A technique for delineating the unfolding requirements for substrate entry into retrotranslocons during endoplasmic reticulum?associated degradation
A technique for delineating the unfolding requirements for substrate entry into retrotranslocons during endoplasmic reticulum?associated degradation
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DOI：
10.1074/jbc.ra119.010019
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发表时间：
2019-12-27
影响因子：
4.8
通讯作者：
Zhang, Zai-Rong
中科院分区：
文献类型：
--
作者：
Shi, Junfen;Hu, Xianyan;Zhang, Zai-Rong
内质网相关降解(ERAD)途径通过逆转录蛋白复合体介导缺陷蛋白从内质网到胞浆的逆转位。有缺陷的蛋白质通常具有复杂的构象和拓扑结构，目前尚不清楚ERAD如何将这些构象不同的蛋白质底物穿过逆转录子。在这里，我们研究了通过ERAD-L、ERAD-M和HIV编码蛋白VPU劫持的ERAD分支上的逆转录转运子运输所需的底物构象灵活性。为此，我们在不同的ERAD底物上添加了特定的结构域，通过与适当的配体结合，这些结构域的构象可以灵活或紧密地调节。利用这项技术，我们可以确定特定的逆转录转运子在解缠非常紧密、不那么紧密但折叠良好和无结构构象方面的能力。ERAD-L支链上的逆转位Hrd1复合体允许底物通过具有抗蛋白酶K？紧密构象的底物，而E3连接酶gp78介导的ERAD-M只允许几乎完全无序的底物通过，而不允许折叠良好的底物通过，因此可能具有最低的展开酶活性。VPU介导的ERAD含有潜在的逆转录转位，可以为成功的逆转录转位打开折叠良好的底物。然而，在VPU介导的ERAD中，底物的逆转位被底物的构象紧密性增强所阻断。在这些发现的基础上，我们提出了多肽通过内质网膜移动的机制。我们预计，我们的生化系统为鉴定膜蛋白逆转移位所必需的因素铺平了道路。
The endoplasmic reticulum?associated degradation (ERAD) pathway mediates the endoplasmic reticulum?to?cytosol retrotranslocation of defective proteins through protein complexes called retrotranslocons. Defective proteins usually have complex conformations and topologies, and it is unclear how ERAD can thread these conformationally diverse protein substrates through the retrotranslocons. Here, we investigated the substrate conformation flexibility necessary for transport via retrotranslocons on the ERAD-L, ERAD-M, and HIV-encoded protein Vpu-hijacked ERAD branches. To this end, we appended various ERAD substrates with specific domains whose conformations were tunable in flexibility or tightness by binding to appropriate ligands. With this technique, we could define the capacity of specific retrotranslocons in disentangling very tight, less tight but well-folded, and unstructured conformations. The Hrd1 complex, the retrotranslocon on the ERAD-L branch, permitted the passage of substrates with a proteinase K?resistant tight conformation, whereas the E3 ligase gp78-mediated ERAD-M allowed passage only of nearly completely disordered but not well-folded substrates and thus may have the least unfoldase activity. Vpu-mediated ERAD, containing a potential retrotranslocon, could unfold well-folded substrates for successful retrotranslocation. However, substrate retrotranslocation in Vpu-mediated ERAD was blocked by enhanced conformational tightness of the substrate. On the basis of these findings, we propose a mechanism underlying polypeptide movement through the endoplasmic reticulum membrane. We anticipate that our biochemical system paves the way for identifying the factors necessary for the retrotranslocation of membrane proteins.