Homologous protein subunits from Escherichia coli NADH:quinone oxidoreductase can functionally replace MrpA and MrpD in Bacillus subtilis

Abstract

The complex I subunits NuoL, NuoM and NuoN are homologous to two proteins, MrpA and MrpD, from one particular class of Na+/H+ antiporters. In many bacteria MrpA and MrpD are encoded by an operon comprising 6-7 conserved genes. In complex I these protein subunits are prime candidates for harboring important parts of the proton pumping machinery. Deletion of either mrpA or mrpD from the Bacillus subtilis chromosome resulted in a Na+ and pH sensitive growth phenotype. The deletion strains could be complemented in trans by their respective Mrp protein, but expression of MrpA in the B. subtilis ΔmrpD strain and vice versa did not improve growth at pH 7.4. This corroborates that the two proteins have unique specific functions. Under the same conditions NuoL could rescue B. subtilis ΔmrpA, but improved the growth of B. subtilis ΔmrpD only slightly. NuoN could restore the wild type properties of B. subtilis ΔmrpD, but had no effect on the ΔmrpA strain. Expression of NuoM did not result in any growth improvement under these conditions. This reveals that the complex I subunits NuoL, NuoM and NuoN also demonstrate functional specializations. The simplest explanation that accounts for all previous and current observations is that the five homologous proteins are single ion transporters. Presumably, MrpA transports Na+ whereas MrpD transports H+ in opposite directions, resulting in antiporter activity. This hypothesis has implications for the complex I functional mechanism, suggesting that one Na+ channel, NuoL, and two H+ channels, NuoM and NuoN, are present.

MrpA Functions in Energy Conversion during Acetate-Dependent Growth of Methanosarcina acetivorans

“…Indeed, it has been proposed that MrpA channels Na ϩ , whereas MrpD channels H ϩ in the complex from B. subtilis (21). The results obtained with methanol-grown M. acetivorans indicated that the Mrp complex is not required for pH homoeostasis or tolerance to Na ϩ stress.…”

“…Roles for Mrp of M. acetivorans of the domain Archaea were addressed by comparing growth parameters of the wild type versus the ⌬mrpA mutant cultured with either methanol or acetate. The MrpA subunit of the Mrp complex was targeted, since it had been shown that the homologous MrpA of Bacillus subtilis is essential for Na ϩ /H ϩ exchange activity and the proposed site of Na ϩ translocation (21,34,35). Figure 1 shows similar growth rates and final optical densities between the wild type and mutant grown with methanol, although the lag phase for both strains was lengthened when cultured at pH 8.0 compared to pH 6.8, indicating a required adaptation period for optimal growth at the higher pH for both the mutant and wild type.…”

“…The homolog is upregulated in M. acetivorans in response to growth with acetate (11,17), a result consistent with an Mrp requirement for optimal acetotrophic growth. Mrp homologs from species in the domain Bacteria function in Na ϩ resistance, pH homeostasis of alkaliphiles, bile salt resistance, and energy-yielding metabolism (18)(19)(20)(21)(22). Although Mrp homologs are encoded in the genomes of species from the domain Archaea (18), none has been investigated.…”

The role of proton and sodium ions in energy transduction by respiratory complex I

“…The functional similarity of MrpA to NuoL and that of MrpD to NuoN was recently corroborated by complementation studies using B. subtilis MrpA and MrpD deletions strains, in which the subunits of complex I could replace those of the Mrp antiporter (43). These observations further support the antiporter activity by complex I.…”

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

“…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).…”