V-type H+-ATPase/synthase from a thermophilic eubacterium, Thermus thermophilus. Subunit structure and operon

Abstract

V-type ATPase (V(o)V1) capable of ATP-driven H+ pumping and of H+ gradient driven ATP synthesis was isolated from a thermophilic eubacterium, Thermus thermophilus. When the enzyme was analyzed by gel electrophoresis in the presence of sodium dodecyl sulfate, it showed eight polypeptide bands of which four were subunits of V1. We also isolated the V(o)V1 operon, containing nine genes in the order of atpG-I-L-E-X-F-AB-D, which encoded proteins with molecular sizes of 13, 43, 10, 20, 35, 11, 64, 53, and 25 kDa, respectively. The last four genes were identified as those for V1 subunits; atpA, B, D, and F encoded the A, B, γ, and δ subunits, respectively. The first five genes, atpG-atpX, were identified as genes for the V(o) subunits. The product of atpL, the proteolipid subunit, lacked a 19-amino acid presequence and, unlike V-type ATPases, contained two membrane-spanning domains rather than four. The hydrophobic 43-kDa product of atpI is the smallest member so far found of the eukaryotic 100-kDa subunit family. Its electrophoretic band overlapped with the band of the A subunit. Therefore, all the gene products were found in our purified V(o)V1. We isolated the A3B3 subcomplex reconstituted from the isolated subunits and the A3B3γ subcomplex from subunit-expressing Escherichia coli. Electron microscopic observation of these subcomplexes revealed that the γ subunit of V1 filled the central cavity of A3B3 and might be central subunit, similar to the γ subunit of F1-ATPase.