Deshar Rakesh, Moon Song, Yoo Wonjin, Cho Eun-Bee, Yoon Sungjoo K, Yoon Jong-Bok
Department of Medical Lifesciences, The Catholic University of Korea, Seoul, Korea.
Department of Biochemistry and Translational Research Center for Protein Function Control, Yonsei University, Seoul, Korea.
FEBS J. 2016 Dec;283(24):4583-4599. doi: 10.1111/febs.13947. Epub 2016 Nov 16.
The protease-associated (PA) domain-containing E3 ubiquitin ligases are transmembrane proteins located in intracellular organelles such as the endoplasmic reticulum, endosomes, or lysosomes. The functional roles of these ubiquitin ligases are not well defined. To understand the function of E3 ubiquitin ligases, identification of their substrates is of critical importance. In this study, we describe a newly devised method based on proximity-dependent biotin labeling to identify substrates of ubiquitin ligases. Application of this method to RING finger protein 167 (RNF167), a member of the PA domain-containing E3 family, led to identification of Arl8B as its substrate. We demonstrated that RNF167 ubiquitinates Arl8B at the lysine residue K141 and reduces the level of the Arl8B protein. Overexpression and knockdown of RNF167 revealed its regulatory role in Arl8B-dependent lysosome positioning and endocytic trafficking to lysosomes. Furthermore, we found that the ubiquitination-defective Arl8B K141R mutant counteracts RNF167 in these cellular events. These results indicate that RNF167 plays a crucial role as an E3 ubiquitin ligase targeting Arl8B to regulate lysosome positioning and endocytic trafficking.
含蛋白酶相关（PA）结构域的E3泛素连接酶是位于内质网、内体或溶酶体等细胞内细胞器中的跨膜蛋白。这些泛素连接酶的功能作用尚未明确界定。为了解E3泛素连接酶的功能，鉴定其底物至关重要。在本研究中，我们描述了一种基于邻近依赖性生物素标记的新方法，用于鉴定泛素连接酶的底物。将该方法应用于含PA结构域的E3家族成员环指蛋白167（RNF167），鉴定出Arl8B为其底物。我们证明RNF167在赖氨酸残基K141处使Arl8B泛素化，并降低Arl8B蛋白水平。RNF167的过表达和敲低揭示了其在Arl8B依赖性溶酶体定位和向溶酶体的内吞运输中的调节作用。此外，我们发现泛素化缺陷型Arl8B K141R突变体在这些细胞事件中抵消了RNF167的作用。这些结果表明，RNF167作为靶向Arl8B的E3泛素连接酶，在调节溶酶体定位和内吞运输中起关键作用。
