Lee Robert J, McCracken Ardythe A, Brodsky Jeffrey L
Department of Biological Sciences, University of Pittsburgh, PA, USA.
Methods Mol Biol. 2005;301:175-84. doi: 10.1385/1-59259-895-1:175.
The first compartment encountered by newly synthesized secreted proteins is the endoplasmic reticulum (ER). Before secreted proteins can traffic beyond the ER they must fold into their final conformations, and components of multiprotein complexes must assemble. Not surprisingly, then, the ER lumen houses a high concentration of molecular chaperones, factors that facilitate protein folding. However, if misfolded secreted proteins arise they may be selected and proteolyzed. This process, which removes potentially toxic proteins from the cell, has been termed ER-associated degradation (ERAD). Surprisingly, ERAD substrates are not degraded within the ER after being selected but are retrotranslocated to the cytoplasm and destroyed by the 26S proteasome. Thus, the ERAD pathway comprises substrate selection, substrate export from the ER, and substrate degradation, and each step in this pathway has been elucidated in part through an in vitro ERAD assay using reagents prepared from a model eukaryote, the yeast Saccharomyces cerevisiae. This chapter describes this in vitro ERAD assay in detail, and special considerations when performing the assay are noted.
新合成的分泌蛋白首先遇到的区室是内质网（ER）。分泌蛋白在能够转运出内质网之前，必须折叠成其最终构象，并且多蛋白复合物的组分必须组装。因此，内质网腔中存在高浓度的分子伴侣，即促进蛋白质折叠的因子，这并不奇怪。然而，如果出现错误折叠的分泌蛋白，它们可能会被挑选出来并进行蛋白水解。这个从细胞中清除潜在毒性蛋白的过程被称为内质网相关降解（ERAD）。令人惊讶的是，ERAD底物在被挑选后并非在内质网内被降解，而是被逆向转运到细胞质并被26S蛋白酶体破坏。因此，ERAD途径包括底物选择、从内质网输出底物以及底物降解，并且该途径中的每个步骤部分是通过使用从模式真核生物酿酒酵母制备的试剂进行的体外ERAD测定来阐明的。本章详细描述了这种体外ERAD测定，并指出了进行该测定时的特殊注意事项。
