Feliciano Daniel, Bultema Jarred J, Ambrosio Andrea L, Di Pietro Santiago M
Department of Biochemistry and Molecular Biology, Colorado State University, USA.
J Vis Exp. 2011 Jan 26(47):2352. doi: 10.3791/2352.
A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes. CCVs are distinguished by a polyhedral lattice of clathrin that coats the vesicle membrane and serves as a mechanical scaffold. Clathrin coats are assembled during vesicle formation from individual clathrin triskelia , the soluble form of clathrin composed of three heavy and three light chain subunits. Because the triskelion does not have the ability to bind to the membrane directly, clathrin-binding adaptors are critical to link the forming clathrin lattice to the membrane through association with lipids and/or membrane proteins. Adaptors also package transmembrane protein cargo, such as receptors, and can interact with each other and with other components of the CCV formation machinery. Over twenty clathrin adaptors have been described, several are involved in clathrin mediated endocytosis and others localize to the trans Golgi network or endosomes. With the exception of HIP1R (yeast Sla2p), all known clathrin adaptors bind to the N-terminal -propeller domain of the clathrin heavy chain. Clathrin adaptors are modular proteins consisting of folded domains connected by unstructured flexible linkers. Within these linker regions, short binding motifs mediate interactions with the clathrin N-terminal domain or other components of the vesicle formation machinery. Two distinct clathrin-binding motifs have been defined: the clathrin-box and the W-box. The consensus clathrin-box sequence was originally defined as L[L/I][D/E/N][L/F][D/E] but variants have been subsequently discovered. The W-box conforms to the sequence PWxxW (where x is any residue). Sla1p (Synthetic Lethal with Actin binding protein-1) was originally identified as an actin associated protein and is necessary for normal actin cytoskeleton structure and dynamics at endocytic sites in yeast cells. Sla1p also binds the NPFxD endocytic sorting signal and is critical for endocytosis of cargo bearing the NPFxD signal. More recently, Sla1p was demonstrated to bind clathrin through a motif similar to the clathrin box, LLDLQ, termed a variant clathrin-box (vCB), and to function as an endocytic clathrin adaptor. In addition, Sla1p has become a widely used marker for the endocytic coat in live cell fluorescence microscopy studies. Here we use Sla1p as a model to describe approaches for adaptor-clathrin interaction studies. We focus on live cell fluorescence microscopy, GST-pull down, and co-immunoprecipitation methods.
一条主要的内吞途径始于网格蛋白包被囊泡（CCV）的形成，这些囊泡将货物从细胞表面运输到内体。CCV的特征是由包裹囊泡膜的网格蛋白多面体晶格构成，它作为一个机械支架。网格蛋白包被在囊泡形成过程中由单个网格蛋白三脚蛋白复合体组装而成，网格蛋白的可溶形式由三个重链和三个轻链亚基组成。由于三脚蛋白复合体没有直接结合膜的能力，网格蛋白结合衔接蛋白对于通过与脂质和/或膜蛋白结合将正在形成的网格蛋白晶格与膜连接起来至关重要。衔接蛋白还包装跨膜蛋白货物，如受体，并且可以相互作用以及与CCV形成机制的其他成分相互作用。已经描述了二十多种网格蛋白衔接蛋白，其中一些参与网格蛋白介导的内吞作用，其他的则定位于反式高尔基体网络或内体。除了HIP1R（酵母Sla2p）之外，所有已知的网格蛋白衔接蛋白都与网格蛋白重链的N端螺旋桨结构域结合。网格蛋白衔接蛋白是模块化蛋白质，由通过无结构的柔性接头连接的折叠结构域组成。在这些接头区域内，短的结合基序介导与网格蛋白N端结构域或囊泡形成机制的其他成分的相互作用。已经定义了两种不同的网格蛋白结合基序：网格蛋白盒和W盒。网格蛋白盒的共有序列最初被定义为L[L/I][D/E/N][L/F][D/E]，但随后发现了变体。W盒符合序列PWxxW（其中x是任何残基）。Sla1p（与肌动蛋白结合蛋白-1合成致死）最初被鉴定为一种肌动蛋白相关蛋白，并对酵母细胞内吞位点的正常肌动蛋白细胞骨架结构和动力学是必需的。Sla1p还结合NPFxD内吞分选信号，并且对携带NPFxD信号的货物的内吞作用至关重要。最近，已证明Sla1p通过与网格蛋白盒类似的基序LLDLQ（称为变体网格蛋白盒（vCB））结合网格蛋白，并作为内吞网格蛋白衔接蛋白发挥作用。此外，在活细胞荧光显微镜研究中，Sla1p已成为内吞包被广泛使用的标记物。在这里，我们以Sla1p为模型描述衔接蛋白-网格蛋白相互作用研究的方法。我们专注于活细胞荧光显微镜、GST下拉和免疫共沉淀方法。
