Real-time fluorescence detection of ERAD substrate retrotranslocation in a mammalian in vitro system.
Real-time fluorescence detection of ERAD substrate retrotranslocation in a mammalian in vitro system.
复制标题
哺乳动物体外系统中 ERAD 底物逆转录的实时荧光检测。
DOI：
10.1016/j.cell.2007.03.046
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发表时间：
2007
期刊：
影响因子：
64.5
通讯作者：
Johnson,ArthurE
中科院分区：
文献类型：
--
作者：
Wahlman,Judit;DeMartino,GeorgeN;Skach,WilliamR;Bulleid,NeilJ;Brodsky,JeffreyL;Johnson,ArthurE
在内质网 (ER) 中无法组装成其天然状态的分泌蛋白被运回或“逆转位”到胞质溶胶中，进行 ER 相关降解 (ERAD)。为了研究不同成分在 ERAD 中的作用，将一种荧光标记的 ERAD 底物与选定的管腔因子一起封装在哺乳动物微粒体内。将微粒体与荧光猝灭剂和选定的胞质蛋白混合后，通过胞质猝灭剂接触染料标记底物时荧光强度的降低来连续实时监测底物流出速率。仅用蛋白质二硫键异构酶替换所有管腔蛋白或仅用 PA700（26S 蛋白酶体的 19S 调节颗粒）替换所有胞浆蛋白，非糖基化 pro-α 因子的逆转位动力学没有显着改变。逆转位被假定的逆转位通道蛋白 derlin-1 抗体阻断，但 Sec61α 则不然。此外，pro-α因子光交联derlin-1，但不是Sec61α。因此，derlin-1 似乎参与了前α因子逆转位。
Secretory proteins unable to assemble into their native states in the endoplasmic reticulum (ER) are transported back or "retrotranslocated" into the cytosol for ER-associated degradation (ERAD). To examine the roles of different components in ERAD, one fluorescence-labeled ERAD substrate was encapsulated with selected lumenal factors inside mammalian microsomes. After mixing microsomes with fluorescence-quenching agents and selected cytosolic proteins, the rate of substrate efflux was monitored continuously in real time by the decrease in fluorescence intensity as cytosolic quenchers contacted dye-labeled substrates. The retrotranslocation kinetics of nonglycosylated pro-α factor were not significantly altered by replacing all lumenal proteins with only protein disulfide isomerase or all cytosolic proteins with only PA700, the 19S regulatory particle of the 26S proteasome. Retrotranslocation was blocked by antibodies against a putative retrotranslocation channel protein, derlin-1, but not Sec61α. In addition, pro-α factor photocrosslinked derlin-1, but not Sec61α. Thus, derlin-1 appears to be involved in pro-α factor retrotranslocation.