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

V-type ATPase (VoV1) capable of ATP-driven H+ pumping and of H+ gradient dr iven ATP synthesis was isolated from a thermophilic eubacterium, Thermus th ermophilus. When the enzyme was analyzed by gel electrophoresis in the pres ence of sodium dodecyl sulfate, it showed eight polypeptide bands of which four were subunits of V-1. We also isolated the VoV1 operon, containing nin e genes in the order of atpG-I-L-E-X-F-A-B-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 V-1 subunits; atpA, B, D , and F encoded the A, B, gamma, and delta subunits, respectively. The firs t 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 presequen ce and, unlike V-type ATPases, contained two membrane-spanning domains rath er 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 pr oducts were found in our purified VoV1. We isolated the A(3)B(3) subcomplex reconstituted from the isolated subunits and the A(3)B(3)gamma subcomplex from subunit-expressing Escherichia coli. Electron microscopic observation of these subcomplexes revealed that the gamma subunit of V-1 filled the cen tral cavity of A,B, and might be central subunit, similar to the gamma subu nit of F-1-ATPase.