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Elife DOI:10.7554/eLife.69142

An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput CRISPRi screens.

Publication TypeJournal Article
Year of Publication2021
AuthorsCoukos, R, Yao, D, Sanchez, MI, Strand, ET, Olive, ME, Udeshi, ND, Weissman, JS, Carr, SA, Bassik, MC, Ting, AY
Date Published2021 08 20

The trafficking of specific protein cohorts to correct subcellular locations at correct times is essential for every signaling and regulatory process in biology. Gene perturbation screens could provide a powerful approach to probe the molecular mechanisms of protein trafficking, but only if protein localization or mislocalization can be tied to a simple and robust phenotype for cell selection, such as cell proliferation or fluorescence-activated cell sorting (FACS). To empower the study of protein trafficking processes with gene perturbation, we developed a genetically encoded molecular tool named HiLITR (High-throughput Localization Indicator with Transcriptional Readout). HiLITR converts protein colocalization into proteolytic release of a membrane-anchored transcription factor, which drives the expression of a chosen reporter gene. Using HiLITR in combination with FACS-based CRISPRi screening in human cell lines, we identified genes that influence the trafficking of mitochondrial and ER tail-anchored proteins. We show that loss of the SUMO E1 component SAE1 results in mislocalization and destabilization of many mitochondrial tail-anchored proteins. We also demonstrate a distinct regulatory role for EMC10 in the ER membrane complex, opposing the transmembrane-domain insertion activity of the complex. Through transcriptional integration of complex cellular functions, HiLITR expands the scope of biological processes that can be studied by genetic perturbation screening technologies.


Alternate JournalElife
PubMed ID34414886
PubMed Central IDPMC8423448
Grant ListDP2 HD084069 / HD / NICHD NIH HHS / United States
R01 MH119353 / MH / NIMH NIH HHS / United States
T32 HG000044 / HG / NHGRI NIH HHS / United States
/ HHMI / Howard Hughes Medical Institute / United States