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One of the early events in the pathway of receptor-mediated endocytosis is the acidification of the newly formed endocytic vesicle. To examine the mechanism of acidification, we used fluorescein-labeled c~2-macroglobulin (F-a2M) as a probe for endocytic vesicle pH. Changes in pH were determined from the change in fluorescein fluorescence at 490-nm excitation as measured with a microscope spectrofluorometer. After endocytosis of F-a2M, mouse fibroblast cells were permeabilized by brief exposure to the detergent digitonin. Treatment with the ionophore monensin or the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) caused a rapid increase in the pH of the endocytic vesicle. Upon removal of the ionophore, the endocytic vesicle rapidly acidified only when MgATP or MgGTP was added. Neither ADP nor the nonhydrolyzable analog, adenosine 5'-(fl,3,-imido)triphosphate (AMP-PNP) could support acidification. The ATPdependent acidification did not require a specific cation or anion in the external media. Acidification was insensitive to vanadate and amiloride but was inhibited by Zn 2÷ and the anion transport inhibitor diisothiocyanostilbene disulfonic acid (DIDS).We also examined the acidification of lysosomes with the permeabilized cell system, using fluorescein isothiocyanate dextran as probe. DIDS inhibited the ATP-dependent reacidification of lysosomes, although at a lower concentration than that for inhibition of endocytic vesicle reacidification. These results demonstrate that endocytic vesicles contain an ATP-dependent acidification mechanism that shares similar characteristics with the previously described lysosomal proton pump.Many different peptide hormones, serum proteins, bacteria toxins, and enveloped viruses bind to receptors on cell surfaces and are internalized by the process of receptor-mediated endocytosis (reviewed in references I and 2). For many ligands this pathway has been shown to involve clustering of occupied receptors over clathrin-coated pits and entry into the cell through endocytic vesicles. It has been demonstrated by fluorescent double labeling studies that the serum protease inhibitor a2-macroglobulin (a2M) is internalized within the same endocytic vesicle as diphtheria toxin (3), epidermal growth factor (4), insulin (4), low density lipoprotein (5), and thyroid hormone (6). These results suggest that ligands share a common entry point in the pathway of endocytosis.The final destination for many of these ligands is lysosomes, where they are degraded. However, it has been demonstrated that a sorting process can take place that enables the receptor to escape degradation and return to the cell surface where it is reused. This has been shown to occur for several receptors, including the a2M receptor (7), asialoglycoprotein receptor (8-10), insulin receptor (11), low density lipoprotein receptor (12), and the mannose receptor (13). For this differential processing to occur, it is evident that the ligand must dissociate from its receptor, allowing the unoccupied rece...

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