THE PERIPHERAL COMPLEX OF THE TOBACCO HORNWORM V-ATPASE CONTAINS A NOVEL 13-KDA SUBUNIT-G

A prominent 16-kDa protein copurifies with the V-ATPase isolated from both posterior midgut and Malpighian tubules of Manduca sexta larvae a nd thus was believed to represent a V-ATPase subunit. [C-14]N,N'-dicyc lohexylcarbodiimide labeling and its position on SDS-electrophoresis g els revealed that this protein was different from the 17-kDa proteolip id. A cDNA clone encoding a highly hydrophilic protein with a calculat ed molecular mass of 13,692 Da was obtained by immunoscreening. Monosp ecific antibodies, affinity-purified to the 13-kDa recombinant protein expressed in Escherichia coli, specifically recognized the 16-kDa pro tein of the purified V-ATPase, confirming that a cDNA encoding this pr otein had been cloned. In vitro translation of the cRNA showed that th e cloned 13-kDa subunit behaved like a 16-kDa protein on SDS-electroph oresis gels. The cloned protein showed 37% amino acid sequence identit y to the 13-kDa V-ATPase subunit Vma10p recently cloned from yeast and some similarity to subunit b of bacterial F-ATPases. In contrast to t he Vma10p protein, which behaved like a V-0 subunit, the M. serta 13-k Da protein behaved like a V-1 subunit, since it could be stripped from the membrane by treatment with the chaotropic salt KI and by cold ina ctivation. When KI dissociated V-ATPase subunits were reassociated by dialysis that removed the KI, a soluble, 450-kDa complex of the M. ser ta V-ATPase could be purified by gel chromatography. This V-1 complex consisted of subunits A, B, E, and the 13-kDa subunit, confirming that the cloned protein is a new V-ATPase subunit and a member of the peri pheral V-1 complex of the V-ATPase. We designate this new V-1 componen t subunit G.