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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM047282-02
Application #
3306738
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1992-05-01
Project End
1996-04-30
Budget Start
1993-05-01
Budget End
1994-04-30
Support Year
2
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Chicago
Department
Type
Schools of Medicine
DUNS #
225410919
City
Chicago
State
IL
Country
United States
Zip Code
60637
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Sarkar, S; Steck, T L (1997) An NADH-dependent disulfide reductase activity in the endoplasmic reticulum of Dictyostelium discoideum. Biochem Biophys Res Commun 234:313-5
Steck, T L; Chiaraviglio, L; Meredith, S (1997) Osmotic homeostasis in Dictyostelium discoideum: excretion of amino acids and ingested solutes. J Eukaryot Microbiol 44:503-10
Padh, H (1995) Electromagnetic purification of endocytic vacuoles and acidosomes from Dictyostelium. Arch Biochem Biophys 316:643-8
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Steck, T L; Lavasa, M (1994) A general method for plasma membrane isolation by colloidal gold density shift. Anal Biochem 223:47-50
Lange, Y; Steck, T L (1994) Cholesterol homeostasis. Modulation by amphiphiles. J Biol Chem 269:29371-4

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