The acidosome, a newly-described organelle, is the principal repository of the vacuolar H+-ATPase in the amoeba, Dictyostelium discoideum, and the source of protons for its endocytic circuit. The principal aims of this project are to characterize this organelle and explore its relationship to the acidic compartments of higher eukaryotes, including man. The integral membrane proteins of purified acidosomes will be enumerated and examined for covalently bound sugar and phosphate as well as for their associations with other proteins. The aqueous contents of acidosomes will be analyzed for inorganic ions, metabolites, and proteins. The ultrastructure and relationships of acidosomes to other organelles will be analyzed in intact cells by immunofluorescence and immunogold electron microscopy and by subcellular fractionation. The occurrence and distribution of acidosome proteins in other organelles will be explored. Similarly, the presence in other living kingdoms of organelles corresponding to acidosomes will be tested. A new approach to identifying acidic vacuoles by electron microscopy will be pursued. Two kinds of transport activity will be examined in intact acidosomes: a) Is there H+ antiport of solutes into the lumen of the organelle? b) Do solutes besides H+ move from acidosomes to endosomes? Magnetic fractionation will be used to isolate endosomes bearing ingested iron oxide particles. Acidosomes and plasma membranes will similarly be subjected to affinity/magnetic purification. An assay for the in vitro reassociation of acidosomes with endosomes will be developed. Then, the cytosolic and membrane proteins involved in the reassociation of acidosomes and endosomes will be pursued using monoclonal antibodies as probes.
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