Conserved structural elements specialize ATAD1 as a membrane protein extraction machine

  1. Lan Wang  Is a corresponding author
  2. Hannah Toutkoushian
  3. Vladislav Belyy
  4. Claire Y Kokontis
  5. Peter Walter  Is a corresponding author
  1. Howard Hughes Medical Institute, University of California, San Francisco, United States
  2. University of California San Francisco Medical Center, United States
  3. University of California San Francisco, United States
  4. Altos labs, United States

Abstract

The mitochondrial AAA protein ATAD1 (in humans; Msp1 in yeast) removes mislocalized membrane proteins, as well as stuck import substrates from the mitochondrial outer membrane, facilitating their re-insertion into their cognate organelles and maintaining mitochondria's protein import capacity. In doing so, it helps to maintain proteostasis in mitochondria. How ATAD1 tackles the energetic challenge to extract hydrophobic membrane proteins from the lipid bilayer and what structural features adapt ATAD1 for its particular function has remained a mystery. Previously, we determined the structure of Msp1 in complex with a peptide substrate (Wang et al., 2020). The structure showed that Msp1's mechanism follows the general principle established for AAA proteins while adopting several structural features that specialize it for its function. Among these features in Msp1 was the utilization of multiple aromatic amino acids to firmly grip the substrate in the central pore. However, it was not clear whether the aromatic nature of these amino acids were required, or if they could be functionally replaced by aliphatic amino acids. In this work, we determined the cryo-EM structures of the human ATAD1 in complex with a peptide substrate at near atomic resolution. The structures show that phylogenetically conserved structural elements adapt ATAD1 for its function while generally adopting a conserved mechanism shared by many AAA proteins. We developed a microscopy-based assay reporting on protein mislocalization, with which we directly assessed ATAD1's activity in live cells and showed that both aromatic amino acids in pore-loop 1 are required for ATAD1’s function and cannot be substituted by aliphatic amino acids. A short α-helix at the C-terminus strongly facilitates ATAD1's oligomerization, a structural feature that distinguishes ATAD1 from its closely related proteins.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting file; The imaging analysis pipeline is described in Figure 3 -source data1. The raw images used for data analysis are deposited into dryad.FOR REVIEW: The structural models and validation reports are available to download from the eLife editorial Dropbox account: https://www.dropbox.com/sh/v4k7wo2vdwg0h58/AAD26fS2-z9-cIQGmfhNUOHka?dl=0

The following data sets were generated

Article and author information

Author details

  1. Lan Wang

    Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States
    For correspondence
    lan@walterlab.ucsf.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8931-7201
  2. Hannah Toutkoushian

    Department of Biochemistry and Biophysics, University of California San Francisco Medical Center, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7461-2005
  3. Vladislav Belyy

    Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Claire Y Kokontis

    Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9397-651X
  5. Peter Walter

    Altos labs, San Francisco, United States
    For correspondence
    pwalter@altoslabs.com
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6849-708X

Funding

National Institutes of Health (R01GM032384)

  • Lan Wang

National Institutes of Health (R01GM032384)

  • Hannah Toutkoushian

National Institutes of Health (R01GM032384)

  • Vladislav Belyy

National Institutes of Health (R01GM032384)

  • Peter Walter

Damon Runyon Cancer Research Foundation (DRG-2312-17)

  • Lan Wang

Damon Runyon Cancer Research Foundation (DRG-2284-17)

  • Vladislav Belyy

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

Reviewing Editor

  1. Franz-Ulrich Hartl, Max Planck Institute for Biochemistry, Germany

Publication history

  1. Received: September 24, 2021
  2. Accepted: May 12, 2022
  3. Accepted Manuscript published: May 12, 2022 (version 1)

Copyright

© 2022, Wang 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

  • 362
    Page views
  • 97
    Downloads
  • 0
    Citations

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

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. Lan Wang
  2. Hannah Toutkoushian
  3. Vladislav Belyy
  4. Claire Y Kokontis
  5. Peter Walter
(2022)
Conserved structural elements specialize ATAD1 as a membrane protein extraction machine
eLife 11:e73941.
https://doi.org/10.7554/eLife.73941

Further reading

    1. Cell Biology
    Robert J Tower et al.
    Research Article

    De novo limb regeneration after amputation is restricted in mammals to the distal digit tip. Central to this regenerative process is the blastema, a heterogeneous population of lineage-restricted, dedifferentiated cells that ultimately orchestrates regeneration of the amputated bone and surrounding soft tissue. To investigate skeletal regeneration, we made use of spatial transcriptomics to characterize the transcriptional profile specifically within the blastema. Using this technique, we generated a gene signature with high specificity for the blastema in both our spatial data, as well as other previously published single-cell RNA-sequencing transcriptomic studies. To elucidate potential mechanisms distinguishing regenerative from non-regenerative healing, we applied spatial transcriptomics to an aging model. Consistent with other forms of repair, our digit amputation mouse model showed a significant impairment in regeneration in aged mice. Contrasting young and aged mice, spatial analysis revealed a metabolic shift in aged blastema associated with an increased bioenergetic requirement. This enhanced metabolic turnover was associated with increased hypoxia and angiogenic signaling, leading to excessive vascularization and altered regenerated bone architecture in aged mice. Administration of the metabolite oxaloacetate decreased the oxygen consumption rate of the aged blastema and increased WNT signaling, leading to enhanced in vivo bone regeneration. Thus, targeting cell metabolism may be a promising strategy to mitigate aging-induced declines in tissue regeneration.

    1. Cell Biology
    Wenyang Li et al.
    Research Article

    Chronic liver injury causes fibrosis, characterized by the formation of scar tissue resulting from excessive accumulation of extracellular matrix (ECM) proteins. Hepatic stellate cell (HSC) myofibroblasts are the primary cell type responsible for liver fibrosis, yet there are currently no therapies directed at inhibiting the activity of HSC myofibroblasts. To search for potential anti-fibrotic compounds, we performed a high-throughput compound screen in primary human HSC myofibroblasts and identified 19 small molecules that induce HSC inactivation, including the polyether ionophore nanchangmycin (NCMC). NCMC induces lipid re-accumulation while reducing collagen expression, deposition of collagen in the extracellular matrix, cell proliferation, and migration. We find that NCMC increases cytosolic Ca2+ and reduces the phosphorylated protein levels of FYN, PTK2 (FAK), MAPK1/3 (ERK2/1), HSPB1 (HSP27), and STAT5B. Further, depletion of each of these kinases suppress COL1A1 expression. These studies reveal a signaling network triggered by NCMC to inactivate HSC myofibroblasts and reduce expression of proteins that compose the fibrotic scar. Identification of the antifibrotic effects of NCMC and the elucidation of pathways by which NCMC inhibits fibrosis provide new tools and therapeutic targets that could potentially be utilized to combat the development and progression of liver fibrosis.