Dynamin at the neck of caveolae mediates their budding to form transport vesicles by GTP-driven fission from the plasma membrane of endothelium.-猫眼课题宝

DOI：

10.1083/jcb.141.1.101

发表时间：

1998-04-06

期刊：

The Journal of cell biology

影响因子：

通讯作者：

中科院分区：

其他

文献类型：

作者：

Oh P;McIntosh DP;Schnitzer JE

来源链接：

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2132716/?report=classic

介导细胞表面运输的小窝的分子机制是未知的。小囊从质膜出芽，通过“夹断”或分裂过程形成游离载体囊泡，这需要胞质溶胶并由GTP水解驱动（Schnitzer，J.E.，P.哦，还有D. P.麦金托什。1996.科学274：239-242）。在这里，我们使用几个独立的技术和功能测定，从无细胞到完整的细胞系统，以建立一个功能的发动蛋白在形成运输囊泡从内皮细胞质膜介导的分裂在颈部的小窝。这种小窝分裂需要与胞质动力蛋白的相互作用以及其对GTP的水解。发动蛋白在胞质溶胶中的表达以及单独纯化的重组发动蛋白在无细胞测定中支持GTP诱导的小窝分裂，而将其从胞质溶胶中去除或向胞质溶胶中添加发动蛋白的特异性抗体抑制这种分裂。缺乏正常GTP酶活性的突变动力蛋白的过表达不仅抑制GTP诱导的小窝分裂和出芽，而且还阻止小窝介导的霍乱毒素B链在完整和透化的内皮细胞中的内化。通过亚细胞分级分离和免疫显微镜对体内内皮细胞的分析表明，发动蛋白集中在小窝上，主要在预期的作用部位，即颈部。因此，通过其寡聚化的能力，发动蛋白似乎在小窝的颈部周围形成结构环，其水解GTP以通过小窝从质膜分裂形成游离运输囊泡来介导内化。

The molecular mechanisms mediating cell surface trafficking of caveolae are unknown. Caveolae bud from plasma membranes to form free carrier vesicles through a “pinching off” or fission process requiring cytosol and driven by GTP hydrolysis (Schnitzer, J.E., P. Oh, and D.P. McIntosh. 1996. Science. 274:239–242). Here, we use several independent techniques and functional assays ranging from cell-free to intact cell systems to establish a function for dynamin in the formation of transport vesicles from the endothelial cell plasma membrane by mediating fission at the neck of caveolae. This caveolar fission requires interaction with cytosolic dynamin as well as its hydrolysis of GTP. Expression of dynamin in cytosol as well as purified recombinant dynamin alone supports GTP-induced caveolar fission in a cell-free assay whereas its removal from cytosol or the addition to the cytosol of specific antibodies for dynamin inhibits this fission. Overexpression of mutant dynamin lacking normal GTPase activity not only inhibits GTP-induced fission and budding of caveolae but also prevents caveolae-mediated internalization of cholera toxin B chain in intact and permeabilized endothelial cells. Analysis of endothelium in vivo by subcellular fractionation and immunomicroscopy shows that dynamin is concentrated on caveolae, primarily at the expected site of action, their necks. Thus, through its ability to oligomerize, dynamin appears to form a structural collar around the neck of caveolae that hydrolyzes GTP to mediate internalization via the fission of caveolae from the plasma membrane to form free transport vesicles.