Cloning and immunolocalization of a 17 kDa protein implicated in V-ATPase mediated proton transport and gap junctional communication in the tobacco budworm, Heliothis virescens

The objective of this project was to establish the structure and localization of a 17kDa protein thought to be the proton conducting subunit c in the vascular proton ATPase (V-ATPase). This 17kDa protein belongs to a family of conserved proteins ranging from 15-18kDa known as ductins. The strategy was to utilize the polymerase chain reaction (PCR) to synthesize a probe that was used for the isolation of the corresponding 17kDa protein cDNA. The published nucleotide sequences of four species were compared and conserved regions were identified. Two oligonucleotide primers were synthesized and used to amplify a region of the putative 17kDa protein from larval midgut and Malpighian (M.) tubules cDNA. The obtained product was used as a probe for screening H. virescens lambda ZAPII cDNA library from larval midgut and M. tubule. Four clones were rescued from the library, one was 1.9kb and the other three, 1.4kb. The 1.91kb clone encodes for a protein of 156 amino acids with a predicted molecular weight of 17.2 kDa. Comparisons of the deduced amino acid sequence revealed high degree of identity with the fruit fly (94.2%), mouse, bovine, oat and yeast proteolipids of V-ATPases. The glutamic acid residue present in position 139 might represent the DCCD binding site. The 1.9kb insert was used as a probe for Southern and northern blotting. Southern blot analysis appears to indicate the existence of only one gene, however, three transcripts were detected, of 1.2 and 1.9kb in the midgut and of 1.9 and 2.2kb in the M. tubules.; Anti-peptide antibodies were developed against the sequence DAPSNNYTLYKG, corresponding to the putative second extramembrane loop. Affinity-purified antibodies labeled the goblet cell apical membranes of midgut and apical membranes of M. tubule in H. virescens feeding larvae, and M. tubule apical membrane of M. sexta. These antibodies identified a 17kDa band in a western blot of M. tubule homogenate.; Immunocytochemistry during the L{dollar}sb4{dollar}-L{dollar}sb5{dollar} larval molt and early post ecdysis (PE) revealed that the temporal and spatial expression of the 17kDa protein varies in the midgut goblet cell apical membrane, and in intercellular areas. During the half head slippage stage and up to 1 h PE into the fifth instar, the labeling of goblet cell cavities with antibodies against both, the 17kDa protein and the V-ATPase peripheral subunit B, was fainter than during the feeding period. Apical valves of newly formed goblet cells were intensely stained with the anti-17kDa antibodies but not with the anti-B subunit antiserum. A punctuated labeling pattern was observed during this time in intercellular areas only with the anti-17kDa antibodies and was confirmed by confocal microscopy.; The results indicate the involvement of the 17kDa protein as the V-ATPase subunit c in larval midgut and Malpighian tubule, and possibly as a component of gap junctions in the midgut.