Biosynthesis of the vacuolar H+-ATPase accessory subunit Ac45 in Xenopus pituitary

Vacuolar H+-ATPases (V-ATPases) mediate the acidification of multiple intra cellular compartments, including secretory granules in which an acidic mili eu is necessary for prohormone processing. A search for genes coordinately expressed with the prohormone proopiomelanocortin (POMC) in the melanotrope cells of Xenopus intermediate pituitary led to the isolation of a cDNA enc oding the complete amino-acid sequence of the type I transmembrane V-ATPase accessory subunit Ac45 (predicted size 48 kDa). Comparison of Xenopus and mammalian Ac45 sequences revealed conserved regions in the protein that may he of functional importance. Western blot analysis showed that immunoreact ive Ac45 represents a approximate to 40-kDa product that is expressed predo minantly in neuroendocrine tissues; deglycosylation resulted in a approxima te to 27-kDa immunoreactive Ac45 product which is smaller than predicted fo r the intact protein. Biosynthetic studies revealed that newly synthesized Xenopus Ac45 is an N-glycosylated protein of approximate to 60 kDa; the non glycosylated, newly synthesized form is approximate to 46 kDa which is simi lar to the predicted size. Immunocytochemical analysis showed that in Xenop us pituitary, Ac45 is highly expressed in the biosynthetically active melan otrope cells. We conclude that the regionally conserved Xenopus Ac45 protei n is synthesized as an N-glycosylated approximate to 60-kDa precursor that is intracellularly cleaved to an approximate to 40-kDa product and speculat e that it may assist in the V-ATPase-mediated acidification of neuroendocri ne secretory granules.