title: Functional reconstitution and characterization of the Arabidopsis Mg(2+) 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.
authors.affiliation: Kyoto Prefectural University, Shimogamo, Kyoto, Japan. ishijima@kpu.ac.jp
authors.forename: Sumio
authors.initials: S
authors.lastname: Ishijima
authors.forename: Zenpei
authors.initials: Z
authors.lastname: Shigemi
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Adachi
authors.forename: Nana
authors.initials: N
authors.lastname: Makinouchi
authors.forename: Ikuko
authors.initials: I
authors.lastname: Sagami
mesh_terms: D000535:Aluminum / Q000737:chemistry; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D001703:Biophysics / Q000379:methods*; D027682:Cation Transport Proteins / Q000737:chemistry / Q000502:physiology*; D002412:Cations; D003035:Cobalt / Q000737:chemistry; D003902:Detergents / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D007477:Ions; D008081:Liposomes / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry; D009154:Mutation; D009532:Nickel / Q000737:chemistry; D011510:Proteolipids / Q000737:chemistry*; D013054:Spectrophotometry, Atomic / Q000379:methods; D013329:Structure-Activity Relationship
chemical_list: D029681:Arabidopsis Proteins; D027682:Cation Transport Proteins; D002412:Cations; D003902:Detergents; D007477:Ions; D008081:Liposomes; C517535:MGT1 protein, Arabidopsis; D011510:Proteolipids; C020485:proteoliposomes; D003035:Cobalt; D009532:Nickel; D000535:Aluminum; D008274:Magnesium