Endocytic cargo traverses endosomal vacuoles before encountering acid hydrolases in lysosomes. Both endosomes and lysosomes have an acidic interior believed created by vacuolar proton pumps resident on their membranes. Lower Ph in these endocytic compartments is crucial for lysosomal hydrolases, and the sorting of ligands, receptors, and membrane components. Where these membranes derive their pumps is unclear. Data from studies on Dictyostelium discoideum indicate that endosomes and lysosomes have undetectable proton pumps on their membranes. In addition, abundant proton pumps are present on a discrete and easily separable vacuole, termed "acidosome." The data suggest that through transient association, in the presence of cytosol and magnesium ions, the acidosomes provide protons but not proton pumps to the endosomes. The proposed transport of protons and counterions but not large molecules from the proton-pumping ATPase rich acidosomes to the endocytic vacuoles might proceed through small channels spanning their apposed membranes, perhaps akin to synaptophysin pores in presynaptic ends or gap junctions. Experiments are proposed to elaborate and test the hypothesis that proton transfer is the mechanism of endosomal acidification. The acidosomes, endosomes, and cytosol factor(s) will be purified. The reconstitution of proton transfer between organelles in vitro using the purified preparations of acidosomes, endosomes, and the required cytosol factor(s) should provide sound evidence for this hypothesis. In addition, dynamic communication between these organelles and the required factor(s) will be probed under differing modes of endocytosis. %%% Endocytosis, the uptake of materials from the cellular environment by invagination of the plasma membrane and pinching off to form a membrane-enclosed intracellular organelle containing the material in question, is a crucial process in the life of a eukaryotic cell. It is key to transport and assimilation of nutrients, signal transduction, and biological defense mechanisms. This study will enhance our understanding of the fundamental process of endocytosis.
