Functional reconstitution and characterization of the Arabidopsis Mg(2+) transporter AtMRS2-10 in proteoliposomes

Biochim Biophys Acta. 2012 Sep;1818(9):2202-8. doi: 10.1016/j.bbamem.2012.04.015. Epub 2012 Apr 26.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aluminum / chemistry
  • Arabidopsis / metabolism*
  • Arabidopsis Proteins / chemistry*
  • Arabidopsis Proteins / metabolism
  • Arabidopsis Proteins / physiology
  • Biophysics / methods*
  • Cation Transport Proteins / chemistry
  • Cation Transport Proteins / physiology*
  • Cations
  • Cobalt / chemistry
  • Detergents / chemistry
  • Dose-Response Relationship, Drug
  • Escherichia coli / metabolism
  • Ions
  • Liposomes / chemistry
  • Magnesium / chemistry
  • Mutation
  • Nickel / chemistry
  • Proteolipids / chemistry*
  • Spectrophotometry, Atomic / methods
  • Structure-Activity Relationship

Substances

  • Arabidopsis Proteins
  • Cation Transport Proteins
  • Cations
  • Detergents
  • Ions
  • Liposomes
  • MGT1 protein, Arabidopsis
  • Proteolipids
  • proteoliposomes
  • Cobalt
  • Nickel
  • Aluminum
  • Magnesium