Schock et al. (2000) identified and named the family AtMRS2 based on the similarity of the genes to the MRS2 gene of yeast. The authors also showed that the AtMRS2-1 gene could complement a Δmrs2 yeast mutant phenotype. Independently, Li et al. (2001) published a report identifying the family and showing that two additional members could complement Mg transport deficient mutants, one in S. typhimurium and the other in S. cerevisiae.
The three genes that have been shown to transport Mg are AtMRS2-1, AtMRS2-10 and AtMRS2-11, and these genes produce proteins 442, 443 and 459 amino acids in size, respectively. Each of the proteins shows significant similarity to Mrs2p of yeast and a weak similarity to CorA of bacteria, contains the conserved GMN amino acid motif at the outside end of the first TM domain, and is predicted to have two TM domains.
The AtMRS2-1 gene, when expressed in yeast from the MRS2 promoter and being fused C-terminally to the first 95 amino acids of the Mrs2p protein, was directed to the mitochondria, where it complemented a Δmrs2 mutant both phenotypically (mitochondrial RNA splicing was restored) and with respect to the Mg content of the organelle. No data on the kinetics of the transport was presented. The AtMRS2-11 gene was analysed in yeast (in the alr1 alr2 strain), where it was shown that expression of the gene significantly increased the rate of Mg uptake into starved cells over the control, as measured using flame atomic absorption spectroscopy of total cellular Mg content. However, Alr1p was shown to be significantly more effective at transporting Mg at low extracellular concentrations, suggesting that the affinity of AtMRS2-11 for Mg is lower than that of Alr1p. An electrophysiological (voltage clamp) analysis of the AtMRS2-11 protein in Xenopus oocytes also showed a Mg-dependent current at membrane potentials (ΔΨ) of –100 – –150 mV inside. 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. 63Ni was used as the substitute ion and Mg was shown to inhibit the uptake of 63Ni with a Ki of 20 μM. Uptake was also inhibited by Co(III)Hex and by other divalent cations. Only Co and Cu inhibited transport with Ki values less than 1 mM.
The AtMRS2-10 protein was fused to GFP, and was shown to be localised to the plasma membrane. A similar experiment was attempted in the Schock et al. (2000) paper, but the observed localisation was not significantly different from that seen with unfused GFP. The most likely reason for the lack of a definitive localisation of AtMRS2-1 in the Schock et al. paper is that the authors removed the TM domains from the protein, thereby precluding its insertion into a membrane.
The exact physiological significance of the AtMRS2-1 and AtMRS2-10 proteins in plants has yet to be clarified. The AtMRS2-11 gene has been overexpressed (from the CaMV 35S promoter) in A. thaliana. The transgenic line has been shown to accumulate high levels of the AtMRS2-11 transcript. A strong Mg deficiency phenotype (necrotic spots on the leaves, see Chapter 1.5 below) was recorded during the screening process (in both the T1 and T2 generations) for a homozygote line, but this phenotype was lost in the T3 generation and could not be reproduced when the earlier generations were screened a second time. The author suggested that environmental effects were the most likely cause of the inconsistent phenotype.