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

View ORCID ProfileRobert Coukos, David Yao, View ORCID ProfileMateo I. Sanchez, View ORCID ProfileEric T. Strand, Jonathan S. Weissman, View ORCID ProfileMichael C. Bassik, View ORCID ProfileAlice Y. Ting
doi: https://doi.org/10.1101/2021.04.11.439362
Robert Coukos
1Department of Genetics, Stanford University, Stanford, CA 94305, USA.
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David Yao
1Department of Genetics, Stanford University, Stanford, CA 94305, USA.
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Mateo I. Sanchez
1Department of Genetics, Stanford University, Stanford, CA 94305, USA.
2Chan Zuckerberg Biohub, San Francisco, CA 94158, USA
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Eric T. Strand
1Department of Genetics, Stanford University, Stanford, CA 94305, USA.
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Jonathan S. Weissman
3Whitehead Institute, Cambridge, MA 02142, USA.
4Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
5Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA
6Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA
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Michael C. Bassik
1Department of Genetics, Stanford University, Stanford, CA 94305, USA.
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  • For correspondence: ayting@stanford.edu bassik@stanford.edu
Alice Y. Ting
1Department of Genetics, Stanford University, Stanford, CA 94305, USA.
2Chan Zuckerberg Biohub, San Francisco, CA 94158, USA
7Department of Biology, Stanford University, Stanford, CA 94305, USA
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  • For correspondence: ayting@stanford.edu bassik@stanford.edu
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Abstract

The trafficking of specific protein cohorts to the correct subcellular location at the correct time 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 FACS. To broadly empower the study of protein trafficking processes with gene perturbation, we developed a genetically-encoded molecular tool named HiLITR. 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 identify genes that influence the trafficking of mitochondrial and ER tail-anchored proteins. We show that loss of the SUMO E1 component SAE1 results in the mislocalization and destabilization of 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.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted April 12, 2021.
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An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput screens
Robert Coukos, David Yao, Mateo I. Sanchez, Eric T. Strand, Jonathan S. Weissman, Michael C. Bassik, Alice Y. Ting
bioRxiv 2021.04.11.439362; doi: https://doi.org/10.1101/2021.04.11.439362
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An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput screens
Robert Coukos, David Yao, Mateo I. Sanchez, Eric T. Strand, Jonathan S. Weissman, Michael C. Bassik, Alice Y. Ting
bioRxiv 2021.04.11.439362; doi: https://doi.org/10.1101/2021.04.11.439362

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