gp78 functions downstream of Hrd1 to promote degradation of misfolded proteins of the endoplasmic reticulum.

The functional relationship between mammalian ubiquitin ligase gp78 and Hrd1 was studied. Hrd1 is one of the essential retrotranslocation regulators conserved in yeast and mammalian cells, whereas gp78 serves an assisting role downstream of Hrd1 and possibly other ubiquitin ligases in mammalian cells. 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.

研究了哺乳动物泛素连接酶gp78和Hrd1之间的功能关系。Hrd1是在酵母和哺乳动物细胞中保守的必需的逆向转运调节因子之一，而gp78在哺乳动物细胞中Hrd1以及可能的其他泛素连接酶的下游起辅助作用。 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的泛素连接酶，它们被认为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，并利用生化方法以及最近开发的基于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介导的基因沉默使gp78复合物瞬时失活会导致腔内和膜内质网相关降解底物显著稳定，但与Hrd1不同，Hrd1在这些内质网相关降解底物的逆向转运和泛素化中起关键作用，而gp78的敲低并不影响这些过程中的任何一个。相反，gp78似乎在Hrd1的下游起作用，通过与BAG6伴侣复合物合作促进内质网相关降解。我们得出结论，Hrd1复合物形成了一个在进化上保守的必需的逆向转运模块，但哺乳动物内质网相关降解系统在逆向转运过程中使用额外的泛素连接酶来辅助Hrd1。