Induction of mutant dynamin specifically blocks endocytic coated vesicle formation.
Induction of mutant dynamin specifically blocks endocytic coated vesicle formation.
复制标题
突变动力蛋白的诱导特异性阻断内吞涂层囊泡的形成。
DOI：
10.1083/jcb.127.4.915
复制
发表时间：
1994-11
影响因子：
7.8
通讯作者：
Schmid, S L
中科院分区：
文献类型：
--
作者：
Damke, H;Baba, T;Warnock, D E;Schmid, S L
发动蛋白是果蝇Shibire基因产物的哺乳动物同源物。在这个100 kD的GT3突变引起内吞多效性缺陷。为了进一步研究其作用，我们产生了稳定的HeLa细胞系，其表达野生型发动蛋白或由严格控制的四环素诱导型启动子驱动的GTP结合和水解缺陷的突变体。过表达野生型发动蛋白没有影响。相比之下，包被坑未能成为收缩和包被囊泡未能在过表达突变发动蛋白的细胞中出芽，从而通过转铁蛋白（Tfn）和EGF受体的内吞作用被有效抑制。包被坑组件，内陷，和招聘的受体到包被坑不受影响。其他囊泡运输途径，包括Tfn受体回收，Tfn受体生物合成，和组织蛋白酶D运输到溶酶体通过高尔基体衍生包被囊泡，不受影响。本体液相摄取也以与野生型相同的初始速率继续。EM免疫定位表明，膜结合发动蛋白是专门与网格蛋白包被的小坑质膜。发动蛋白也与孤立的包被囊泡，这表明它在囊泡出芽中发挥作用。像果蝇shibire突变体一样，过度表达突变发动蛋白的HeLa细胞积累了长小管，其中许多仍与质膜连接。我们的结论是，发动蛋白是专门需要内吞包衣囊泡的形成，其GTP结合和水解活性所需的形成收缩包衣坑，随后，包衣囊泡出芽。
Dynamin is the mammalian homologue to the Drosophila shibire gene product. Mutations in this 100-kD GTPase cause a pleiotropic defect in endocytosis. To further investigate its role, we generated stable HeLa cell lines expressing either wild-type dynamin or a mutant defective in GTP binding and hydrolysis driven by a tightly controlled, tetracycline- inducible promoter. Overexpression of wild-type dynamin had no effect. In contrast, coated pits failed to become constricted and coated vesicles failed to bud in cells overexpressing mutant dynamin so that endocytosis via both transferrin (Tfn) and EGF receptors was potently inhibited. Coated pit assembly, invagination, and the recruitment of receptors into coated pits were unaffected. Other vesicular transport pathways, including Tfn receptor recycling, Tfn receptor biosynthesis, and cathepsin D transport to lysosomes via Golgi-derived coated vesicles, were unaffected. Bulk fluid-phase uptake also continued at the same initial rates as wild type. EM immunolocalization showed that membrane-bound dynamin was specifically associated with clathrin-coated pits on the plasma membrane. Dynamin was also associated with isolated coated vesicles, suggesting that it plays a role in vesicle budding. Like the Drosophila shibire mutant, HeLa cells overexpressing mutant dynamin accumulated long tubules, many of which remained connected to the plasma membrane. We conclude that dynamin is specifically required for endocytic coated vesicle formation, and that its GTP binding and hydrolysis activities are required to form constricted coated pits and, subsequently, for coated vesicle budding.