Conserved structural elements specialize ATAD1 as a membrane protein extraction machine
- Howard Hughes Medical Institute, University of California, San Francisco, United States
- University of California San Francisco Medical Center, United States
- University of California San Francisco, United States
- 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
-
Conserved structural elements specialize ATAD1 as a membrane protein extraction machineDryad Digital Repository, doi:10.5061/dryad.q2bvq83n3.
Article and author information
Author details
Hannah Toutkoushian
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.
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
-
- 2,625
- views
-
- 478
- downloads
-
- 10
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
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)
Conserved structural elements specialize ATAD1 as a membrane protein extraction machine
eLife 11:e73941.
https://doi.org/10.7554/eLife.73941
Further reading
-
- Cell Biology
- Developmental Biology
A study in mice reveals key interactions between proteins involved in fibroblast growth factor signaling and how they contribute to distinct stages of eye lens development.
-
- Cell Biology
- Evolutionary Biology
Maintenance of rod-shape in bacterial cells depends on the actin-like protein MreB. Deletion of mreB from Pseudomonas fluorescens SBW25 results in viable spherical cells of variable volume and reduced fitness. Using a combination of time-resolved microscopy and biochemical assay of peptidoglycan synthesis, we show that reduced fitness is a consequence of perturbed cell size homeostasis that arises primarily from differential growth of daughter cells. A 1000-generation selection experiment resulted in rapid restoration of fitness with derived cells retaining spherical shape. Mutations in the peptidoglycan synthesis protein Pbp1A were identified as the main route for evolutionary rescue with genetic reconstructions demonstrating causality. Compensatory pbp1A mutations that targeted transpeptidase activity enhanced homogeneity of cell wall synthesis on lateral surfaces and restored cell size homeostasis. Mechanistic explanations require enhanced understanding of why deletion of mreB causes heterogeneity in cell wall synthesis. We conclude by presenting two testable hypotheses, one of which posits that heterogeneity stems from non-functional cell wall synthesis machinery, while the second posits that the machinery is functional, albeit stalled. Overall, our data provide support for the second hypothesis and draw attention to the importance of balance between transpeptidase and glycosyltransferase functions of peptidoglycan building enzymes for cell shape determination.
