Distinct mechanisms of clathrin-independent endocytosis have unique sphingolipid requirements
Distinct mechanisms of clathrin-independent endocytosis have unique sphingolipid requirements
复制标题
DOI：
10.1091/mbc.e05-12-1101
复制
发表时间：
2006-07-01
影响因子：
3.3
通讯作者：
Pagano, Richard E.
中科院分区：
文献类型：
--
作者：
Cheng, Zhi-Jie;Singh, Raman Deep;Pagano, Richard E.
鞘脂（Sphingolipids, SLs）在膜结构和细胞功能中起重要作用。在这里，我们研究了各种内吞机制对SL的需求，使用突变细胞系和药物抑制剂来破坏SL的生物合成。首先，我们证明了在中国仓鼠卵巢细胞中，我们可以区分出三种不同的不依赖网格蛋白的内吞机制（洞穴，RhoA和Cdc42依赖），它们在货物，对药物的敏感性和显性阴性蛋白方面存在差异。单链蛋白的普遍耗竭通过每一种不依赖网格蛋白的机制抑制胞吞作用，而依赖网格蛋白的摄取则不受影响。鞘糖脂（GSLs）的耗竭；SW的一个亚群选择性地阻断了小泡内吞作用，减少了质膜上的小泡蛋白-1和小泡。急性添加外源gsl可恢复gsl缺失细胞的小泡内吞作用和PM小泡。经证实，SL耗竭对RhoA-和cdc42调节的内噬作用的破坏与这些Rho蛋白对质膜的靶向性降低有关，外源性鞘磷脂可以部分恢复，但gsl不能。RhoA和Cdc42的体内膜靶向和体外与人工脂质囊泡的结合均依赖于鞘磷脂。这些结果提供了第一个证据，表明在不依赖网格蛋白的胞吞作用的不同机制中需要不同的SLs。
Sphingolipids (SLs) play important roles in membrane structure and cell function. Here, we examine the SL requirements of various endocytic mechanisms using a mutant cell line and pharmacological inhibitors to disrupt SL biosynthesis. First, we demonstrated that in Chinese hamster ovary cells we could distinguish three distinct mechanisms of clathrin-independent endocytosis (caveolar, RhoA, and Cdc42 dependent) which differed in cargo, sensitivity to pharmacological agents, and dominant negative proteins. General depletion of SLs inhibited endocytosis by each clathrin-independent mechanism, whereas clathrin-dependent uptake was unaffected. Depletion of glycosphingolipids (GSLs; a subgroup of SW selectively blocked caveolar endocytosis and decreased caveolin-1 and caveolae at the plasma membrane. Caveolar endocytosis and PM caveolae could be restored in GSL-depleted cells by acute addition of exogenous GSLs. Disruption of RhoA- and Cdc42-regulated endocytosis by SL depletion was shown to be related to decreased targeting of these Rho proteins to the plasma membrane and could be partially restored by exogenous sphingomyelin but not GSLs. Both the in vivo membrane targeting and in vitro binding to artificial lipid vesicles of RhoA and Cdc42 were shown to be dependent upon sphingomyelin. These results provide the first evidence that SLs are differentially required for distinct mechanisms of clathrin-independent endocytosis.