Abstract

Many important cellular membrane fission reactions are driven by ESCRT pathways, which culminate in disassembly of ESCRT-III polymers by the AAA ATPase Vps4. We report a 4.3 Å resolution cryo-EM structure of the active Vps4 hexamer with its cofactor Vta1, ADP•BeFx, and an ESCRT-III substrate peptide. Four Vps4 subunits form a helix whose interfaces are consistent with ATP-binding, is stabilized by Vta1, and binds the substrate peptide. The fifth subunit approximately continues this helix but appears to be dissociating. The final Vps4 subunit completes a notched-washer configuration as if transitioning between the ends of the helix. We propose that ATP binding propagates growth at one end of the helix while hydrolysis promotes disassembly at the other end, so that Vps4 'walks' along ESCRT-III until it encounters the ordered N-terminal domain to destabilize the ESCRT-III lattice. This model may be generally applicable to other protein-translocating AAA ATPases.

Data availability

The following data sets were generated
    1. Monroe N
    2. Han H
    3. Shen PS
    4. Sundquist WI
    5. Hill CP
    (2017) Vps4-Vta1 complex
    Publicly available at the RCSB Protein Data Bank (accession no: 5UIE).
    1. Monroe N
    2. Han H
    3. Shen PS
    4. Sundquist WI
    5. Hill CP
    (2017) Vps4-Vta1 complex
    Publicly available at the EMBL-EBI Protein Data Bank (accession no: EMD-8549).
    1. Monroe N
    2. Han H
    3. Shen PS
    4. Sundquist WI
    5. Hill CP
    (2017) Vps4-Vta1 complex_sharpened map
    Publicly available at the EMBL-EBI Protein Data Bank (accession no: EMD-8550).
    1. Monroe N
    2. Han H
    3. Shen PS
    4. Sundquist WI
    5. Hill CP
    (2017) Vps4-HCP hexamer
    Publicly available at the EMBL-EBI Protein Data Bank (accession no: EMD-8551).
    1. Monroe N
    2. Han H
    3. Shen PS
    4. Sundquist WI
    5. Hill CP
    (2017) Vps4-Vta1 complex_VSL_A
    Publicly available at the EMBL-EBI Protein Data Bank (accession no: EMD-8552).
    1. Monroe N
    2. Han H
    3. Shen PS
    4. Sundquist WI
    5. Hill CP
    (2017) Vps4-Vta1 complex_VSL_B
    Publicly available at the EMBL-EBI Protein Data Bank (accession no: EMD-8553).
    1. Monroe N
    2. Han H
    3. Shen PS
    4. Sundquist WI
    5. Hill CP
    (2017) Vps4-Vta1 complex_VSL_C
    Publicly available at the EMBL-EBI Protein Data Bank (accession no: EMD-8554).
    1. Monroe N
    2. Han H
    3. Shen PS
    4. Sundquist WI
    5. Hill CP
    (2017) Vps4-Vta1 complex_VSL_D
    Publicly available at the EMBL-EBI Protein Data Bank (accession no: EMD-8555).
    1. Monroe N
    2. Han H
    3. Shen PS
    4. Sundquist WI
    5. Hill CP
    (2017) Vps4-Vta1 complex_VSL_E
    Publicly available at the EMBL-EBI Protein Data Bank (accession no: EMD-8556).
    1. Monroe N
    2. Han H
    3. Shen PS
    4. Sundquist WI
    5. Hill CP
    (2017) Vps4-Vta1 complex_VSL_F
    Publicly available at the EMBL-EBI Protein Data Bank (accession no: EMD-8557).
    1. Monroe N
    2. Han H
    3. Shen PS
    4. Sundquist WI
    5. Hill CP
    (2017) Vps4-Vta1 complex, State 3 of subunitF
    Publicly available at the EMBL-EBI Protein Data Bank (accession no: EMD-8570).
    1. Monroe N
    2. Han H
    3. Shen PS
    4. Sundquist WI
    5. Hill CP
    (2017) Vps4-Vta1 complex, State 2 of subunitF
    Publicly available at the EMBL-EBI Protein Data Bank (accession no: EMD-8571).
    1. Monroe N
    2. Han H
    3. Shen PS
    4. Sundquist WI
    5. Hill CP
    (2017) Vps4-Vta1 complex, State 1 of subunitF
    Publicly available at the EMBL-EBI Protein Data Bank (accession no: EMD-8572).

Article and author information

Author details

  1. Nicole Monroe

    Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7678-4997
  2. Han Han

    Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, United States
    Competing interests
    No competing interests declared.
  3. Peter S Shen

    Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, United States
    For correspondence
    peter.shen@biochem.utah.edu
    Competing interests
    No competing interests declared.
  4. Wesley I Sundquist

    Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, United States
    For correspondence
    wes@biochem.utah.edu
    Competing interests
    Wesley I Sundquist, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9988-6021
  5. Christopher P Hill

    Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, United States
    For correspondence
    chris@biochem.utah.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6796-7740

Funding

National Institutes of Health (P50 GM082545)

  • Nicole Monroe
  • Han Han
  • Peter S Shen
  • Wesley I Sundquist
  • Christopher P Hill

National Institutes of Health (Microbial Pathogenesis Training Grant T32 AI055434)

  • Nicole Monroe

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

Reviewing Editor

  1. Sriram Subramaniam, National Cancer Institute, United States

Version history

  1. Received: December 21, 2016
  2. Accepted: April 4, 2017
  3. Accepted Manuscript published: April 5, 2017 (version 1)
  4. Version of Record published: May 2, 2017 (version 2)

Copyright

© 2017, Monroe 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

  • 6,144
    views
  • 1,209
    downloads
  • 120
    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. Nicole Monroe
  2. Han Han
  3. Peter S Shen
  4. Wesley I Sundquist
  5. Christopher P Hill
(2017)
Structural Basis of Protein Translocation by the Vps4-Vta1 AAA ATPase
eLife 6:e24487.
https://doi.org/10.7554/eLife.24487

Share this article

https://doi.org/10.7554/eLife.24487

Further reading

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Timothy A Bates, Mila Trank-Greene ... Fikadu G Tafesse
    Research Article

    Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6’s mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH-dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome.

    1. Biochemistry and Chemical Biology
    2. Evolutionary Biology
    Eva Pyrihová, Martin S King ... Edmund RS Kunji
    Research Article

    Stramenopiles form a clade of diverse eukaryotic organisms, including multicellular algae, the fish and plant pathogenic oomycetes, such as the potato blight Phytophthora, and the human intestinal protozoan Blastocystis. In most eukaryotes, glycolysis is a strictly cytosolic metabolic pathway that converts glucose to pyruvate, resulting in the production of NADH and ATP (Adenosine triphosphate). In contrast, stramenopiles have a branched glycolysis in which the enzymes of the pay-off phase are located in both the cytosol and the mitochondrial matrix. Here, we identify a mitochondrial carrier in Blastocystis that can transport glycolytic intermediates, such as dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, across the mitochondrial inner membrane, linking the cytosolic and mitochondrial branches of glycolysis. Comparative analyses with the phylogenetically related human mitochondrial oxoglutarate carrier (SLC25A11) and dicarboxylate carrier (SLC25A10) show that the glycolytic intermediate carrier has lost its ability to transport the canonical substrates malate and oxoglutarate. Blastocystis lacks several key components of oxidative phosphorylation required for the generation of mitochondrial ATP, such as complexes III and IV, ATP synthase, and ADP/ATP carriers. The presence of the glycolytic pay-off phase in the mitochondrial matrix generates ATP, which powers energy-requiring processes, such as macromolecular synthesis, as well as NADH, used by mitochondrial complex I to generate a proton motive force to drive the import of proteins and molecules. Given its unique substrate specificity and central role in carbon and energy metabolism, the carrier for glycolytic intermediates identified here represents a specific drug and pesticide target against stramenopile pathogens, which are of great economic importance.