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

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...

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Autores principales:	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
Formato:	article
Lenguaje:	EN
Publicado:	eLife Sciences Publications Ltd 2021
Materias:	protein engineering CRISPR high-throughput screens tail-anchored proteins Medicine R Science Q Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/6d814cb3a91342cea81c16b1402d48aa
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spelling	oai:doaj.org-article:6d814cb3a91342cea81c16b1402d48aa2021-11-26T11:14:06ZAn engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput CRISPRi screens10.7554/eLife.691422050-084Xe69142https://doaj.org/article/6d814cb3a91342cea81c16b1402d48aa2021-08-01T00:00:00Zhttps://elifesciences.org/articles/69142https://doaj.org/toc/2050-084XThe 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.Robert CoukosDavid YaoMateo I SanchezEric T StrandMeagan E OliveNamrata D UdeshiJonathan S WeissmanSteven A CarrMichael C BassikAlice Y TingeLife Sciences Publications Ltdarticleprotein engineeringCRISPRhigh-throughput screenstail-anchored proteinsMedicineRScienceQBiology (General)QH301-705.5ENeLife, Vol 10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	protein engineering CRISPR high-throughput screens tail-anchored proteins Medicine R Science Q Biology (General) QH301-705.5
spellingShingle	protein engineering CRISPR high-throughput screens tail-anchored proteins Medicine R Science Q Biology (General) QH301-705.5 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
description	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.
format	article
author	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
author_facet	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
author_sort	Robert Coukos
title	An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput CRISPRi screens
title_short	An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput CRISPRi screens
title_full	An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput CRISPRi screens
title_fullStr	An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput CRISPRi screens
title_full_unstemmed	An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput CRISPRi screens
title_sort	engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and er membrane protein trafficking in high-throughput crispri screens
publisher	eLife Sciences Publications Ltd
publishDate	2021
url	https://doaj.org/article/6d814cb3a91342cea81c16b1402d48aa
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An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput CRISPRi screens

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