The long-range objectives of these studies are to understand mechanisms involved in the exchange of membrane between the plasma membrane and internal membrane systems. We use the small soil ameba, Acanthamoeba, as a model system. Acanthamoeba feeds solely by endocytosis, resulting in a high volume of membrane internalization and recycling. The current experimental question was whether newly internalized endocytic membrane is randomized with the existing intracellular pool of vacuolar membrane by internal fusions. Radioactive yeast were fed to amebas and the rate of digestion determined as a function of yeast load. The results showed a """"""""saturation"""""""" of digestive capacity by large loads and suggested that hydrolases were limiting. Cytochemical studies showed that fewer phagosomes acquire hydrolases in heavily loaded cells. Examination of the cells in the electron microscope showed virtually no evidence of phagosome-phagosome fusion within 60 min. We demonstrated that later-formed phagosomes are less likely to obtain hydrolases by feeding amebas varying loads of yeast followed by latex beads. The latex bead phagosomes were isolated and their hydrolase specific activity determined as a function of yeast load. When the yeast load was heavy, hydrolase content of the later-formed bead phagosomes was much reduced. Thus using the hydrolase content of phagosomes as an internal marker, we find no morphological or biochemical evidence for fusion of newly formed phagosomes. We conclude that the membrane entering the cell through endocytosis does not randomize by rapid internal fusions.
