Assembly of ER-associated protein degradation in vitro: Dependence on cytosol, calnexin, and ATP
Assembly of ER-associated protein degradation in vitro: Dependence on cytosol, calnexin, and ATP
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DOI：
10.1083/jcb.132.3.291
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发表时间：
1996-02-01
影响因子：
7.8
通讯作者：
Brodsky, JL
中科院分区：
文献类型：
--
作者：
McCracken, AA;Brodsky, JL
为了研究 ER 相关蛋白降解 (ERAD) 的机制，在体外重建了该过程。将放射性标记的前原α因子翻译后易位到分离的酵母微粒体中的既定程序进行了修改，以抑制糖基化并包括易位后“追逐”孵育期以监测降解。用糖受体肽竞争核心碳水化合物，或使用经过基因工程消除所有三个糖基化位点的放射性标记α因子前体来抑制糖基化。糖基化的抑制导致未糖基化的前α因子（pαF）的产生，这是α因子前体的加工形式，被证明是体内ERAD的底物。使用该系统，糖基化和非糖基化形式的 pro-alpha 因子在 90 分钟的追踪孵育中均保持稳定。然而，在追逐孵育反应中添加胞质溶胶诱导了 p α F 的选择性和快速降解。这些结果直接反映了 α 因子前体在体内的行为；即，p alpha F 是 ERAD 的底物，而糖基化 pro-alpha 因子则不是。胞质溶胶的热灭活和胰蛋白酶处理，以及在追踪孵育中添加 ATP gamma S，导致 p α F 稳定。在 sec12 微粒体中观察到 ERAD，表明不需要通过转运囊泡输出 p α F。此外，在追逐孵育反应的上清液中发现了 p α F 而不是糖基化的 pro-α 因子，这表明该 ERAD 底物有一个特定的转运系统。最后，当检查含有被破坏的钙联蛋白基因的酵母菌株的微粒体时，pelf 的降解受到抑制。总之，这些结果表明胞浆蛋白因子、ATP 水解和钙连接蛋白是酵母中 ER 相关蛋白降解所必需的，并表明胞浆是降解位点。
To investigate the mechanisms of ER-associated protein degradation (ERAD), this process was reconstituted in vitro. Established procedures for posttranslational translocation of radiolabeled prepro-alpha factor into isolated yeast microsomes were modified to inhibit glycosylation and to include a posttranslocation ''chase'' incubation period to monitor degradation. Glycosylation was inhibited with a glyco-acceptor peptide to compete for core carbohydrates, or by using a radiolabeled alpha factor precursor that had been genetically engineered to eliminate all three glycosylation sites. Inhibition of glycosylation led to the production of unglycosylated pro-alpha factor (p alpha F), a processed form of the alpha factor precursor shown to be a substrate of ERAD in vivo. With this system, both glycosylated and unglycosylated forms of pro-alpha factor were stable throughout a 90-min chase incubation. However, the addition of cytosol to the chase incubation reaction induced a selective and rapid degradation of p alpha F. These results directly reflect the behavior of alpha factor precursor in vivo; i.e., p alpha F is a substrate for ERAD, while glycosylated pro-alpha factor is not. Heat inactivation and trypsin treatment of cytosol, as well as addition of ATP gamma S to the chase incubations, led to a stabilization of p alpha F. ERAD was observed in sec12 microsomes, indicating that export of p alpha F via transport vesicles was not required. Furthermore, p alpha F but not glycosylated pro-alpha factor was found in the supernatant of the chase incubation reactions, suggesting a specific transport system for this ERAD substrate. Finally, the degradation of pelf was inhibited when microsomes from a yeast strain containing a disrupted calnexin gene were examined. Together, these results indicate that cytosolic protein factor(s), ATP hydrolysis, and calnexin are required for ER-associated protein degradation in yeast, and suggest the cytosol as the site for degradation.