The multigene family of the tobacco hornworm V-ATPase: novel subunits A, C, D, H, and putative isoforms

The plasma membrane V-ATPase from Manduca sexta (Lepidoptera, Sphingidae) l arval midgut is composed of at least 12 subunits, eight of which have alrea dy been identified molecularly [Wieczorek et al., J. Bioenerg. Biomembr. 31 (1999) 67-74]. Here we report primary sequences of subunits C, D, H and a, which previously had not been identified in insects. Expression of recombi nant proteins, immunostaining and protein sequencing demonstrated that the corresponding proteins are subunits of the Manduca V-ATPase. Genomic Southe rn blot analysis indicated the existence of multiple genes encoding subunit s G, a, c, d and e. Moreover. multiple transcripts were detected in Norther n blots from midgut poly(A) RNA for subunits B, G, c and d. Thus, these pol ypeptides appear to exist as multiple isoforms that could be expressed eith er in different tissues or at distinct locations within a cell. By contrast subunits A, C, D, E, F and Fl appear to be encoded by single transcripts a nd therefore should be present in any Manduca V-ATPase, independent of its subcellular or cell specific origin. (C) 2000 Elsevier Science B.V. All rig hts reserved.