We have examined the ameba Dictyostelium discoideum for evidence of a discrete, prelysosomal, acidic receiving compartment in endocytosis. We observed in the cytoplasm abundant round vacuoles with diameters up to 2 microns that concentrated acridine orange by a process inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). They were therefore taken to be acidic. The vacuoles were observed to fuse nearly quantitatively with primary phagosomes over 30 min and thereby to confer upon them the ability to accumulate acridine orange. The entry into lysosomes of phagocytic cargo occurred later. In the absence of phagocytosis, almost all of the acidic vacuoles rapidly accumulated fluorescent markers that had either been covalently coupled to the cell surface or fed as the soluble dextran conjugate. Therefore, these vacuoles also lie on the pathway of pinocytosis. A prominent subcellular ATPase activity inhibited by 25 microM NBD-Cl co-distributed on sucrose equilibrium density gradients with vacuoles capable of concentrating acridine orange in vitro. The peak was broad and more buoyant than that bearing lysosomal acid hydrolases, which contained only a minor amount of this ATPase. Also migrating in the buoyant peak were internalized plasma membrane markers; e.g., 3H-galactose had been covalently coupled to the surface of intact cells and allowed to enter pinosomes. We conclude that in D. discoideum an extensive prelysosomal vacuolar compartment provides the proton pumps that acidify both phagosomes and pinosomes.

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