eIF2B conformation and assembly state regulates the integrated stress response

  1. Michael Schoof
  2. Morgane Boone
  3. Lan Wang
  4. Rosalie Lawrence
  5. Adam Frost  Is a corresponding author
  6. Peter Walter  Is a corresponding author
  1. University of California, San Francisco, United States
  2. Howard Hughes Medical Institute, University of California, San Francisco, United States

Abstract

The integrated stress response (ISR) is activated by phosphorylation of the translation initiation factor eIF2 in response to various stress conditions. Phosphorylated eIF2 (eIF2-P) inhibits eIF2's nucleotide exchange factor eIF2B, a two-fold symmetric heterodecamer assembled from subcomplexes. Here, we monitor and manipulate eIF2B assembly in vitro and in vivo. In the absence of eIF2B's α-subunit, the ISR is induced because unassembled eIF2B tetramer subcomplexes accumulate in cells. Upon addition of the small-molecule ISR inhibitor ISRIB, eIF2B tetramers assemble into active octamers. Surprisingly, ISRIB inhibits the ISR even in the context of fully assembled eIF2B decamers, revealing allosteric communication between the physically distant eIF2, eIF2-P, and ISRIB binding sites. Cryo-EM structures suggest a rocking motion in eIF2B that couples these binding sites. eIF2-P binding converts eIF2B decamers into 'conjoined tetramers' with diminished substrate binding and enzymatic activity. Canonical eIF2-P-driven ISR activation thus arises due to this change in eIF2B's conformational state.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Michael Schoof

    Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
  2. Morgane Boone

    Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7807-5542
  3. Lan Wang

    Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8931-7201
  4. Rosalie Lawrence

    Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
  5. Adam Frost

    Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    For correspondence
    adam.frost@ucsf.edu
    Competing interests
    Adam Frost, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2231-2577
  6. Peter Walter

    Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States
    For correspondence
    peter@walterlab.ucsf.edu
    Competing interests
    Peter Walter, PW is an inventor on U.S. Patent 9708247 held by the Regents of the University of California that describes ISRIB and its analogs. Rights to the invention have been licensed by UCSF to Calico..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6849-708X

Funding

Howard Hughes Medical Institute (Investigator Grant)

  • Peter Walter

Howard Hughes Medical Institute (HHMI Faculty Scholar Grant)

  • Adam Frost

Calico Life Sciences LLC

  • Peter Walter

The George and Judy Marcus Family Foundation

  • Peter Walter

Damon Runyon Cancer Research Foundation (Postdoctoral Fellowship)

  • Lan Wang

Jane Coffin Childs Memorial Fund for Medical Research (Postdoctoral Fellowship)

  • Rosalie Lawrence

Belgian-American Educational Foundation (Postdoctoral Fellowship)

  • Morgane Boone

Chan Zuckerberg Initiative (Investigator Grant)

  • Adam Frost

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

Reviewing Editor

  1. Nahum Sonenberg, McGill University, Canada

Publication history

  1. Received: December 12, 2020
  2. Accepted: March 9, 2021
  3. Accepted Manuscript published: March 10, 2021 (version 1)
  4. Version of Record published: March 24, 2021 (version 2)
  5. Version of Record updated: April 7, 2021 (version 3)

Copyright

© 2021, Schoof 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.

Metrics

  • 3,672
    Page views
  • 585
    Downloads
  • 24
    Citations

Article citation count generated by polling the highest count across the following sources: Scopus, Crossref, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Michael Schoof
  2. Morgane Boone
  3. Lan Wang
  4. Rosalie Lawrence
  5. Adam Frost
  6. Peter Walter
(2021)
eIF2B conformation and assembly state regulates the integrated stress response
eLife 10:e65703.
https://doi.org/10.7554/eLife.65703

Further reading

    1. Biochemistry and Chemical Biology
    Ziyue Wang, Michael Mülleder ... Markus Ralser
    Research Article

    The possibility to record proteomes in high throughput and at high quality has opened new avenues for biomedical research, drug discovery, systems biology, and clinical translation. However, high-throughput proteomic experiments often require high sample amounts and can be less sensitive compared to conventional proteomic experiments. Here, we introduce and benchmark Zeno SWATH MS, a data-independent acquisition technique that employs a linear ion trap pulsing (Zeno trap pulsing) to increase the sensitivity in high-throughput proteomic experiments. We demonstrate that when combined with fast micro- or analytical flow-rate chromatography, Zeno SWATH MS increases protein identification with low sample amounts. For instance, using 20 min micro-flow-rate chromatography, Zeno SWATH MS identified more than 5000 proteins consistently, and with a coefficient of variation of 6%, from a 62.5 ng load of human cell line tryptic digest. Using 5 min analytical flow-rate chromatography (800 µl/min), Zeno SWATH MS identified 4907 proteins from a triplicate injection of 2 µg of a human cell lysate, or more than 3000 proteins from a 250 ng tryptic digest. Zeno SWATH MS hence facilitates sensitive high-throughput proteomic experiments with low sample amounts, mitigating the current bottlenecks of high-throughput proteomics.

    1. Biochemistry and Chemical Biology
    2. Neuroscience
    Jinli Geng, Yingjun Tang ... Xiaodong Liu
    Research Article Updated

    Dynamic Ca2+ signals reflect acute changes in membrane excitability, and also mediate signaling cascades in chronic processes. In both cases, chronic Ca2+ imaging is often desired, but challenged by the cytotoxicity intrinsic to calmodulin (CaM)-based GCaMP, a series of genetically-encoded Ca2+ indicators that have been widely applied. Here, we demonstrate the performance of GCaMP-X in chronic Ca2+ imaging of cortical neurons, where GCaMP-X by design is to eliminate the unwanted interactions between the conventional GCaMP and endogenous (apo)CaM-binding proteins. By expressing in adult mice at high levels over an extended time frame, GCaMP-X showed less damage and improved performance in two-photon imaging of sensory (whisker-deflection) responses or spontaneous Ca2+ fluctuations, in comparison with GCaMP. Chronic Ca2+ imaging of one month or longer was conducted for cultured cortical neurons expressing GCaMP-X, unveiling that spontaneous/local Ca2+ transients progressively developed into autonomous/global Ca2+ oscillations. Along with the morphological indices of neurite length and soma size, the major metrics of oscillatory Ca2+, including rate, amplitude and synchrony were also examined. Dysregulations of both neuritogenesis and Ca2+ oscillations became discernible around 2–3 weeks after virus injection or drug induction to express GCaMP in newborn or mature neurons, which were exacerbated by stronger or prolonged expression of GCaMP. In contrast, neurons expressing GCaMP-X were significantly less damaged or perturbed, altogether highlighting the unique importance of oscillatory Ca2+ to neural development and neuronal health. In summary, GCaMP-X provides a viable solution for Ca2+ imaging applications involving long-time and/or high-level expression of Ca2+ probes.