In vitro dissection of protein translocation into the mammalian endoplasmic reticulum.
In vitro dissection of protein translocation into the mammalian endoplasmic reticulum.
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DOI：
10.1007/978-1-60327-412-8_20
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发表时间：
2010
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影响因子：
--
通讯作者：
Hegde, Ramanujan S
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文献类型：
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作者：
Sharma, Ajay;Mariappan, Malaiyalam;Appathurai, Suhila;Hegde, Ramanujan S
在真核细胞中，大约四分之一的mRNA编码分泌蛋白和膜蛋白。这类蛋白质必须首先被分离到内质网，在那里它们要么被移位到内腔中，要么被插入到脂质双层中。对这些过程的研究长期以来依赖于它们在无细胞系统中的成功重构。这种体外系统的高度可操作性允许识别关键机器，阐明它们在易位中的功能作用，并解剖它们的作用机制。在这里，我们提供了基本的方法（一）建立强大的基于拟南芥的体外翻译和易位系统，（二）蛋白质易位，插入和拓扑结构的测定，和（iii）方法来溶解，破碎，和重建ER膜。这些方法的变化不仅适用于正向蛋白质易位系统，而且也适用于解剖其他知之甚少的膜相关过程，如逆向易位。
In eukaryotic cells, roughly one-fourth of all mRNAs code for secretory and membrane proteins. This class of proteins must first be segregated to the endoplasmic reticulum, where they are either translocated into the lumen or inserted into the lipid bilayer. The study of these processes has long relied on their successful reconstitution in cell-free systems. The high manipulability of such in vitro systems has allowed the identification of key machinery, elucidation of their functional roles in translocation, and dissection of their mechanisms of action. Here, we provide the basic methodology for (i) setting up robust mammalian-based in vitro translation and translocation systems, (ii) assays for protein translocation, insertion, and topology, and (iii) methods to solubilize, fractionate, and reconstitute ER membranes. Variations of these methods should be applicable not only to forward protein translocation systems but also for dissecting other poorly understood membrane-associated processes such as retrotranslocation.