1. Biochemistry and Chemical Biology
  2. Cell Biology
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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

  1. Robert W Coukos
  2. David Yao
  3. Mateo Lopez Sanchez
  4. Eric T Strand
  5. Meagan E Olive
  6. Namrata D Udeshi
  7. Jonathan S Weissman
  8. Steven A Carr
  9. Michael C Bassik  Is a corresponding author
  10. Alice Y Ting  Is a corresponding author
  1. Stanford University, United States
  2. Broad Institute of MIT and Harvard, United States
  3. Whitehead Institute, United States
Research Article
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Cite this article as: eLife 2021;10:e69142 doi: 10.7554/eLife.69142

Abstract

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. 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.

Data availability

Lead contact: Further information and requests for resources or reagents should be directed to the lead contact, Alice Ting (ayting@stanford.edu)Materials availability: Plasmids generated in the study have been deposited to Addgene or are available upon request (Supplementary File 1)Data and code availability: HiLITR screen sequencing data has been deposited to Dryad (doi:10.5061/dryad.tb2rbp00n). The original mass spectra and the protein sequence database used for searches have been deposited in the public proteomics repository MassIVE (http://massive.ucsd.edu) and are accessible at ftp://massive.ucsd.edu/MSV000087769/.

The following data sets were generated
    1. Yao D
    (2021) HiLITR CRISPR screens
    https://creativecommons.org/publicdomain/zero/1.0/.

Article and author information

Author details

  1. Robert W Coukos

    Genetics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7307-8293
  2. David Yao

    Department of Genetics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Mateo Lopez Sanchez

    Genetics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1359-6969
  4. Eric T Strand

    Genetics, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1488-1043
  5. Meagan E Olive

    Broad Institute of MIT and Harvard, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Namrata D Udeshi

    Broad Institute of MIT and Harvard, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Jonathan S Weissman

    Department of Biology, Whitehead Institute, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Steven A Carr

    Broad Institute of MIT and Harvard, Cambridge, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Michael C Bassik

    Department of Genetics, Stanford University, Stanford, United States
    For correspondence
    bassik@stanford.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5185-8427
  10. Alice Y Ting

    Department of Biology, Stanford University, Stanford, United States
    For correspondence
    ayting@stanford.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8277-5226

Funding

National Institute of Mental Health (MH119353)

  • Alice Y Ting

NIH Office of the Director (1DP2HD084069-01)

  • Michael C Bassik

National Science Foundation (1656518)

  • David Yao

Stanford Bio-X

  • Robert W Coukos

National Institute of Standards and Technology

  • Robert W Coukos

National Human Genome Research Institute (2T32HG000044)

  • Robert W Coukos
  • David Yao

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Heedeok Hong, Michigan State University, United States

Publication history

  1. Received: April 6, 2021
  2. Preprint posted: April 12, 2021 (view preprint)
  3. Accepted: August 18, 2021
  4. Accepted Manuscript published: August 20, 2021 (version 1)
  5. Version of Record published: September 7, 2021 (version 2)

Copyright

© 2021, Coukos et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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