The role of dynamin and its binding partners in coated pit invagination and scission.
The role of dynamin and its binding partners in coated pit invagination and scission.
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DOI：
10.1083/jcb.152.2.309
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发表时间：
2001-01-22
影响因子：
7.8
通讯作者：
Smythe, E
中科院分区：
文献类型：
--
作者：
Hill, E;van Der Kaay, J;Downes, C P;Smythe, E
胞浆中的网状蛋白和接头复合体AP2直接组装到膜上后，形成包被于细胞膜上的囊泡。除了这些结构成分外，还有其他几种蛋白质与笼蛋白包裹的囊泡的形成有关。这些蛋白包括大分子量GTPase、Dynamin和几个含有Src Homology 3(SH3)结构域的蛋白质，它们通过与Dynamin的COOH末端的Pro/Arg富含结构域(PRD)相互作用而与Dynamin结合。为了了解包膜囊泡的形成机制，必须确定单个组分所针对的层级，并在包膜小孔的组装、内陷和断裂中发挥作用。为了解决动力蛋白及其结合伙伴在内吞作用早期阶段的作用，我们使用了成熟的体外检测方法，用于涂层小凹内陷和涂层囊泡破裂的后期阶段。动力蛋白此前已被证明在包膜囊泡的断裂中起作用。我们表明，动力也是需要的后期阶段的内陷笼状蛋白涂层的坑。此外，动力素必须结合和水解GTP，因为它在将配体隔离到深度凹陷的涂层凹坑中的作用。我们还证明了亲内素的SH3结构域，它结合了突触素和动力素，在体外既抑制了内陷的后期阶段，也抑制了断裂。这种抑制是由于磷酸肌醇4，5-二磷酸水平的降低，导致AP2、网状蛋白和动力素从质膜上解离。亲内素SH3结构域的巨大效应使我们提出了亲内素及其结合伙伴突触素在包被囊泡周期中添加的时间顺序的模型。
Plasma membrane clathrin-coated vesicles form after the directed assembly of clathrin and the adaptor complex, AP2, from the cytosol onto the membrane. In addition to these structural components, several other proteins have been implicated in clathrin-coated vesicle formation. These include the large molecular weight GTPase, dynamin, and several Src homology 3 (SH3) domain–containing proteins which bind to dynamin via interactions with its COOH-terminal proline/arginine-rich domain (PRD). To understand the mechanism of coated vesicle formation, it is essential to determine the hierarchy by which individual components are targeted to and act in coated pit assembly, invagination, and scission. To address the role of dynamin and its binding partners in the early stages of endocytosis, we have used well-established in vitro assays for the late stages of coated pit invagination and coated vesicle scission. Dynamin has previously been shown to have a role in scission of coated vesicles. We show that dynamin is also required for the late stages of invagination of clathrin-coated pits. Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand into deeply invaginated coated pits. We also demonstrate that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro. This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane. The dramatic effects of the SH3 domain of endophilin led us to propose a model for the temporal order of addition of endophilin and its binding partner synaptojanin in the coated vesicle cycle.