Functional analysis of whether the glycine residue of the GMN motif of the Arabidopsis MRS2/MGT/CorA-type Mg²⁺ channel protein AtMRS2-11 is critical for Mg²⁺ transport activity

Highlights

• AtMRS2-11 is a Mg²⁺ channel protein mediating rapid Mg²⁺ transport.

• The Gly residue in the GMN motif of AtMRS2-11 is critical at low Mg²⁺ concentrations.

• Gly mutations in the GMN motif of AtMRS2-11 are tolerated at high Mg²⁺ concentrations.

• MRS2-like proteins with WMN motifs may be highly functional.

Abstract

Magnesium (Mg²⁺) 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 Mg²⁺ uptake into proteoliposomes at extraliposomal Mg²⁺ concentrations of 10 and 20 mM. Mutations in the GMN motif, G417 to A, S or V, did not show a significant change in Mg²⁺ uptake relative to the wild-type protein. The G417W mutant exhibited a significant increase in Mg²⁺ 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 Mg²⁺ supplementation, while growth was observed in LB medium supplemented with 0.5 mM Mg²⁺; 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 Mg²⁺ transport activity of the AtMRS2-11 GMN-motif mutants was low at low physiological Mg²⁺ concentrations; thus, the Gly residue is critical for Mg²⁺ transport, and the Mg²⁺ transport activity of the GMN-motif mutants was increased at high Mg²⁺ concentrations.

Introduction

Magnesium is one of the most important and abundant divalent cations in living cells. Mg²⁺ is an essential cofactor for hundreds of enzymes involved in numerous biological reactions [1]. In plants, Mg²⁺ critically contributes to the process of photosynthesis. Mg²⁺ functions as a reaction center in photosynthetic reactions, and Mg²⁺ regulates key enzymes that are involved in carbon fixation in chloroplasts [2,3]. The Mg²⁺ concentration in the chloroplast stroma is lower in the dark than that upon illumination [3,4]. Within plants, most metabolically active Mg²⁺ is bound or incorporated into cellular compartments [5], with the highest concentrations in chloroplasts [6]. As with any cation, the cellular concentration of Mg²⁺ is regulated by transmembrane pathways. In plants, a family of proteins that has been designated MRS2 [7] or MGT proteins [8] appears to play a major role in Mg²⁺ transport across diverse biological membranes. We refer to this family as MRS2 for simplicity.

Plant MRS2 proteins are structurally similar to bacterial CorA proteins. CorA is the primary Mg²⁺ uptake system in the domains Bacteria and Archaea [9]. All CorA/MRS2-like proteins are universally characterized by two C-terminal transmembrane segments. The two transmembrane segments are joined by the loop, which contains the conserved signature Gly-Met-Asn (GMN) sequence. In Arabidopsis thaliana, a family of nine genes (and two pseudogenes) has been annotated as AtMRS2 or AtMGT because the members share the CorA GMN signature motif [10]. Phylogenetic analysis of AtMRS2 proteins has revealed that MRS2 proteins belong to five clades, and AtMRS2-11/MGT10 is the sole member of clade A [10]. The expression and activity of the putative Mg²⁺ transporter AtMRS2s have been studied in Arabidopsis thaliana. AtMRS2-11, a member of the AtMRS2 family, is likely localized within the inner chloroplast envelope membrane, where it mediates Mg²⁺ import into the chloroplast stroma [11,12]. Attempts to obtain insertional knockout mutant plants of AtMRS2-11 have not been successful, suggesting that the AtMRS2-11 gene may be essential in Arabidopsis [10,12].

Bacterial CorA is homopentameric and acts as an Mg²⁺ import channel [13]. For influx, a hydrated Mg²⁺ ion first binds at the pore entrance via a conserved GMN motif, and the Mg²⁺ ion is subsequently dehydrated for transmembrane movement [14,15]. Consequently, mutations within the GMN signature sequence of CorA abolish Mg²⁺ movement, indicating that this motif is essential for the function of CorA [16,17].

We previously purified and characterized AtMRS2-1 and AtMRS2-10 reconstituted into liposomes [18,19]. Our previous Mg²⁺ transport assay showed that these proteins had the ability to transport Mg²⁺ without any additional protein partners. Functional complementation assays using Escherichia coli mutants also showed that AtMRS2-1, AtMRS2-10 and AtMRS2-11 were capable of mediating Mg²⁺ uptake [19,20]. We purified AtMRS2-11 and reconstituted it into liposomes. Our Mg²⁺ transport assay of AtMRS2-11 in reconstituted proteoliposomes showed that this protein has the ability to transport Mg²⁺ without any additional protein partners. Here, we mutated the Gly residue in the GMN signature motif in an attempt to abolish the contribution of the GMN motif to protein function. All AtMRS2 family proteins except for the putative products of the pseudogenes possess the GMN motif. We previously showed that the Met to Ile and Met to Ala mutations in the GMN motifs of AtMRS2-10 [18] and AtMRS2-1 [19], respectively, reduced the Mg²⁺ transport activity of these proteins but compared to the bacterial protein CorA, little is known about the role of the GMN motif of AtMRS2 proteins. Here, we showed that the Gly residue in the GMN motif of AtMRS2-11 was critical for Mg²⁺ transport activity at low physiological Mg²⁺ concentrations but was not critical at high Mg²⁺ concentrations.