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 heterooligomeric 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 protontranslocating 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 F o F 1 -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 ؉ .

Functional Role of MrpA in the MrpABCDEFG Na + /H + Antiporter Complex from the Archaeon Methanosarcina acetivorans

“…1, supporting the conclusion that MrpA is essential for Na ϩ /H ϩ antiport activity. The results further show that activity is independent of an intact MrpABCDEFG complex, contrary to previous assertions that intact complexes from the domain Bacteria are required for activity (2,3,11,20,25,26).…”

“…Most cation/proton antiporters are monomers, in contrast to the cation/proton antiporter 3 (CPA3) family, also called Mrp complexes, which are comprised of six or seven subunits, depending on whether subunits MrpA and MrpB are fused (1)(2)(3). Analyses of genomic sequences show Mrp complexes are broadly distributed among species with diverse physiologies from the domains Bacteria and Archaea, although investigations have focused on Bacteria to the exclusion of Archaea.…”

“…The seven-subunit MrpABCDEFG complexes from Bacillus species have been model systems for investigation, with a focus on the MrpA and MrpD subunits that contain transmembrane acidic residues, consistent with ion translocation functions (24). Although MrpA and MrpD subunits from the domain Bacteria are implicated in translocation of Na ϩ and H ϩ , respectively, a consensus has emerged that all seven subunits are required for antiport activity (2,3,11,20,25,26). Specifically, expression of an MrpAD subcomplex in the antiporterdeficient Escherichia coli strain KNabc showed no antiport activity (27).…”

Adaptation of Staphylococcus xylosus to Nutrients and Osmotic Stress in a Salted Meat Model

“…The PMF can expel the protons, resulting from the activity of the two S. xylosus Mnh antiporters, from the cytoplasm to maintain pH homeostasis. In Bacillus pseudofirmus , proton pumping by the F 0 F 1 -ATPase generated a proton motive force across the membrane that powered Mnh antiporter activity after addition of Na + (Morino et al, 2014 ).…”

Proteomic and Mutant Analysis of the CO 2 Concentrating Mechanism of Hydrothermal Vent Chemolithoautotroph Thiomicrospira crunogena

“…Included in this family are the proton-translocating subunit from both the NADH dehydrogenase complex (synonyms include NuoL, ND5, and NdhF [25]) and a subunit from Mrp-type Na ϩ /H ϩ antiporters (26). Also included are the transmembrane subunits from the CO 2 uptake systems present in some cyanobacteria (27).…”