| Abstract: | We have recently described a mutant of Chinese hamster ovary cells, termed G.7.1, that contains a temperature-sensitive, conditionally lethal mutation resulting in defective vacuolar acidification (Marnell, M. H., Mathis, L. S., Stookey, M., Shia, S.-P., Stone, D.K., and Draper, R. K. (1984) J. Cell Biol. 99, 1907-1916). To further characterize the lesion, clathrin-coated vesicles were partially purified from wild type and G.7.1 cells, and the thermolabilities of vanadate and oligomycin-insensitive, N-ethylmaleimide-sensitive, H+-ATPase activity, 32Pi-ATPase exchange activity, and proton pumping were compared. All three parameters of H+ pump activity were markedly diminished by preincubation at 44 degrees C for vesicles harvested from the G.7.1 cells, but not for those from wild type cells. Phosphatidylserine did not protect against heat inactivation in vesicle fractions prepared from G.7.1 cells. The results suggest that the mutation responsible for defective acidification in G.7.1 cells is expressed at the level of the proton pump of organelles present in our clathrin-coated vesicle-enriched preparation. |
