Immunolocalization of the 17 kDa vacuolar H(+)-ATPase subunit c in Heliothis virescens midgut and malpighian tubules with an anti-peptide antibody.

The transmembrane sector of V-ATPases is involved in proton conduction across the membrane where a 15-17 kDa proteolipid forms a putative proton channel. An affinity-purified rabbit polyclonal antibody was developed to an antigenic and putatively extracellular region of a cloned 17 kDa proteolipid. In larval tissue sections, this antibody labeled the midgut goblet cell apical membrane in Heliothis virescens (Lepidoptera: Noctuidae) and the apical membrane in Malpighian tubules from H. virescens and Manduca sexta (Lepidoptera: Sphingidae). The antibody also recognized the 17 kDa protein in an immunoblot of H. virescens Malpighian tubule homogenate. Northern blot analysis revealed the presence of two transcript sizes in the midgut (1.9 and 1.2 kb) and Malpighian tubules (2.2 and 1.9 kb). Our results strongly support the hypothesis that the 17 kDa protein is a component of the V-ATPase, where it is thought to be the proton-conducting subunit. This polyclonal antibody may provide a powerful tool for V-ATPase regulation studies, while the use of the anti-peptide antibody approach may be helpful for the immunolocalization of other ductins.