A novel accessory subunit for vacuolar H(+)-ATPase from chromaffin granules.
Three subunits, Ac115, Ac39, and the proteolipid, were positively identified 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 medulla library. The cDNA sequence contains an open reading frame encoding a protein of 468 amino acids with a calculated molecular mass of 51,786 daltons. A potential signal sequence comprised of the first 35 amino acids and a potential transmembrane domain at the C terminus of the protein were identified. There exist seven potential glycosylation sites between the aforementioned protein motifs. Experiments with a specific antibody against Ac45 demonstrated that it is copurifying with the V-ATPase from chromaffin granules. Immunological cross-reactivity was observed with purified V-ATPase from bovine kidney microsomes but not from plasma membranes of epithelial cells. Cell-free expression of the protein from synthetic mRNA produced a single protein band at about 50 kDa on SDS gels. Upon inclusion of dog pancreas microsomes in the reaction mixture, a slow migrating band sensitive to peptide:N-glycosidase F was observed.
Supek
F
F
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
Supekova
L
L
Mandiyan
S
S
Pan
Y C
YC
Nelson
H
H
Nelson
N
N
D000313:Adrenal Medulla / Q000201:enzymology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002837:Chromaffin Granules / Q000201:enzymology*; D018076:DNA, Complementary; D004285:Dogs; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney / Q000201:enzymology; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases; D014617:Vacuoles / Q000201:enzymology*
C089598:ATP6AP1 protein, human; D018076:DNA, Complementary; D008565:Membrane Proteins; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases