelope membrane, and structure of the proteins (X ray crystal or cryo TEM) might allow the determination of the basic mechanisms involved in the recognition and transport of the Mg2+ ion. However, perhaps the best advance would be the development of methods allowing the measurement of the protein's function in the patch clamp system using artificial membranes.

In 1968, Lusk described the limitation of bacterial (Escherichia coli) growth on Mg2+ poor media, suggesting that bacteria required Mg2+ and were likely to actively take this ion from the environment. The following year, the same group and another group, Silver, independently described the uptake and efflux of Mg2+ in metabolically active E. coli cells using 28Mg2+. By the end of 1971, two papers had been published describing the interference of Co2+, Ni2+ and Mn2+ on the transport of Mg2+ in E. coli and in Aerobacter aerogenes and Bacillus megaterium. In the last major development before the cloning of the genes encoding the transporters, it was discovered that there was a second Mg2+ uptake system that showed similar affinity and transport kinetics to the first system, but had a different range of sensitivities to interfering cations. This system was also repressible by high extracellular concentrations of Mg2+
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The CorA gene and its corresponding protein are the most exhaustively studied Mg2+ transport system in any organism. Most of the published literature on the CorA gene comes from the laboratory of M. E. Maguire. Recently the group of R. J. Schweyen made a significant impact on the understanding of Mg2+ transport by CorA. The gene was originally named after the cobalt resistant phenotype in E. coli that was caused by the gene's inactivation. that the CorA gene was present in 17 gram negative bacteria. With the large number of complete genome sequences now available for prokaryotes, CorA has been shown to be virtually ubiquitous among the Eubacteria, as well as being widely distributed among the Archaea. The CorA locus in E. coli contains a single open reading frame of 948 nucleotides, producing a protein of 316 amino acids. This protein is well conserved amongst the Eubacteria and Archaea. Between E. coli and S. typhimurium, the proteins are 98% identical, but in more distantly related species, the similarity falls to between 15 and 20%. However, recent evidence suggests that the activity of the protein may be regulated by the PhoPQ two component regulatory system. This sensor responds to low external Mg2+ concentrations during the infection process of S. typhimurium in humans. In low external Mg2+ conditions, the PhoPQ system was reported to suppress the function of CorA and it has been previously shown that the transcription of the alternative Mg2+ transporters MgtA and MgtB is activated in these conditions. The figure (not to scale) shows the originally published transmembrane (TM) domain topology of the S. typhimurium CorA protein, which was said to have three membrane spanning regions in the C terminal part of the protein (shown in blue), as determined by Smith et al Evidence for CorA acting as a homotetramer was published by Warren et al. in 2004. In December 2005 the crystal structure of the CorA channel was posted to the RSCB protein structure database. The results showed that the protein has two TM domains and exists as a homopentamer, in direct conflict with the earlier reports. Follow this link to see the structure in 3D. The soluble intracellular parts of the protein are highly charged, containing 31 positively charged and 53 negatively charged residues. Conversely, the TM domains contain only one charged amino acid, which has been shown to be unimportant in the activity of the transporter. From mutagenesis experiments, it appears that the chemistry of the Mg2+ transport relies on the hydroxyl groups lining the inside of the transport pore; there is also an absolute requirement for the GMN motif (shown in red). Before the activity of CorA could be studied in vivo, any other Mg2+ transport systems in the bacterial host had to be identified and inactivated or deleted (see below). A strain of S. typhimurium containing a functional CorA gene but lacking MgtA and MgtB was constructed(also see below), and the uptake kinetics of the transporter were analysed. This strain showed nearly normal growth rates on standard media (50 μM Mg2+), but the removal of all three genes created a bacterial strain requiring 100 mM external Mg2+ for normal growth. although with lower affinities (km) and velocities (Vmax) than for Mg2+ (see table). The km values for Co2+ and Ni2+ are significantly above those expected to be encountered by the cells in their normal environment, so it is unlikely that the CorA transport system mediates the uptake of these ions under natural conditions. Cobalt (III) hexaammine, Co(III)Hex, is a covalently bound (non labile) analog for the first shell of hydration for several divalent cations, including Mg2+. The radius of the Co(III)Hex molecule is 244 pm, very similar to the 250 pm radius of the first hydration shell of Mg2+. This analog is a potent inhibitor of the CorA transport system, more so than Mg2+, Co2+ or Ni2+. The additional strength of the Co(III)Hex inhibition might come from the blocking of the transport pore due to the inability of the protein to ‘dehydrate’ the substrate. It was also shown that Co(III)Hex was not transported into the cells, Although neither gene is required for pathogenicity, the MgtB protein does enhance the long term survival of the pathogen in the cell. The genes are also upregulated in vitro when the Mg2+ concentration falls below 50 μM (Snavely et al., 1991a). Although the proteins have km values similar to CorA and transport rates approximately 10 times less, the genes may be part of a Mg2+ scavenging system. Chamnongpol and Groisman (2002) presents evidence that the role of these proteins may be to compensate for the inactivation of the CorA protein by the PhoPQ regulon. and neither gene shows any similarity to CorA. The MgtA and MgtB proteins are 75% similar (50% identical), although it seems that MgtB may have been acquired by horizontal gene transfer as part of Salmonella Pathogenicity Island 3. The TM topology of the MgtB protein has been experimentally determined, showing that the protein has ten TM spanning helices with the termini of the protein in the cytoplasm (see figure
). MgtA is present in widely divergent bacteria, but is not nearly as common as CorA, while MgtB appears to have a quite restricted distribution. No hypotheses for the unusual distribution have been suggested. The figure, adapted from Smith et al. (1993b), shows the experimentally determined membrane topology of the MgtB protein in S. typhimurium. The TM domains are shown in light blue and the orientation in the membrane and the positions of the N and C termini are indicated. The figure is not drawn to scale. While the MgtA and MgtB proteins are very similar, they do show some minor differences in activity. MgtB is very sensitive to temperature, losing all activity (with regard to Mg2+ transport) at a temperature of 20 °C. Further experiments will be required to define the physiological function of these proteins.

Two papers describe MgtE, a fourth Mg2+ uptake protein in bacteria unrelated to MgtA/B or CorA. The figure (right), adapted from Smith et al. (1995) The orientation in the membrane and the positions of the N and C termini are indicated. This figure is not drawn to scale. This transporter has recently had its structure solved by x ray crystallography. The MgtE gene was first identified by Smith et al. (1995) during a screen for CorA like proteins in bacteria and complements the Mg2+ uptake deficient S. typhimurium strain MM281 (corA mgtA mgtB), restoring wild type growth on standard media. shifted the focus more towards the uptake of the metal cations, showing that yeast take up cations with the following affinity series; Mg2+, Co2+, Zn2+ > Mn2+ > Ni2+ > Ca2+ > Sr2+. Additionally, it was suggested that the transport of the different cations is mediated by the same transport system — a situation very much like that in bacteria. In 1998, MacDiarmid and Gardner finally identified the proteins responsible for the observed cation transport phenotype in Saccharomyces cerevisiae. on the strength of mutagenesis studies, making the TM topology of these proteins more like that of CorA (see figure). Also, Alr1p contains the conserved GMN motif at the outside end of TM 2 (TM 2') and the mutation of the methionine (M) in this motif to a leucine (L) led to the loss of transport capability. The localisation of the Alr1p was observed to be internalised and degraded in the vacuole in response to extracellular cations. Mg2+, at very low extracellular concentrations (100 μM; < 10% of the standard media Mg2+ content), and Co2+ and Mn2+ at relatively high concentrations (> 20× standard media), induced the change in Alr1p protein localisation, and the effect was dependent on functional ubiquitination, endocytosis and vacuolar degradation. Salih et al., in prep. The kinetics of Mg2+ uptake by Alr1p have been investigated by electrophysiology techniques on whole yeast cells. suggesting that multiple systems inside the yeast are transporting Mg2+ into storage compartments. This internal transport will very likely mask the uptake process. The expression of ALR1 in S. typhimurium without Mg2+ uptake genes may be an alternative, but, as stated earlier, the effects of a heterologous expression system would need to be taken into account.

The MNR2 gene encodes a protein closely related to the Alr proteins, but includes conserved features that define a distinct subgroup of CorA proteins in fungal genomes, suggesting a distinct role in Mg2+ homeostasis. Like an alr1 mutant, growth of an mnr2 mutant was sensitive to Mg2+ deficient conditions, but the mnr2 mutant was observed to accumulate more Mg2+ than a wild type strain under these conditions. These phenotypes suggested that Mnr2 may regulate Mg2+ storage within an intracellular compartment. Consistent with this interpretation, the Mnr2 protein was localized to the membrane of the vacuole, an internal compartment implicated in the storage of excess mineral nutrients by yeast. A direct role of Mnr2 in Mg2+ transport was suggested by the observation that increased Mnr2 expression, which redirected some Mnr2 protein to the cell surface, also suppressed the Mg2+ requirement of an alr1 alr2 double mutant strain. The mnr2 mutation also altered accumulation of other divalent cations, suggesting this mutation may increase Alr gene expression or protein activity. Recent work and was cloned and sequenced by Wiesenberger et al. (1992). Mrs2p was not identified as a putative Mg2+ transporter until Bui et al. (1999). Mg2+ transport has been shown to be directly mediated by Mrs2p, before the gene was cloned by Haynes et al. (2002).) and SLC24 (K+ dependent Na+/Ca2+ exchanger) human solute transport proteins. The XntAp is equally similar to the SLC8 and SLC24 protein families by amino acid sequence, but the predicted TM topology is more like that of SLC24, but the similarity is at best weak and the relationship is very distant. The TM domains are shown in light blue, the α1 and α2 domains are shown in green. The orientation in the membrane and the positions of the N and C termini are indicated and the figure is not drawn to scale. The Mg2+ dependent currents carried by XntAp are kinetically like that of a channel protein and have an ion selectivity order of Mg2+ > Co2+, Mn2+ > Ca2+ — a series again very similar to that of CorA.

A bioinformatic search of the sequence databases identified one homologue of the MRS2 gene of yeast in a range of metazoans. have reviewed the research into the TRP genes, and a second review by Montell (2003) has reviewed the research into the TRPM genes. The TRPM family of ion channels has members throughout the metazoa. The TRPM6 and TRPM7 proteins are highly unusual, containing both an ion channel domain and a kinase domain (Figure 1.7), the role of which brings about the most heated debate. but in the presence of TRPM6 the affinity series of transported cations places Mg2+ above Ca2+. The differences in reported conductance were caused by the expression patterns of these genes. TRPM7 is expressed in all cell types tested so far, while TRPM6 shows a more restricted pattern of expression. An unfortunate choice of experimental system by Voets et al., (2004) led to the conclusion that TRPM6 is a functional Mg2+ transporter. However, later work by Chubanov et al. (2004) and Montell et al. (2002),

The gene Claudin 16 was cloned by Simon et al. (1999), The expression pattern of the gene was determined by RT PCR, and was shown to be very tightly confined to a continuous region of the kidney tubule running from the medullary thick descending limb to the distal convoluted tubule. strengthening the links between the gene and the uptake of Mg2+.

The current knowledge of the molecular mechanisms for Mg2+ transport in plants is very limited, with only three publications reporting a molecular basis for Mg2+ transport in plants. These values are physiologically significant, as several membranes in plants maintain ΔΨ in this range. However, the author had difficulty reproducing these results due to an apparent "death" of oocytes containing the AtMRS2 11 protein, and therefore these results should be viewed with caution. The AtMRS2 10 transporter has been analysed using radioactive tracer uptake analysis. How the transport of Mg2+ by the protein is regulated was not determined. Currents were observed to pass through the protein in both directions, but the Mg2+ out current required a ‘cytoplasmic’ pH of 5.5, a condition not found in plant cells under normal circumstances. In addition to the transport of Mg2+, Shaul et al. (1999) also showed that the protein could transport Zn2+ and Fe2+, but did not report on the capacity of the protein to transport other divalent cations (e. g. Co2+ and Ni2+) or its susceptibility to inhibition by cobalt (III) hexaammine. The detailed kinetics of Mg2+ transport have not been determined for AtMHX. However, physiological effects have been demonstrated. When A. thaliana plants were transformed with overexpression constructs of the AtMHX gene driven by the CaMV 35S promoter, the plants over accumulated the protein and showed a phenotype of necrotic lesions in the leaves, which the authors suggest is caused by a disruption in the normal function of the vacuole, given their observation that the total Mg2+ (or Zn2+) content of the plants was not altered in the transgenic plants. The image has been adapted from Shaul et al. (1999) and Quednau et al. (2004), and combined with an analysis using HMMTOP, this figure shows the computer predicted membrane topology of the AtMHX protein in Arabidopsis thaliana. At this time the topology shown should be considered a tentative hypothesis. The TM domains are shown in light blue, the orientation in the membrane and the positions of the N and C termini are indicated, and the figure is not drawn to scale. The α1 and α2 domains, shown in green, are both quite hydrophobic and may both be inserted into the membrane.