STAGE-SPECIFIC ASSAYS FOR COATED PIT FORMATION AND COATED VESICLE BUDDING INVITRO

被引:135
作者
SCHMID, SL [1 ]
SMYTHE, E [1 ]
机构
[1] Scripps Res Inst, RES INST, DEPT MOLEC BIOL, LA JOLLA, CA 92037 USA
关键词
D O I
10.1083/jcb.114.5.869
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
Internalization of biotin-S-S-I-125-transferrin (I-125-BSST) into semiintact A431 cells were assessed by two different criteria which have allowed us to distinguish partial reactions in the complex overall process of receptor-mediated endocytosis. Early events resulting in the sequestration of ligand into deeply invaginated coated pits were measured by inaccessibility of I-125-BSST to exogenously added antibodies. Later events involving coated vesicle budding and membrane fission were measured by resistance of I-125-BSST to reduction by the membrane impermeant-reducing agent, MesNa. Acquisition of Ab inaccessibility occurred very efficiently in this cell-free system (approximately 50% of total cell-associated I-125-BSST became inaccessible) and could be inhibited by anti-clathrin mAbs and by antibodies directed against the cytoplasmic domain of the transferrin-receptor. In contrast, acquisition of MesNa resistance occurred less efficiently (approximately 10-20% of total cell-associated I-125-BSST) and showed differential sensitivity to inhibition by anti-clathrin and anti-transferrin receptor mAbs. Both partial reactions were stimulated by ATP and cytosol; indicating at least two ATP-requiring events in receptor-mediated endocytosis. The temperature dependence of both reactions was similar to that for I-125-BSST internalization in intact cells with no activity being observed below 10-degrees-C. Morphological studies using gold-labeled ligands confirmed that internalization of transferrin receptors into semiintact A431 cell occurred via coated pits and coated vesicles and resulted in delivery of ligand to endosomal structures.
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页码:869 / 880
页数:12
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