IMMUNOLOCALIZATION OF THE 17-KDA VACUOLAR H-ATPASE SUBUNIT-C IN HELIOTHIS-VIRESCENS MIDGUT AND MALPIGHIAN TUBULES WITH AN ANTIPEPTIDE ANTIBODY()

The transmembrane sector of V-ATPases is involved in proton conduction across the membrane where a 15-17 kDa proteolipid forms a putative pr oton channel. An affinity-purified rabbit polyclonal antibody was deve loped to an antigenic and putatively extracellular region of a cloned 17kDa proteolipid. In larval tissue sections, this antibody labeled th e midgut goblet cell apical membrane in Heliothis virescens (Lepidopte ra: Noctuidae) and the apical membrane in Malpighian tubules from H. v irescens and Manduca sexta (Lepidoptera: Sphingidae). The antibody als o recognized the 17kDa protein in an immunoblot of H. virescens Malpig hian tubule homogenate. Northern blot analysis revealed the presence o f two transcript sizes in the midgut (1.9 and 1.2kb) and Malpighian tu bules (2.2 and 1.9kb). Our results strongly support the hypothesis tha t the 17kDa protein is a component of the V-ATPase, where it is though t to be the proton-conducting subunit. This polyclonal antibody may pr ovide a powerful tool for V-ATPase regulation studies, while the use o f the anti-peptide antibody approach may be helpful for the immunoloca lization of other ductins.