Biosynthesis of the vacuolar H+-ATPase accessory subunit Ac45 in Xenopus pituitary.
Vacuolar H+-ATPases (V-ATPases) mediate the acidification of multiple intracellular compartments, including secretory granules in which an acidic milieu 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 encoding 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 be of functional importance. Western blot analysis showed that immunoreactive Ac45 represents a approximately 40-kDa product that is expressed predominantly in neuroendocrine tissues; deglycosylation resulted in a approximately 27-kDa immunoreactive Ac45 product which is smaller than predicted for the intact protein. Biosynthetic studies revealed that newly synthesized Xenopus Ac45 is an N-glycosylated protein of approximately 60 kDa; the nonglycosylated, newly synthesized form is approximately 46 kDa which is similar to the predicted size. Immunocytochemical analysis showed that in Xenopus pituitary, Ac45 is highly expressed in the biosynthetically active melanotrope cells. We conclude that the regionally conserved Xenopus Ac45 protein is synthesized as an N-glycosylated approximately 60-kDa precursor that is intracellularly cleaved to an approximately 40-kDa product and speculate that it may assist in the V-ATPase-mediated acidification of neuroendocrine secretory granules.
Holthuis
J C
JC
Department of Animal Physiology, University of Nijmegen, Toernooiveld, The Netherlands.
Jansen
E J
EJ
Schoonderwoert
V T
VT
Burbach
J P
JP
Martens
G J
GJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005075:Biological Evolution; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D006868:Hydrolysis; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D025262:Vacuolar Proton-Translocating ATPases; D014982:Xenopus laevis
D018076:DNA, Complementary; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases