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A genome‐wide CRISPR interference screen using a novel reporter for endo‐lysosomal trafficking reveals a role for RME‐8 in opioid receptor regulation
Author(s) -
Lobingier Braden,
Novy Brandon,
Maria Monica,
Tsvetanova Nikoleta,
Zastrow Mark
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.03224
Subject(s) - endocytic cycle , microbiology and biotechnology , endosome , endocytosis , lysosome , biology , membrane protein , transport protein , rab , crispr , chemistry , receptor , gtpase , membrane , biochemistry , gene , enzyme , intracellular
Membrane protein trafficking is essential for cellular function and mistrafficking can alter or disrupt membrane protein activity. Here we describe a highly sensitive chemical biology method which allows for quantitative single‐cell analysis of membrane protein expression and trafficking. This approach is based on the engineered peroxidase called APEX2 and activation of a fluorogenic substrate. When genetically fused to a membrane protein, we demonstrate that the catalytic activity of APEX2 is rapidly quenched upon trafficking to the lysosome. This characteristic allows APEX2‐based genetic screening of the preceding trafficking steps including movement through the secretory, endocytic, and endosomal pathways. We applied this approach to understand the mechanism by which the delta opioid receptor traffics to the lysosome. Using a genome‐wide CRISPR interference screen, we identified novel genes regulating the trafficking of the delta opioid receptor including receptor‐mediated endocytosis 8 (RME‐8). We show that RME‐8 plays a critical role in the post‐endocytic trafficking of the delta opioid receptor. Together, this chemical biology approach for studying membrane protein trafficking provides a rapid and highly sensitive way to interrogate the critical processes which mediate movement of membrane proteins between organelles.