Zhang Ting, Xu Yue, Liu Yanfen, Ye Yihong
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892
Mol Biol Cell. 2015 Dec 1;26(24):4438-50. doi: 10.1091/mbc.E15-06-0354. Epub 2015 Sep 30.
Eukaryotic cells eliminate misfolded proteins from the endoplasmic reticulum (ER) via a conserved process termed ER-associated degradation (ERAD). Central regulators of the ERAD system are membrane-bound ubiquitin ligases, which are thought to channel misfolded proteins through the ER membrane during retrotranslocation. Hrd1 and gp78 are mammalian ubiquitin ligases homologous to Hrd1p, an ubiquitin ligase essential for ERAD in Saccharomyces cerevisiae. However, the functional relevance of these proteins to Hrd1p is unclear. In this paper, we characterize the gp78-containing ubiquitin ligase complex and define its functional interplay with Hrd1 using biochemical and recently developed CRISPR-based genetic tools. Our data show that transient inactivation of the gp78 complex by short hairpin RNA-mediated gene silencing causes significant stabilization of both luminal and membrane ERAD substrates, but unlike Hrd1, which plays an essential role in retrotranslocation and ubiquitination of these ERAD substrates, knockdown of gp78 does not affect either of these processes. Instead, gp78 appears to act downstream of Hrd1 to promote ERAD via cooperation with the BAG6 chaperone complex. We conclude that the Hrd1 complex forms an essential retrotranslocation module that is evolutionarily conserved, but the mammalian ERAD system uses additional ubiquitin ligases to assist Hrd1 during retrotranslocation.
真核细胞通过一种称为内质网相关降解（ERAD）的保守过程，从内质网（ER）中清除错误折叠的蛋白质。ERAD系统的核心调节因子是膜结合泛素连接酶，人们认为这些酶在逆向转运过程中引导错误折叠的蛋白质穿过内质网膜。Hrd1和gp78是与Hrd1p同源的哺乳动物泛素连接酶，Hrd1p是酿酒酵母中ERAD所必需的一种泛素连接酶。然而，这些蛋白质与Hrd1p的功能相关性尚不清楚。在本文中，我们对含gp78的泛素连接酶复合物进行了表征，并使用生化和最近开发的基于CRISPR的基因工具确定了它与Hrd1的功能相互作用。我们的数据表明，通过短发夹RNA介导的基因沉默对gp78复合物进行瞬时失活，会导致内质网腔和膜上的ERAD底物都显著稳定，但与在这些ERAD底物的逆向转运和泛素化中起关键作用的Hrd1不同，敲低gp78不会影响这两个过程中的任何一个。相反，gp78似乎在Hrd1的下游起作用，通过与BAG6伴侣复合物合作来促进ERAD。我们得出结论，Hrd1复合物形成了一个进化上保守的必需逆向转运模块，但哺乳动物的ERAD系统在逆向转运过程中使用额外的泛素连接酶来协助Hrd1。
