Padh H, Lavasa M, Steck T L
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.
J Biol Chem. 1991 Mar 25;266(9):5514-20.
The endocytic compartment in the amoeba Dictyostelium discoideum was labeled by feeding fluorescein 5-isothiocyanate-dextran. In homogenates containing 2 mM Mg2+, the compartments so labeled copurified with all of the vacuolar H(+)-ATPase activity in a dense peak. The fluorescence properties of the probe showed that these dense vacuoles were inherently acidic. Furthermore, after purging their residual acidity, they could be re-acidified by the addition of ATP. These data suggest that the H(+)-ATPase was structurally and functionally coupled to the endocytic space. The association of the H(+)-ATPase and endocytic compartment was reversed by the removal of either Mg2+ or traces of the cytosol. Endocytic vacuoles prepared in this way were deficient in vacuolar H(+)-ATPase activity and were not acidified upon addition of MgATP. The missing proton pumps were recovered in large buoyant vacuoles that lacked ingested fluorescein 5-isothiocyanate-dextran, acid hydrolases, and residual acidity. These vacuoles were also less susceptible than endosomes to disruption by digitonin, suggesting that their bilayers were low in sterols. These results indicate that the endocytic circuit in Dictyostelium is acidified by a discrete and separable proton-pumping organelle.
通过喂食异硫氰酸荧光素 - 葡聚糖对盘基网柄菌变形虫中的内吞区室进行标记。在含有2 mM Mg2+的匀浆中，如此标记的区室与所有液泡H(+)-ATP酶活性一起在一个致密峰中共纯化。探针的荧光特性表明这些致密液泡本质上是酸性的。此外，在清除其残余酸度后，通过添加ATP它们可以重新酸化。这些数据表明H(+)-ATP酶在结构和功能上与内吞空间偶联。去除Mg2+或微量胞质溶胶会逆转H(+)-ATP酶与内吞区室的结合。以这种方式制备的内吞液泡缺乏液泡H(+)-ATP酶活性，并且在添加MgATP时不会酸化。缺失的质子泵在缺乏摄入的异硫氰酸荧光素 - 葡聚糖、酸性水解酶和残余酸度的大的浮力液泡中回收。这些液泡也比内体更不易被洋地黄皂苷破坏，表明它们的双层膜中固醇含量低。这些结果表明，盘基网柄菌中的内吞循环是由一个离散且可分离的质子泵细胞器酸化的。
