DISPOSITION AND ORIENTATION OF DUCTIN (DCCD-REACTIVE VACUOLAR H+-ATPASE SUBUNIT) IN MAMMALIAN MEMBRANE COMPLEXES

The disposition and orientation of mouse duct in (the subunit c of the vacuolar H+-ATPase) in gap junctions has been examined. Like the Nephrops norvegicus (arthropod) form, mouse ductin in the intact junctional structure is resistant to high levels of nonspecific proteinase, suggesting that it is for the most part buried in the bilayer. Antisera to an octapeptide near the N-terminus cross-react with ductins in gap junction preparations from four different mouse tissues, from chicken and Xenopus laevis liver, and from N. norvegicus hepatopancreas. The antisera and antibodies, affinity purified against the octapeptide, agglutinate isolated gap junctions, suggesting that the N-terminus is located on the exposed surface, equivalent to the cytoplasmic face of an intercellular gap junction. The antibodies also block dye coupling when injected into cells in culture, confirming the cytoplasmic location of the epitope. The lipophylic reagent dicylohexyl carbodiimide (DCCD), which targets carboxyl groups within the membrane and selectively reacts with ductin in N. norvegicus gap junction preparations, rapidly inhibits junctional communication. Bafilomycin A1, which inhibits V-ATPase and stops vacuolar acidification, does not affect dye coupling, showing that the inhibition seen with antibodies and DCCD is not an indirect consequence of their action on the ductin of V-ATPase. Consistent with this interpretation the anti-peptide antibodies do not bind to intact chromaffin granules or inhibit their V-ATPase activity, but do bind to osmotically disrupted granule membrane. This suggests that ductin has an orientation (N-terminus pointing away from the cytoplasm) in the vacuolar membrane opposite to that in the gap junction membrane. © 1993 Academic Press, Inc.