Subcellular Fractionation Analysis of the Extraction of Ubiquitinated Polytopic Membrane Substrate during ER-Associated Degradation.
Subcellular Fractionation Analysis of the Extraction of Ubiquitinated Polytopic Membrane Substrate during ER-Associated Degradation.
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DOI：
10.1371/journal.pone.0148327
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Kamura T
中科院分区：
文献类型：
--
作者：
Nakatsukasa K;Kamura T
在内质网相关降解（ERAD）过程中，错误折叠的多位膜蛋白被泛素化并逆转运到细胞质中进行蛋白酶体降解。然而，我们的理解，多位膜蛋白是如何从ER提取到胞质溶胶仍然在很大程度上不清楚。为了更好地定义泛素化的多位膜底物在体内的定位和物理性质，我们进行了亚细胞分级分离分析的Ste 6 *，十二跨膜蛋白，主要是由Doa 10 E3连接酶在酵母中的泛素化。与先前的体外研究一致，以Cdc 48/p97依赖性方式将泛素化的Ste 6 * 从P20（20，000 g沉淀）级分提取到S20（20，000 g上清液）级分。类似地，Ubx 2 p将Cdc 48/p97募集到ER，促进了Ste 6 * 的提取。相比之下，脂滴的形成，这被认为是在酵母中的Hrd 1底物的降解所必需的，是不需要的Ste 6 * 的降解。有趣的是，我们发现S20级分中的泛素化Ste 6 * 可以通过以100，000 g进一步离心来富集。虽然目前还不确定P100组分中的泛素化Ste 6 * 是否完全不含任何脂质，但膜浮选分析表明存在两种不同的泛素化Ste 6 * 群体，其具有不同的膜缔合状态。总之，这些结果意味着泛素化的Ste 6 * 可能被Cdc 48/p97隔离到一个推定的质量控制子结构中。在本研究中开发的分级分析提供了一种手段，以进一步剖析不明确的后泛素化步骤ERAD的多定位膜基板。
During ER-associated degradation (ERAD), misfolded polytopic membrane proteins are ubiquitinated and retrotranslocated to the cytosol for proteasomal degradation. However, our understanding as to how polytopic membrane proteins are extracted from the ER to the cytosol remains largely unclear. To better define the localization and physical properties of ubiquitinated polytopic membrane substrates in vivo, we performed subcellular fractionation analysis of Ste6*, a twelve transmembrane protein that is ubiquitinated primarily by Doa10 E3 ligase in yeast. Consistent with previous in vitro studies, ubiquitinated Ste6* was extracted from P20 (20,000 g pellet) fraction to S20 (20,000 g supernatant) fraction in a Cdc48/p97-dependent manner. Similarly, Ubx2p, which recruits Cdc48/p97 to the ER, facilitated the extraction of Ste6*. By contrast, lipid droplet formation, which was suggested to be dispensable for the degradation of Hrd1-substrates in yeast, was not required for the degradation of Ste6*. Intriguingly, we found that ubiquitinated Ste6* in the S20 fraction could be enriched by further centrifugation at 100,000 g. Although it is currently uncertain whether ubiquitinated Ste6* in P100 fraction is completely free from any lipids, membrane flotation analysis suggested the existence of two distinct populations of ubiquitinated Ste6* with different states of membrane association. Together, these results imply that ubiquitinated Ste6* may be sequestered into a putative quality control sub-structure by Cdc48/p97. Fractionation assays developed in the present study provide a means to further dissect the ill-defined post-ubiquitination step during ERAD of polytopic membrane substrates.