Padh H, Lavasa M, Steck T L
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.
J Biol Chem. 1991 Jul 5;266(19):12123-6.
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+.
在变形虫盘基网柄菌（Dictyostelium discoideum）中，内吞泡通过质子泵酸化，质子泵并不存在于内吞泡膜上，而是存在于一种相关的细胞器中，我们将其称为酸小体。这两种细胞器在体外可以解离，我们现在描述它们功能重新结合的条件。将异硫氰酸荧光素标记的葡聚糖喂给变形虫，以报告其内吞泡的pH值。匀浆后，通过去除Mg2+和胞质溶胶，使内吞泡与酸小体解离，并通过短暂暴露于尼日利亚菌素去除其天然酸度。如果先在以下优化条件下预孵育，内吞泡随后可以被ATP重新酸化：在酸小体存在下于30℃孵育30分钟，胞质溶胶过量4倍，pH 7.4时5 mM Mg2+。早期而非晚期内吞区室观察到重新酸化。Mn2+和Ca2+不能很好地替代Mg2+；白蛋白不能替代胞质溶胶。Ca2+、ATP或腺苷5'-O-(3-硫代三磷酸)均未对重组产生明显影响；在预孵育期间以0.1 mM存在时，鸟苷5'-O-(3-硫代三磷酸)可使重新酸化抑制50%。将胞质溶胶加热至50℃或使其暴露于蛋白酶会使其失活，但N-乙基马来酰亚胺不会。分子筛分析表明胞质因子是一种大分子。我们得出结论，酸小体和内吞泡的特异性功能结合可以在体外通过可溶性蛋白质加Mg2+进行重组。
