A NOVEL ACCESSORY SUBUNIT FOR VACUOLAR H-ATPASE FROM CHROMAFFIN GRANULES()

Three subunits, Ac115, Ac39, and the proteolipid, were positively iden tified in the membrane sectors of V-ATPases from different sources. We searched for organelle-specific protein in purified preparations of V -ATPase from bovine chromaffin granules. A diffused protein band at a position of about 45 kDa was identified in SDS-polyacrylamide gels of the above preparation. Following digestion with endopeptidase Glu-C (V -8), a polypeptide of about 10 kDa was isolated and subjected to amino acid sequencing. Hence, the cDNA encoding the protein Ac45 was cloned from a bovine adrenal me dulla library. The cDNA sequence contains an open read ing frame encoding a protein of 468 amino acids with a calc ulated molecular mass of 51,786 daltons. A potential signal sequence c omprised of the first 35 amino acids and a potential transmembrane dom ain at the C terminus of the protein were identified. There exist seve n potential glycosylation sites between the aforementioned protein mot ifs. Experiments with a specific antibody against Ac45 demonstrated th at it is copurifying with the V-ATPase from chromaffin granules. Immun o logical cross-reactivity was observed with purified V-ATPase from bo vine kidney microsomes but not from plasma membranes of epithelial cel ls. Cell-free expression of the protein from synthetic mRNA produced a single protein band at about 50 kDa on SDS gels. Upon inclusion of do g pancreas microsomes in the reaction mixture, a slow migrating band s ensitive to peptide:N-glycosidase F was observed.