An empirical energy landscape reveals mechanism of proteasome in polypeptide translocation
Abstract
The ring-like ATPase complexes in the AAA+ family perform diverse cellular functions that require coordination between the conformational transitions of their individual ATPase subunits1,2. How the energy from ATP hydrolysis is captured to perform mechanical work by these coordinated movements is unknown. In this study, we developed a novel approach for delineating the nucleotide-dependent free-energy landscape (FEL) of the proteasome's heterohexameric ATPase complex based on complementary structural and kinetic measurements. We used the FEL to simulate the dynamics of the proteasome and quantitatively evaluated the predicted structural and kinetic properties. The FEL model predictions are consistent with a wide range of experimental observations in this and previous studies and suggested novel mechanistic features of the proteasomal ATPases. We find that the cooperative movements of the ATPase subunits result from the design of the ATPase hexamer entailing a unique free-energy minimum for each nucleotide-binding status. ATP hydrolysis dictates the direction of substrate translocation by triggering an energy-dissipating conformational transition of the ATPase complex.
Data availability
All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 3,4,5. The original images for the single-molecule experiments are available on Dryad (https://doi.org/10.5061/dryad.t1g1jwt2t). The source code is available in GitHub (https://github.com/luyinghms/Proteasome-FEL-model.git)
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Single molecule images for: An empirical energy landscape reveals mechanism of proteasome in polypeptide translocationDryad Digital Repository, doi:10.5061/dryad.t1g1jwt2t.
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cryo-structures of substrate-engaged human 26s proteasome6MSE, 6MSD, 6MSG, 6MSH, 6MSK, 6MSJ, 6MSB.
Article and author information
Author details
Funding
National Institute of General Medical Sciences (GM134064-01)
- Ying Lu
Edward Mallinckrodt, Jr. Foundation
- Ying Lu
Harvard Medical School (Dean's initiative award)
- Ying Lu
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- James M Berger, Johns Hopkins University School of Medicine, United States
Version history
- Preprint posted: June 22, 2021 (view preprint)
- Received: July 3, 2021
- Accepted: January 17, 2022
- Accepted Manuscript published: January 20, 2022 (version 1)
- Version of Record published: February 17, 2022 (version 2)
Copyright
© 2022, Fang 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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Further reading
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- Cell Biology
- Physics of Living Systems
As cells migrate and experience forces from their surroundings, they constantly undergo mechanical deformations which reshape their plasma membrane (PM). To maintain homeostasis, cells need to detect and restore such changes, not only in terms of overall PM area and tension as previously described, but also in terms of local, nano-scale topography. Here we describe a novel phenomenon, by which cells sense and restore mechanically induced PM nano-scale deformations. We show that cell stretch and subsequent compression reshape the PM in a way that generates local membrane evaginations in the 100 nm scale. These evaginations are recognized by I-BAR proteins, which triggers a burst of actin polymerization mediated by Rac1 and Arp2/3. The actin polymerization burst subsequently re-flattens the evagination, completing the mechanochemical feedback loop. Our results demonstrate a new mechanosensing mechanism for PM shape homeostasis, with potential applicability in different physiological scenarios.
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- Cell Biology
- Physics of Living Systems
Cell-generated forces play a major role in coordinating the large-scale behavior of cell assemblies, in particular during development, wound healing, and cancer. Mechanical signals propagate faster than biochemical signals, but can have similar effects, especially in epithelial tissues with strong cell–cell adhesion. However, a quantitative description of the transmission chain from force generation in a sender cell, force propagation across cell–cell boundaries, and the concomitant response of receiver cells is missing. For a quantitative analysis of this important situation, here we propose a minimal model system of two epithelial cells on an H-pattern (‘cell doublet’). After optogenetically activating RhoA, a major regulator of cell contractility, in the sender cell, we measure the mechanical response of the receiver cell by traction force and monolayer stress microscopies. In general, we find that the receiver cells show an active response so that the cell doublet forms a coherent unit. However, force propagation and response of the receiver cell also strongly depend on the mechano-structural polarization in the cell assembly, which is controlled by cell–matrix adhesion to the adhesive micropattern. We find that the response of the receiver cell is stronger when the mechano-structural polarization axis is oriented perpendicular to the direction of force propagation, reminiscent of the Poisson effect in passive materials. We finally show that the same effects are at work in small tissues. Our work demonstrates that cellular organization and active mechanical response of a tissue are key to maintain signal strength and lead to the emergence of elasticity, which means that signals are not dissipated like in a viscous system, but can propagate over large distances.