ATP is required for receptor-mediated endocytosis in intact cells.
ATP is required for receptor-mediated endocytosis in intact cells.
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DOI：
10.1083/jcb.111.6.2307
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发表时间：
1990-12
期刊：
影响因子：
--
通讯作者：
Carter LL
中科院分区：
文献类型：
--
作者：
Schmid SL;Carter LL
我们已经证明转铁蛋白的受体介导的内吞作用需要细胞ATP。这已经完成了使用一种新的测定的基础上收购的抗膜渗透性还原剂，谷胱甘肽（GSH）的内吞作用。二铁-转铁蛋白通过可裂解的二硫键与生物素缀合并碘化。用GSH处理细胞后，通过亲和素-琼脂糖凝胶吸附定量125 I-BSST的内化。受体介导的125 I-BSST的内吞作用在ATP耗竭的细胞中被严重抑制。当通过在N2气氛下或在NaN 3和NaF存在下孵育细胞来耗尽ATP时，获得了类似的结果。后一种治疗单独也导致表面转铁蛋白受体的损失，这与细胞ATP的减少无关。与GSH抗性的获得相反，通过抗转铁蛋白抗体的不可及性评估的125 I-BSST的表观内化在ATP耗尽的细胞中达到对照水平。我们的生物化学和形态学数据表明，虽然ATP是必需的受体介导的内吞作用，在ATP耗尽的细胞配体可以成为有效地隔离到深深陷入的坑，是不可接近的大探针，如抗体，但仍然可以访问小分子，如GSH。
We have demonstrated a requirement for cellular ATP in the receptor- mediated endocytosis of transferrin. This has been accomplished using a novel assay for endocytosis based on acquisition of resistance to the membrane impermeable reducing agent, glutathione (GSH). Diferric- transferrin was conjugated to biotin via a cleavable disulfide bond and iodinated. Internalization of 125I-biotin-S-S-transferrin (125I-BSST) was quantitated by adsorption to avidin-Sepharose after treatment of cells with GSH. Receptor-mediated endocytosis of 125I-BSST was severely inhibited in ATP-depleted cells. Similar results were obtained when ATP was depleted by incubation of cells either under a N2-atmosphere or in the presence of NaN3 and NaF. The latter treatment, alone, also resulted in a loss of surface transferrin receptors which could not be correlated to reductions in cellular ATP. In contrast to the acquisition of GSH resistance, the apparent internalization of 125I- BSST as assessed by inaccessibility to antitransferrin antibodies reached control levels in ATP-depleted cells. Our biochemical and morphological data suggested that, although ATP is required for receptor-mediated endocytosis, in ATP-depleted cells ligands can become efficiently sequestered into deeply invaginated pits that are inaccessible to large probes such as antibodies, but remain accessible to small molecules such as GSH.