RAT-LIVER ENDOCYTIC COATED VESICLES DO NOT EXHIBIT ATP-DEPENDENT ACIDIFICATION IN-VITRO

Coated vesicle fractions from a variety of tissues have been found to contain a vacuolar proton ATPase. Since these fractions contain both p lasma membrane- and Golgi-derived coated vesicles, we sought to determ ine specifically whether endocytic coated vesicles from rat liver cont ain an active vacuolar proton ATPase. Endocytic vesicles (coated vesic les and endosomes) were selectively labeled with pH-sensitive endocyti c tracers (fluorescein isothiocyanate-dextran or -asialoorsomucoid). C oated vesicles were then separated from endosomes by sucrose density g radient centrifugation. Although the endosomal fractions were found to exhibit significant ATP-dependent acidification activity, highly puri fied coated vesicles containing pH-sensitive endocytic tracers were un able to generate a pH gradient in response to ATP addition. The coated vesicles could be passively acidified, however, by creating potassium diffusion potentials, indicating that they were in fact capable of ma intaining proton gradients. Moreover, significant ATP-dependent acidif ication activity was observed when the coated vesicle fractions were a ssayed using the nonselective externally added pH probe acridine orang e. Thus, it appears that rat liver endocytic coated vesicles do not co ntain a functional proton pump. The active vacuolar proton ATPase foun d in these fractions instead reflected the presence of Golgi-derived c oated vesicles or contaminating membranes.