1. Structural Biology and Molecular Biophysics
  2. Cell Biology

Structural inhibition of dynamin-mediated membrane fission by endophilin

  1. Annika Hohendahl
  2. Nathaniel Talledge
  3. Valentina Galli
  4. Peter S Shen
  5. Frédéric Humbert
  6. Pietro De Camilli
  7. Adam Frost  Is a corresponding author
  8. Aurélien Roux  Is a corresponding author
  1. University of Geneva, Switzerland
  2. University of California, San Francisco, United States
  3. University of Utah, United States
  4. Howard Hughes Medical Institute, Yale University School of Medicine, United States
https://doi.org/10.7554/eLife.26856
Share this article
Cite this article
  1. Annika Hohendahl
  2. Nathaniel Talledge
  3. Valentina Galli
  4. Peter S Shen
  5. Frédéric Humbert
  6. Pietro De Camilli
  7. Adam Frost
  8. Aurélien Roux
(2017)
Structural inhibition of dynamin-mediated membrane fission by endophilin
eLife 6:e26856.
https://doi.org/10.7554/eLife.26856
  2. Download BibTeX
  3. Download .RIS

Abstract

Dynamin, which mediates membrane fission during endocytosis, binds endophilin and other members of the Bin-Amphiphysin-Rvs (BAR) protein family. How endophilin influences endocytic membrane fission is still unclear. Here we show that dynamin-mediated membrane fission is potently inhibited in vitro when an excess of endophilin co-assembles with dynamin around membrane tubules. We further show by electron microscopy that endophilin intercalates between turns of the dynamin helix and impairs fission by preventing trans interactions between dynamin rungs that are thought to play critical roles in membrane constriction. In living cells, overexpression of endophilin delayed both fission and transferrin uptake. Together, our observations suggest that while endophilin helps shape endocytic tubules and recruit dynamin to endocytic sites, it can also block membrane fission when present in excess by inhibiting inter-dynamin interactions. The sequence of recruitment and the relative stoichiometry of the two proteins may be critical to regulated endocytic fission.

Article and author information

Author details

  1. Annika Hohendahl

    Department of Biochemistry, University of Geneva, Geneva, Switzerland
    Competing interests
    The authors declare that no competing interests exist.

  2. Nathaniel Talledge

    Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.

  3. Valentina Galli

    Department of Biochemistry, University of Geneva, Geneva, Switzerland
    Competing interests
    The authors declare that no competing interests exist.

  4. Peter S Shen

    Department of Biochemistry, University of Utah, Salt Lake City, United States
    Competing interests
    The authors declare that no competing interests exist.

  5. Frédéric Humbert

    Department of Biochemistry, University of Geneva, Geneva, Switzerland
    Competing interests
    The authors declare that no competing interests exist.

  6. Pietro De Camilli

    Department of Neuroscience, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.

  7. Adam Frost

    Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    For correspondence
    adam.frost@ucsf.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2231-2577

  8. Aurélien Roux

    Department of Biochemistry, University of Geneva, Geneva, Switzerland
    For correspondence
    aurelien.roux@unige.ch
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6088-0711

Funding

H2020 European Research Council (311536)

  • Aurélien Roux

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (31003A_130520)

  • Aurélien Roux

Human Frontier Science Program (CDA-0061-08)

  • Aurélien Roux

National Institutes of Health (1DP2GM110772-01)

  • Adam Frost

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (31003A_149975)

  • Aurélien Roux

Howard Hughes Medical Institute (55108523)

  • Pietro De Camilli

Searle scholars award (13SSP218)

  • Adam Frost

National Institutes of Health (NS036251)

  • Pietro De Camilli

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

Reviewing Editor

  1. Jenny Hinshaw, National Institutes of Health, United States

Version history

  1. Received: March 16, 2017
  2. Accepted: September 20, 2017
  3. Accepted Manuscript published: September 21, 2017 (version 1)
  4. Version of Record published: October 31, 2017 (version 2)

Copyright

© 2017, Hohendahl 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

  • 2,980
    views
  • 692
    downloads
  • 42
    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)

  1. Annika Hohendahl
  2. Nathaniel Talledge
  3. Valentina Galli
  4. Peter S Shen
  5. Frédéric Humbert
  6. Pietro De Camilli
  7. Adam Frost
  8. Aurélien Roux
(2017)
Structural inhibition of dynamin-mediated membrane fission by endophilin
eLife 6:e26856.
https://doi.org/10.7554/eLife.26856

Share this article

https://doi.org/10.7554/eLife.26856

Categories and tags

Further reading

    1. Structural Biology and Molecular Biophysics

    The mechanism of mammalian proton-coupled peptide transporters

    Simon M Lichtinger, Joanne L Parker ... Philip C Biggin
    Research Article

    Proton-coupled oligopeptide transporters (POTs) are of great pharmaceutical interest owing to their promiscuous substrate binding site that has been linked to improved oral bioavailability of several classes of drugs. Members of the POT family are conserved across all phylogenetic kingdoms and function by coupling peptide uptake to the proton electrochemical gradient. Cryo-EM structures and alphafold models have recently provided new insights into different conformational states of two mammalian POTs, SLC15A1, and SLC15A2. Nevertheless, these studies leave open important questions regarding the mechanism of proton and substrate coupling, while simultaneously providing a unique opportunity to investigate these processes using molecular dynamics (MD) simulations. Here, we employ extensive unbiased and enhanced-sampling MD to map out the full SLC15A2 conformational cycle and its thermodynamic driving forces. By computing conformational free energy landscapes in different protonation states and in the absence or presence of peptide substrate, we identify a likely sequence of intermediate protonation steps that drive inward-directed alternating access. These simulations identify key differences in the extracellular gate between mammalian and bacterial POTs, which we validate experimentally in cell-based transport assays. Our results from constant-PH MD and absolute binding free energy (ABFE) calculations also establish a mechanistic link between proton binding and peptide recognition, revealing key details underpining secondary active transport in POTs. This study provides a vital step forward in understanding proton-coupled peptide and drug transport in mammals and pave the way to integrate knowledge of solute carrier structural biology with enhanced drug design to target tissue and organ bioavailability.

    1. Structural Biology and Molecular Biophysics

    Plasticity of the proteasome-targeting signal Fat10 enhances substrate degradation

    Hitendra Negi, Aravind Ravichandran ... Ranabir Das
    Research Article

    The proteasome controls levels of most cellular proteins, and its activity is regulated under stress, quiescence, and inflammation. However, factors determining the proteasomal degradation rate remain poorly understood. Proteasome substrates are conjugated with small proteins (tags) like ubiquitin and Fat10 to target them to the proteasome. It is unclear if the structural plasticity of proteasome-targeting tags can influence substrate degradation. Fat10 is upregulated during inflammation, and its substrates undergo rapid proteasomal degradation. We report that the degradation rate of Fat10 substrates critically depends on the structural plasticity of Fat10. While the ubiquitin tag is recycled at the proteasome, Fat10 is degraded with the substrate. Our results suggest significantly lower thermodynamic stability and faster mechanical unfolding in Fat10 compared to ubiquitin. Long-range salt bridges are absent in the Fat10 structure, creating a plastic protein with partially unstructured regions suitable for proteasome engagement. Fat10 plasticity destabilizes substrates significantly and creates partially unstructured regions in the substrate to enhance degradation. NMR-relaxation-derived order parameters and temperature dependence of chemical shifts identify the Fat10-induced partially unstructured regions in the substrate, which correlated excellently to Fat10-substrate contacts, suggesting that the tag-substrate collision destabilizes the substrate. These results highlight a strong dependence of proteasomal degradation on the structural plasticity and thermodynamic properties of the proteasome-targeting tags.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    Special Issue: Allosteric regulation of kinase activity

    Amy H Andreotti, Volker Dötsch
    Editorial

    The articles in this special issue highlight how modern cellular, biochemical, biophysical and computational techniques are allowing deeper and more detailed studies of allosteric kinase regulation.