Functional involvement of membrane-embedded and conserved acidic residues in the ShaA subunit of the multigene-encoded Na+/H+ antiporter in Bacillus subtilis.
ShaA, a member of a multigene-encoded Na+/H+ antiporter in B. subtilis, is a large integral membrane protein consisting of 20 transmembrane helices (TM). Conservation of ShaA-like protein subunits in several cation-coupled enzymes, including the NuoL (ND5) subunit of the H+-translocating complex I, suggests the involvement of ShaA in cation transport. Bacillus subtilis ShaA contains six acidic residues that are conserved in ShaA homologues and are located in putative transmembrane helices. We examined the functional involvement of the six transmembrane acidic residues of ShaA by site-directed mutagenesis. Mutation in glutamate (Glu)-113 in TM-4, Glu-657 in TM-18, aspartate (Asp)-734 and Glu-747 in TM-20 abolished the antiport activity, suggesting that these residues play important roles in the ion transport of Sha. The acidic group was necessary and sufficient in Glu-657 and Asp-743, while it was not true of Glu-113 and Glu-747. Mutation in Asp-103 in TM-3, which is conserved in ShaA-types but not in ShaAB-types, partially affected on the antiport activity. Mutation in Asp-50 in TM-2 resulted in a unexpected phenotype: mutants retained the wild type level of ability to confer NaCl resistance to the Na+/H+ antiporter-deficient E. coli KNabc, but showed a very low antiport activity. The acidic group of Asp-50 and Asp-103 was not essential for the function. Our results suggested that these acidic residues are functionally involved in the ion transport of Sha, and some of them probably in cation binding and/or translocation.
Kosono
Saori
S
Environmental Molecular Biology Laboratory, RIKEN, Wako, Saitama 351-0198, Japan. kosono@riken.jp
Kajiyama
Yusuke
Y
Kawasaki
Shin
S
Yoshinaka
Toko
T
Haga
Koki
K
Kudo
Toshiaki
T
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics* / Q000254:growth & development; D001483:Base Sequence; D015153:Blotting, Western; D017931:DNA Primers; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000235:genetics*
D017931:DNA Primers; D017923:Sodium-Hydrogen Exchangers