Deletion Analysis of the Subunit Genes of V-Type Na⁺-ATPase from Enterococcus hirae

• Published in: Journal of biochemistry (Tokyo) Vol. 139; no. 6; pp. 1045 - 1052
• Main Authors: Hosaka, Toshiaki, Takase, Kazuma, Murata, Takeshi, Kakinuma, Yoshimi, Yamato, Ichiro
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.06.2006
• Subjects: Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - metabolism; ATPase activity; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Blotting, Western; carbonyl cyanide m-chlorophenylhydrazone; Cation Transport Proteins - genetics; Cation Transport Proteins - metabolism; CCCP; Cell Membrane - metabolism; DCCD; deletion mutant; dithiothreitol; DTT; EDTA; Electrophoresis, Polyacrylamide Gel; Enterococcus - enzymology; Enterococcus - genetics; Enterococcus - growth & development; ethylenediaminetetraacetic acid; Gene Deletion; Genetic Complementation Test; Hydrogen-Ion Concentration; Immunohistochemistry; Ion Transport - genetics; keyhole limpet hemocyanin; KLH; Models, Genetic; Mutagenesis, Insertional - genetics; Mutagenesis, Insertional - methods; Mutation - genetics; Na+ efflux activity; N′-dicyclohexylcarbodiimide; Operon - genetics; Protein Subunits - genetics; Protein Subunits - metabolism; Sodium - metabolism; subunit composition; V-type ATPase; Vacuolar Proton-Translocating ATPases - genetics; Vacuolar Proton-Translocating ATPases - metabolism
• DOI: 10.1093/jb/mvj108
• ISSN: 0021-924X

The V₁Vo-ATPase from Enterococcus hirae catalyzes ATP hydrolysis coupled with sodium translocation. Mutants with deletions of each of 10 subunits (NtpA, B, C, D, E, F, G, H, I, and K) were constructed by insertion of a chloramphenicol acetyltransferase gene into the corresponding subunit gene in the genome. Measurements of cell growth rates, ²²Na⁺ efflux activities, and ATP hydrolysis activities of the membranes of the deletion mutants indicated that V-ATPase requires nine of the subunits, the exception being the NtpH subunit. The results of Western blotting and V₁-ATPase dissociation analysis suggested that the A, B, C, D, E, F, and G subunits constitute the V₁ moiety, whereas the V₀ moiety comprises the I and K subunits.