title: Purification and functional reconstitution of a seven-subunit mrp-type na+/h+ antiporter.
abstract: Mrp antiporters and their homologues in the cation/proton antiporter 3 family of the Membrane Transporter Database are widely distributed in bacteria. They have major roles in supporting cation and cytoplasmic pH homeostasis in many environmental, extremophilic, and pathogenic bacteria. These antiporters require six or seven hydrophobic proteins that form hetero-oligomeric complexes, while most other cation/proton antiporters require only one membrane protein for their activity. The resemblance of three Mrp subunits to membrane-embedded subunits of the NADH:quinone oxidoreductase of respiratory chains and to subunits of several hydrogenases has raised interest in the evolutionary path and commonalities of their proton-translocating domains. In order to move toward a greater mechanistic understanding of these unusual antiporters and to rigorously demonstrate that they function as secondary antiporters, powered by an imposed proton motive force, we established a method for purification and functional reconstitution of the seven-subunit Mrp antiporter from alkaliphilic Bacillus pseudofirmus OF4. Na(+)/H(+) antiporter activity was demonstrated by a fluorescence-based assay with proteoliposomes in which the Mrp complex was coreconstituted with a bacterial FoF1-ATPase. Proton pumping by the ATPase upon addition of ATP generated a proton motive force across the membranes that powered antiporter activity upon subsequent addition of Na(+).
authors.affiliation: Department of Pharmacology and System Therapeutics, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Masato
authors.initials: M
authors.lastname: Morino
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Suzuki
authors.forename: Masahiro
authors.initials: M
authors.lastname: Ito
authors.forename: Terry Ann
authors.initials: TA
authors.lastname: Krulwich
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization; D021122:Protein Subunits / Q000302:isolation & purification / Q000378:metabolism; D018892:Proton-Motive Force; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D008081:Liposomes; D021122:Protein Subunits; D017923:Sodium-Hydrogen Exchangers; D012964:Sodium