Reconstitution of the association of endocytic vacuoles and acidosomes from Dictyostelium.
In the amoeba, Dictyostelium discoideum, endocytic vacuoles are acidified by proton pumps which reside not in their membranes but in an associated organelle which we call the acidosome. These two organelles can be dissociated in vitro, and we now describe conditions for their functional reassociation. Fluorescein 5-isothiocyanate-dextran was fed to amoebae to report on the pH of their endocytic vacuoles. Following homogenization, the endocytic vacuoles were dissociated from acidosomes by removing Mg2+ and cytosol and purged of their native acidity by transient exposure to nigericin. The endocytic vacuoles could then be reacidified by ATP if first preincubated under these optimized conditions: 30 degrees C for 30 min in the presence of acidosomes, a 4-fold excess of cytosol, and 5 mM Mg2+ at pH 7.4. Reacidification was observed with early but not late endocytic compartments. Mn2+ and Ca2+ were poor substitutes for Mg2+; albumin did not substitute for cytosol. Neither Ca2+, ATP, nor adenosine 5'-O-(3-thiotriphosphate) affected reconstitution appreciably; guanosine 5'-O-(3-thiotriphosphate) inhibited reacidification by 50% when present during preincubation at 0.1 mM. Warming the cytosol to 50 degrees C or exposing it to protease abolished its activity but N-ethylmaleimide did not. Molecular sieving indicated that the cytosolic factor was a macromolecule. We conclude that the specific functional association of acidosomes and endocytic vacuoles can be reconstituted in vitro with soluble proteins plus Mg2+.
Padh
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H
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.
Lavasa
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Steck
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TL
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001692:Biological Transport; D004023:Dictyostelium / Q000166:cytology* / Q000201:enzymology; D004705:Endocytosis; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D011522:Protons; D014617:Vacuoles / Q000378:metabolism*
D011522:Protons; D008345:Manganese; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D008274:Magnesium