Functional reconstitution and characterization of the Arabidopsis Mg2+ transporter AtMRS2-10 in proteoliposomes

Abstract

Magnesium (Mg(2+)) plays critical role in many physiological processes. The mechanism of Mg(2+) transport has been well documented in bacteria; however, less is known about Mg(2+) transporters in eukaryotes. The AtMRS2 family, which consists of 10 Arabidopsis genes, belongs to a eukaryotic subset of the CorA superfamily proteins. Proteins in this superfamily have been identified by a universally conserved GlyMetAsn motif and have been characterized as Mg(2+) transporters. Some members of the AtMRS2 family, including AtMRS2-10, may complement bacterial mutants or yeast mutants that lack Mg(2+) transport capabilities. Here, we report the purification and functional reconstitution of AtMRS2-10 into liposomes. AtMRS2-10, which contains an N-terminal His-tag, was expressed in Escherichia coli and solubilized with sarcosyl. The purified AtMRS2-10 protein was reconstituted into liposomes. AtMRS2-10 was inserted into liposomes in a unidirectional orientation. Direct measurement of Mg(2+) uptake into proteoliposomes revealed that reconstituted AtMRS2-10 transported Mg(2+) without any accessory proteins. Mutation in the GMN motif, M400 to I, inactivated Mg(2+) uptake. The AtMRS2-10-mediated Mg(2+) influx was blocked by Co(III)hexamine, and was independent of the external pH from 5 to 9. The activity of AtMRS2-10 was inhibited by Co(2+) and Ni(2+); however, it was not inhibited by Ca(2+), Fe(2+), or Fe(3+). While these results indicate that AtMRS2-10 has similar properties to the bacterial CorA proteins, unlike bacterial CorA proteins, AtMRS2-10 was potently inhibited by Al(3+). These studies demonstrate the functional capability of the AtMRS2 proteins in proteoliposomes to study structure-function relationships.

A novel mitochondrial carrier protein Mme1 acts as a yeast mitochondrial magnesium exporter

“…Were the orientation opposite, little or only part of the amino acids between the two predicted transmembrane domains of Mme1 would have been digested. Worth noting is that this predicted orientation of Mme1 in the membrane is opposite to that of AtMrs2-10, which was preferentially inserted into proteoliposomes with most of it exposed to the interior of the liposomes [22]. This difference is consistent with their opposite functions in Mg 2+ transport with AtMrs2-10 as Mg 2+ importer whereas Mme1 as a candidate of Mg 2+ exporter.…”

“…There are only two Mg 2+ transport proteins that have been previously purified and reconstituted into liposomes-T. maritima CorA and Arabidopsis AtMrs2-10 [22,30]. In this work, through the use of proteoliposome, we provide compelling evidence showing Mme1 as a mitochondrial Mg 2+ exporter.…”

“…Therefore, it may also be the same with Mrs2. However, only the importing activity of the AtMrs2 has been measured while the exporting activity of it has not yet been measured successfully so far [22]. Another possible explanation is that in vivo there might be other accessory proteins interacting with Mme1, acting as molecular switches to control the orientation of Mg 2+ transportation.…”

“…The column-bound protein was eluted with 5 column volumes of buffer C (20 mM Tris, 150 mM NaCl, 2 mM phenylmethylsulfonylfluoride, 300 mM imidazole, 0.15% FC12; pH = 7.5). To further purify the protein and dissolve the protein in buffer suitable for the following proteoliposome experiments, the eluted protein was passed through a Sephadex 200 column with buffer D (20 mM HEPES, 100 mM KCl, 15 mM MgCl 2 , 0.5 mM ATP-Mg, 0.15% FC12; pH = 7.0) [22].…”

“…Assays of Mg 2+ transport activity of Mme1 in proteoliposomes were accomplished according to the published methods with some modifications [22,23]. Chloroform-methanol (3:1) was added to dissolve the yeast total lipids (Avanti Polar Lipids, Inc. Alabama, USA), and the lipids were subsequently dried in a rotary evaporator at 30°C for another 30 min to remove the methanol and traces of chloroform.…”