Rapid acidification of endocytic vesicles containing asialoglycoprotein in cells of a human hepatoma line.

Abstract

Acidification of endocytic vesicles has been implicated as a necessary step in various processes including receptor recycling, virus penetration and the entry of diphtheria toxin into cells. However, there have been few accurate pH measurements in morphologically and biochemically defined endocytic compartments. Prelysosomal endocytic vesicles in HepG2 human hepatoma cells have an internal pH of .apprx. 5.4. Similar vesicles in mouse fibroblasts have a pH of 5.0. The pH values were obtained from the fluorescence excitation profile after internalization of fluorescein labeled asialo-orosomucoid (ASOR). To make fluorescence measurements against the high autofluorescence background, digital image analysis methods were developed for estimating the pH within individual endocytic vesicles or lysosomes. Ultrastructural localization with colloidal gold ASOR demonstrated that the pH measurements were made when ligand was in tubulovesicular structures lacking acid phosphatase activity. Biochemical studies with 125I-ASOR demonstrated that acidification precedes degradation by more than 30 min at 37.degree. C. At 23.degree. C ligand degradation ceases almost entirely, but endocytic vesicle acidification and receptor recycling continue. Acidification of endocytic vesicles, which causes ligand dissociation, occurs without fusion of endocytic vesicles with lysosomes. Methylamine and monensin raise the pH of endocytic vesicles and cause a ligand-independent loss of receptors. The effects on endocytic vesicle pH are rapidly reversible upon removal of the perturbant, but the effects on cell surface receptors are slowly reversible with methylamine and essentially irreversible with monensin. Monensin apparently can block receptor recycling at a highly sensitive step beyond the acidification of endocytic vesicles. Taken together with other direct and indirect estimates of endocytic vesicle pH, these studies indicate that endocytic vesicles in many cell types reapidly acidify below pH 5.5, a pH sufficiently acidic to allow receptor-ligand dissociation and the penetration of some toxin chains and enveloped virus nucleocapsids into the cytoplasm.