Name: in vivo and in vitro studies of adaptor-clathrin interaction
Uploaded: 2011-01-26T23:30:00
Description: Watch this Scientific Journal Video about In vivo and in vitro Studies of Adaptor-clathrin Interaction at JoVE.com

DF是由美国国家科学基金会桥梁博士奖学金的支持。在这项工作中所使用的显微镜​​是由科罗拉多州立大学的显微镜成像网络核心基础设施授予部分支持。在作者的实验室的网格蛋白适配器的工作是支持CSU的启动资金和美国心脏协会奖09SDG2280525到SD

1。 SLA1基因选择标记的GFP标记和团以融合的GFP标记直接SLA1基因的开放阅读框3&#39;（Sla1p彗星端），同时标记基因与大肠杆菌应用Longtine方法17 大肠杆菌根R基因，允许G418筛选。 <ol><li>生成一个用PCR的DNA片段作为模板使用的pFA6a - GFP的质粒（S65T）kanMX6 18及以下的引物：正向，5&#39; - CA AGG民航局海湾合作委员会的AAC阿拉木图TTC亚洲空运中?...

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网格蛋白包被小泡（CCV）参与内吞作用和跨高尔基网络运输和内涵体，保守的途径是真核细胞生物学的基础。此方法文件中所描述的方法是有用的负责CCV的形成，特别是适配器蛋白和网格蛋白之间的相互作用的分子机制研究。 GST融合蛋白的亲和力和免疫共沉淀实验，让测试适配器（候选人）和网格蛋白之间的物理协会。绿色荧光蛋白标记和荧光显微镜成像允许在活细胞的动态研究。尤其是...

<table border="1"><th>材料</th><th>公司</th><tbody><tr><td>快速转换- XL定点突变试剂盒</td><td> Stratagene公司</td></tr><tr><td>蛋白酶抑制剂的鸡尾酒</td><td>西格玛</td></tr></tbody></table>

in vivo and in vitro studies of adaptor-clathrin interaction

一个主要的内吞途径启动，形成网格蛋白包被小泡（CCVS），从细胞表面到内涵体运输货物1-6。 CCVS的区别在于一个多面体的笼形蛋白，大衣囊膜的晶格，并担任机械支架。从个别网格蛋白triskelia，三个重型和轻链亚基7,8组成的笼形蛋白的可溶形式在囊泡形成网格蛋白大衣组装。由于triskelion没有直接绑定到膜的能力，网格蛋白结合适配器协会通过与血脂和/或膜蛋白9的链接，形成网格蛋白晶格膜的关键。适配器还包跨膜蛋白的货物，如受体，而且可以互相交流，与其他组件的CCV形成机械 9 ​​。 有超过20个网格蛋白适配器，一些参与网格蛋白介导的内吞作用和其他本地化跨高尔基网络或内涵体 9 。 HIP1R（酵母Sla2p）的异常，所有已知的网格蛋白适配器绑定到网格蛋白重链 9 N -末端螺旋桨域。网格蛋白适配器非结构化灵活的连接器连接的折叠域组成的模块化的蛋白质。在这些连接器的地区中，短期结合基序调解的网格蛋白的N端结构域或其他组件的囊泡形成机械9 ​​的交互。两个不同的网格蛋白结合的图案已被定义：网格蛋白框和框的W - 9 。的共识网格蛋白- box序列的最初定义为L [L / I] [D / E / L / F]，[D / E] 10，但变种已被后来发现11。符合的W - box序列PWxxW（其中x是任何残留物）。 Sla1p（与肌动蛋白结合蛋白1合成致命的）最初确定为一个肌动蛋白相关蛋白在酵母细胞的12内吞网站是正常的肌动蛋白细胞骨架的结构和动力学的必要。 Sla1p还结合NPFxD吞分拣信号NPFxD信号13,14轴承货物的内吞作用的关键。最近，Sla1p被证明绑定通过类似网格蛋白中，LLDLQ的一个Motif网格蛋白，被称为网格蛋白盒（VCB）的一个变种，和功能吞网格蛋白适配器15。此外，Sla1p已成为广泛使用的标记为在活细胞荧光显微镜研究 16的内吞大衣。这里我们使用Sla1p作为一个模型来描述适配器网格蛋白的相互作用研究方法。我们重点对活细胞的荧光显微镜，消费税拔下来，和免疫共沉淀方法。

Watch this Scientific Journal Video about In vivo and in vitro Studies of Adaptor-clathrin Interaction at JoVE.com

In vivo and in vitro Studies of Adaptor-clathrin Interaction

网格蛋白介导的内吞作用取决于适配器的蛋白质，协调货物选择和网格蛋白外衣大会。在这里，我们描述的程序来研究网格蛋白适配器的物理互动和活细胞成像的方法，作为一种模式使用酵母吞适配器蛋白Sla1p。

适配器，网格蛋白相互作用在体内和体外研究

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs ...

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