Cytosolic aspartate aminotransferase moonlights as a ribosome binding modulator of Gcn2 activity during oxidative stress

  1. Robert A Crawford
  2. Mark Peter Ashe
  3. Simon J Hubbard
  4. Graham D Pavitt  Is a corresponding author
  1. University of Manchester, United Kingdom

Abstract

Regulation of translation is a fundamental facet of the cellular response to rapidly changing external conditions. Specific RNA-binding proteins (RBPs) co-ordinate the translational regulation of distinct mRNA cohorts during stress. To identify RBPs with previously under-appreciated roles in translational control, we used polysome profiling and mass spectrometry to identify and quantify proteins associated with translating ribosomes in unstressed yeast cells and during oxidative stress and amino acid starvation, which both induce the integrated stress response (ISR). Over 800 proteins were identified across polysome gradient fractions, including ribosomal proteins, translation factors and many others without previously described translation-related roles, including numerous metabolic enzymes. We identified variations in patterns of polysome enrichment in both unstressed and stressed cells and identified proteins enriched in heavy polysomes during stress. Genetic screening of polysome-enriched RBPs identified the cytosolic aspartate aminotransferase, Aat2, as a ribosome-associated protein whose deletion conferred growth sensitivity to oxidative stress. Loss of Aat2 caused aberrantly high activation of the ISR via enhanced eIF2a phosphorylation and GCN4 activation. Importantly, non-catalytic AAT2 mutants retained polysome association and did not show heightened stress sensitivity. Aat2 therefore has a separate ribosome-associated translational regulatory or 'moonlighting' function that modulates the ISR independent of its aspartate aminotransferase activity.

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al. 2019) partner repository with the dataset identifier PXD027903.

Article and author information

Author details

  1. Robert A Crawford

    Division of Molecular and Cellular Function, University of Manchester, Manchester, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9788-5137
  2. Mark Peter Ashe

    Division of Molecular and Cellular Function, University of Manchester, Manchester, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4457-7851
  3. Simon J Hubbard

    Division of Evolution, Infection and Genomics, University of Manchester, Manchester, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8601-9524
  4. Graham D Pavitt

    Division of Molecular and Cellular Function, University of Manchester, Manchester, United Kingdom
    For correspondence
    graham.pavitt@manchester.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8593-2418

Funding

BBSRC (BB/M011208/1)

  • Robert A Crawford

Weizmann UK (2020/129488)

  • Graham D Pavitt

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

Reviewing Editor

  1. Ivan Topisirovic, Jewish General Hospital, Canada

Version history

  1. Received: August 30, 2021
  2. Preprint posted: September 10, 2021 (view preprint)
  3. Accepted: May 25, 2022
  4. Accepted Manuscript published: May 27, 2022 (version 1)
  5. Version of Record published: June 13, 2022 (version 2)

Copyright

© 2022, Crawford 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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  1. Robert A Crawford
  2. Mark Peter Ashe
  3. Simon J Hubbard
  4. Graham D Pavitt
(2022)
Cytosolic aspartate aminotransferase moonlights as a ribosome binding modulator of Gcn2 activity during oxidative stress
eLife 11:e73466.
https://doi.org/10.7554/eLife.73466

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https://doi.org/10.7554/eLife.73466

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    Background:

    Fetal growth restriction (FGR) is a pregnancy complication in which a newborn fails to achieve its growth potential, increasing the risk of perinatal morbidity and mortality. Chronic maternal gestational hypoxia, as well as placental insufficiency are associated with increased FGR incidence; however, the molecular mechanisms underlying FGR remain unknown.

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    Conclusions:

    This study suggests a possible involvement of placental BPGM in maternal-fetal oxygen transfer, and in the pathophysiology of FGR.

    Funding:

    This work was supported by the Weizmann Krenter Foundation and the Weizmann – Ichilov (Tel Aviv Sourasky Medical Center) Collaborative Grant in Biomedical Research, by the Minerva Foundation, by the ISF KillCorona grant 3777/19.