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 . In this paper, we show that prelysosomal endocytic vesicles in HepG2 human hepatoma cells have an internal pH of approximately 5.4 . (We previously reported that 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, we developed digital image analysis methods 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 "'I-ASOR demonstrated that acidification precedes degradation by more than 30 min at 37°C . At 23°C ligand degradation ceases almost entirely, but endocytic vesicle acidification and receptor recycling continue . These results demonstrate that 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 . This suggests that monensin 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 rapidly 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.We recently demonstrated that endocytic vesicles in mouse fibroblasts become acidified shortly after formation (31), and suggested that this early acidification of endocytic vesicles may be a necessary step in processes such as receptor recycling and the penetration of enveloped viruses and diphtheria toxin into the cytoplasm . While there have been several recent studies that support this hypothesis through indirect evidence, there have been very few direct measurements of the pH of endocytic vesicles. In addition to our measurement in mouse fibroblasts using fluorescein-labeled a 2 -macroglobulin as a pH probe, van Renswoude et al . (34) have shown that fluoresceinlabeled transferrin taken up by erythroleukemic cells is in nonlysomal intracellular compartments with an average pH of 5 .5 ...

Legionella pneumophila inhibits acidification of its phagosome in human monocytes.

“…The fluorescence intensities of a 40-vm-diam circle containing one to three monocytes that had phagocytized fluorescent bacteria or erythrocytes was measured using a Leitz MPV microscope spectrofluorometer mounted on a Diavert inverted microscope as previously described (16,18). A similar method has been used with other cells (19,20).…”

“…Four frames of all images were averaged to improve the signal-tonoise ratio. Local background fluorescence was determined using previously described programs (18) and was taken as the 25th percentile of intensity in a 32 x 64 picture element (~6.5 x 13 urn) region.…”

“…In some cases, it has been reported that endocytic compartments undergo further acidification as iysosomal fusion occurs (27)(28)(29). However, in other cell types, it has been found that the pH of prelysosomal endocytic compartments is about the same as the pH of lysosomes (18,21,27). We do not know if phagosomes containing formalin-killed L. pneumophila become more acidic after they fuse with lysosomes (see below).…”

Acidification of morphologically distinct endosomes in mutant and wild-type Chinese hamster ovary cells.

“…Video output from the recorder was passed through a model CCDHPS time base corrector (Fortel Inc., Norcross, GA), and images were digitized on a model 1P8500 image processor (Gould, Inc., San Jose, CA), with a MicroVAX II (Digital Equipment Corp., Marlboro, MA) as host computer. Digitized images were further processed as described (10,18,37,41). Briefly, 490-and 450-nm images were divided into 64 pixel • 64 pixel regions (13.4 gm • 13.4 gm), and the background fluorescence (defined as the median [50th percentile] intensity of the region) was subtracted from each pixel.…”

“…To measure the pH of specific endocytic compartments we have used fluorescence microscopy and digital image analysis (18,35,37). We have previously shown in wild-type CHO cells that the recycling endosomes have a pH of 6.4, while the large endosomes have a pH of 5.2 (41).…”