Functional analysis of whether the glycine residue of the GMN motif of the Arabidopsis MRS2/MGT/CorA-type Mg channel protein AtMRS2-11 is critical for Mg transport activity.

Magnesium (Mg) plays a critical role in many physiological processes. The AtMRS2/MGT family, which contains nine Arabidopsis genes (and two pseudogenes), belongs to a eukaryotic subset of the CorA superfamily of divalent cation transporters. AtMRS2-11/MGT10 possesses the signature GlyMetAsn sequence (the GMN motif) conserved in the CorA superfamily; however, little is known about the role of the GMN motif in AtMRS2. Direct measurement using the fluorescent dye mag-fura-2 revealed that reconstituted AtMRS2-11 mediated rapid Mg uptake into proteoliposomes at extraliposomal Mg concentrations of 10 and 20 mM. Mutations in the GMN motif, G417 to A, S or V, did not show a significant change in Mg uptake relative to the wild-type protein. The G417W mutant exhibited a significant increase in Mg uptake. The functional complementation assay in Escherichia coli strain TM2 showed that E. coli cells expressing AtMRS2-11 with mutations in G of the GMN motif did not grow in LB medium without Mg supplementation, while growth was observed in LB medium supplemented with 0.5 mM Mg; no difference was observed between the growth of TM2 cells expressing the AtMRS2-11 G417W mutant and that of cells expressing wild-type AtMRS2-11. These results suggested that the Mg transport activity of the AtMRS2-11 GMN-motif mutants was low at low physiological Mg concentrations; thus, the Gly residue is critical for Mg transport, and the Mg transport activity of the GMN-motif mutants was increased at high Mg concentrations.