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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\u2010dependent biotin labeling to identify substrates of ubiquitin ligases. Application of this method to RING finger protein 167 (<jats:styled-content style=\"fixed-case\">RNF<\/jats:styled-content>167), a member of the <jats:styled-content style=\"fixed-case\">PA<\/jats:styled-content> domain\u2010containing E3 family, led to identification of Arl8B as its substrate. We demonstrated that <jats:styled-content style=\"fixed-case\">RNF<\/jats:styled-content>167 ubiquitinates Arl8B at the lysine residue K141 and reduces the level of the Arl8B protein. Overexpression and knockdown of <jats:styled-content style=\"fixed-case\">RNF<\/jats:styled-content>167 revealed its regulatory role in Arl8B\u2010dependent lysosome positioning and endocytic trafficking to lysosomes. Furthermore, we found that the ubiquitination\u2010defective Arl8B K141R mutant counteracts <jats:styled-content style=\"fixed-case\">RNF<\/jats:styled-content>167 in these cellular events. These results indicate that <jats:styled-content style=\"fixed-case\">RNF<\/jats:styled-content>167 plays a crucial role as an E3 ubiquitin ligase targeting Arl8B to regulate lysosome positioning and endocytic trafficking.<\/jats:p>","DOI":"10.1111\/febs.13947","type":"journal-article","created":{"date-parts":[[2016,11,3]],"date-time":"2016-11-03T10:38:26Z","timestamp":1478169506000},"page":"4583-4599","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":28,"title":["<scp>RNF<\/scp>167 targets Arl8B for degradation to regulate lysosome positioning and endocytic trafficking"],"prefix":"10.1111","volume":"283","author":[{"given":"Rakesh","family":"Deshar","sequence":"first","affiliation":[{"name":"Department of Medical Lifesciences The Catholic University of Korea Seoul 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