ASSEMBLY OF THE PERIPHERAL DOMAIN OF THE BOVINE VACUOLAR H-ADENOSINE TRIPHOSPHATASE()

The biosynthesis and assembly of the peripheral sector (V1) of the vac uolar proton-translocating adenosine triphosphatase (V-ATPase) was stu died in a bovine kidney epithelial cell line. Monolayer cultures of ce lls were metabolically radiolabeled with Tran S-35-label and the V-ATP ase subsequently immunoprecipitated using a monoclonal antibody raised against the bovine brain-coated vesicle proton pump. The V-ATPase imm unoprecipitated from the bovine kidney cell line has a subunit composi tion very similar to that of the bovine brain-coated vesicle proton pu mp and the V-ATPase prepared from other kidney tissues. Radiolabeling the cells for increasing times showed that the V1 or peripheral portio n of the V-ATPase is assembled within 10-15 min; the intact V1V0 compl ex is also detectable within 10-15 min. Fractionation of the cells int o cytosolic and membrane components prior to immunoprecipitation revea led that there is a significant pool of V1 in the cytosol; a similar c omplex is also found in bovine brain cytosol. Pulse-chase studies sugg est that this cytosolic pool is not an obligate precursor for membrane -bound V1V0 and does not exchange with the membrane V1 population at l ater times. No qualitative differences in assembly were observed when pulse-chase studies were performed at 15-degrees-C or in the presence of brefeldin A. This suggests that assembly of V1V0 is probably comple ted in the endoplasmic reticulum prior to distribution of the enzyme t hroughout the cell, with a cytosolic pool of V1 of unknown function ex isting in parallel with the fully assembled complex. (C) 1993 Wiley-Li ss, Inc.