Rapid acidification of endocytic vesicles containing [alpha]2-macroglobulin

Author: Tycko, B.; Maxfield, F.R.

Description: We have used fluorescein-labeled α₂-macroglobulin (α₂M) to measure pH changes in the microenvironment of internalized ligands following receptor-mediated endocytosis. Fluorescence intensities of single BALB/c 3T3 mouse fibroblasts were measured by using a microscope spectrofluorometer with narrow bandpass excitation filters. The pH was determined from the ratio of fluorescein fluorescence intensities with 450 nm and 490 nm excitation. A standard pH curve was obtained by incubating cells with F-α₂M for 30 min at 37°C followed by fixation and incubation in buffers of varying pH. To measure the pH of endocytic vesicles, cells were incubated with F-α₂M for 15 min at 37°C. Fluorescence intensities were measured on living cells within 5 min of rinsing. Under these conditions, the pH of the F-α₂M microenvironment was 5.0 ± 0.2. Using colloidal gold-α₂M for electron microscopic localizations we have verified that, under these conditions, α₂M is predominantly in uncoated vesicles that are negative for acid phosphatase activity. With further incubation for hr, we obtained a pH of 5.0 ± 0.2 for the F-α2M. Using fluorescein dextran, we obtained a lysosomal pH of 4.6 ± 0.2. These results indicate that endocytic vesicles become acidic prior to fusion with lysosomes.