1. Biochemistry and Chemical Biology
  2. Chromosomes and Gene Expression
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In vitro analysis of RQC activities provides insights into the mechanism and function of CAT tailing

  1. Beatriz A Osuna
  2. Conor J Howard
  3. Subheksha KC
  4. Adam Frost  Is a corresponding author
  5. David E Weinberg  Is a corresponding author
  1. University of California, San Francisco, United States
Research Article
  • Cited 19
  • Views 2,644
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Cite this article as: eLife 2017;6:e27949 doi: 10.7554/eLife.27949

Abstract

Ribosomes can stall during translation due to defects in the mRNA template or translation machinery, leading to the production of incomplete proteins. The Ribosome-associated Quality control Complex (RQC) engages stalled ribosomes and targets nascent polypeptides for proteasomal degradation. However, how each RQC component contributes to this process remains unclear. Here we demonstrate that key RQC activities-Ltn1p-dependent ubiquitination and Rqc2p-mediated Carboxy-terminal Alanine and Threonine (CAT) tail elongation-can be recapitulated in vitro with a yeast cell-free system. Using this approach, we determined that CAT tailing is mechanistically distinct from canonical translation, that Ltn1p-mediated ubiquitination depends on the poorly characterized RQC component Rqc1p, and that the process of CAT tailing enables robust ubiquitination of the nascent polypeptide. These findings establish a novel system to study the RQC and provide a framework for understanding how RQC factors coordinate their activities to facilitate clearance of incompletely synthesized proteins.

Article and author information

Author details

  1. Beatriz A Osuna

    Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Conor J Howard

    Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Subheksha KC

    Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Adam Frost

    Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    For correspondence
    adam.frost@ucsf.edu
    Competing interests
    The authors declare that no competing interests exist.
  5. David E Weinberg

    Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
    For correspondence
    david.weinberg@ucsf.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9348-1709

Funding

National Science Foundation (Graduate Research Fellowship)

  • Beatriz A Osuna

UCSF Mortiz-Heyman Discovery Fellowship (Graduate Student Research Fellowship)

  • Beatriz A Osuna

UCSF Hillblom Fellowship (Graduate Student Research Fellowship)

  • Conor J Howard

Searle Scholars Program (13SSP218)

  • Adam Frost

NIH Office of the Director (DP2GM110772)

  • Adam Frost

UCSF Program for Breakthrough Biomedical Research funded in part by the Sandler Foundation

  • Adam Frost
  • David E Weinberg

NIH Office of the Director (DP5OD017895)

  • David E Weinberg

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

Reviewing Editor

  1. Alan G Hinnebusch, National Institutes of Health, United States

Publication history

  1. Received: April 20, 2017
  2. Accepted: July 11, 2017
  3. Accepted Manuscript published: July 18, 2017 (version 1)
  4. Version of Record published: August 18, 2017 (version 2)

Copyright

© 2017, Osuna 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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