Podbilewicz B, Mellman I
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510.
EMBO J. 1990 Nov;9(11):3477-87. doi: 10.1002/j.1460-2075.1990.tb07556.x.
We have developed an in vitro system for studying membrane transport during receptor-mediated endocytosis. Using nitrocellulose disruption to permeabilize selectively the apical domain of filter-grown MDCK cells, the recycling of receptor-bound transferrin (Tfn) from an intracellular pool was reconstituted in vitro with a rate and efficiency similar to that of intact cells. Tfn and Tfn receptor recycling from endosomes back to the cell surface was dependent on added ATP and cytosol-derived proteins. Thus, incubation of intact cells under conditions of ATP depletion resulted in the clearance of Tfn receptors from the basolateral membrane, this was reversible upon removal of the energy poisons. Reappearance of previously internalized receptors could also be obtained in disrupted cells but required the addition of both ATP and cytosol to the assay mixture. Similarly, when intact cells were allowed to internalize labeled Tfn prior to disruption, efficient and rapid release of ligand back into the medium was markedly stimulated by ATP and cytosol. Recycling was judged to be both selective and vectorial since only the expected small fraction of a previously internalized horseradish peroxidase was released after addition of ATP and cytosol, and release was primarily into the basal medium. While the cytosol contributed one or more protein factors, none was sensitive to N-ethylmaleimide. Alkylation of the disrupted cells, however, did inactivate recycling.
我们开发了一种体外系统，用于研究受体介导的内吞作用过程中的膜转运。利用硝酸纤维素破坏法选择性地使滤膜培养的MDCK细胞的顶端区域通透化，从细胞内池回收受体结合的转铁蛋白（Tfn）的过程在体外得以重建，其速率和效率与完整细胞相似。Tfn和Tfn受体从内体循环回到细胞表面依赖于添加的ATP和胞质溶胶衍生的蛋白质。因此，在ATP耗尽的条件下孵育完整细胞会导致Tfn受体从基底外侧膜清除，去除能量毒物后这种清除是可逆的。在破裂细胞中也可以获得先前内化受体的重新出现，但需要在测定混合物中添加ATP和胞质溶胶。同样，当完整细胞在破裂前被允许内化标记的Tfn时，ATP和胞质溶胶会显著刺激配体高效快速地释放回培养基中。由于添加ATP和胞质溶胶后仅释放了预期的一小部分先前内化的辣根过氧化物酶，且释放主要进入基底培养基，因此循环被认为是选择性的和定向的。虽然胞质溶胶贡献了一种或多种蛋白质因子，但没有一种对N - 乙基马来酰亚胺敏感。然而，对破裂细胞进行烷基化处理确实会使循环失活。
