Assembly of the peripheral domain of the bovine vacuolar H(+)-adenosine triphosphatase.
The biosynthesis and assembly of the peripheral sector (V1) of the vacuolar proton-translocating adenosine triphosphatase (V-ATPase) was studied in a bovine kidney epithelial cell line. Monolayer cultures of cells were metabolically radiolabeled with Tran35S-label and the V-ATPase subsequently immunoprecipitated using a monoclonal antibody raised against the bovine brain-coated vesicle proton pump. The V-ATPase immunoprecipitated from the bovine kidney cell line has a subunit composition very similar to that of the bovine brain-coated vesicle proton pump and the V-ATPase prepared from other kidney tissues. Radiolabeling the cells for increasing times showed that the V1 or peripheral portion of the V-ATPase is assembled within 10-15 min; the intact V1V0 complex is also detectable within 10-15 min. Fractionation of the cells into cytosolic and membrane components prior to immunoprecipitation revealed that there is a significant pool of V1 in the cytosol; a similar complex is also found in bovine brain cytosol. Pulse-chase studies suggest that this cytosolic pool is not an obligate precursor for membrane-bound V1V0 and does not exchange with the membrane V1 population at later 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 completed in the endoplasmic reticulum prior to distribution of the enzyme throughout the cell, with a cytosolic pool of V1 of unknown function existing in parallel with the fully assembled complex.
Myers
M
M
Department of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
Forgac
M
M
D000818:Animals; D002417:Cattle; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007668:Kidney; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000648:ultrastructure; D008970:Molecular Weight; D006180:Proton-Translocating ATPases / Q000648:ultrastructure*; D014617:Vacuoles / Q000201:enzymology*
D046911:Macromolecular Substances; D008565:Membrane Proteins; D006180:Proton-Translocating ATPases