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

Robert Coukos, David Yao, Mateo I Sanchez, Eric T Strand, Meagan E Olive, Namrata D Udeshi, Jonathan S Weissman, Steven A Carr, Michael C Bassik, Alice Y Ting

eLife 2021 Aug 20

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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. © 2021, Coukos et al.

Citation

Robert Coukos, David Yao, Mateo I Sanchez, Eric T Strand, Meagan E Olive, Namrata D Udeshi, Jonathan S Weissman, Steven A Carr, Michael C Bassik, Alice Y Ting. An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput CRISPRi screens. eLife. 2021 Aug 20;10