Ductin, a component of the V-ATPase, is developmentally regulated in Heliothis virescens midgut, and anti-ductin antibodies label lateral membranes

Abstract.

We previously cloned from Heliothis virescens a 16-kDa protein that is homologous to other ductin sequences. We also reported its immunolocalization with a specific affinity-purified anti-peptide antibody in the midgut and Malpighian tubule of feeding larvae, and concluded that the cloned proteolipid encodes the V-ATPase proton-transporting subunit c from the V₀ sector. We now present the immunolocalization of this protein in the midgut during the L₄-L₅ larval molt and early post-ecdysis into the fifth instar in H. virescens. The results show that the spatial expression of the 16-kDa protein is developmentally regulated. Labeling by anti-peptide antibody varies during the molt in the midgut goblet cell apical plasma membrane and the goblet cell apical valve. Epifluorescence and confocal microscopy revealed strong anti-ductin labeling in areas of cell-to-cell contact during the molt, and during early post-ecdysis into the fifth larval instar. The characteristic labeling pattern observed in areas of cell-to-cell contact is consistent with the claimed involvement of ductins in gap junctions. Conclusive evidence for the presence of the 16-kDa protein in areas of cell-to-cell contact in the midgut of feeding larvae is, however, lacking. V-ATPase regulation during the molt was also investigated by simultaneous immunohistochemistry with an anti-B subunit antiserum, a probe for the V₁ sector.