1. Cell Biology
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A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex

  1. Edward A Partlow
  2. Richard W Baker  Is a corresponding author
  3. Gwendolyn M Beacham
  4. Joshua S Chappie
  5. Andres E Leschziner  Is a corresponding author
  6. Gunther Hollopeter  Is a corresponding author
  1. Cornell University, United States
  2. University of California, San Diego, United States
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Cite this article as: eLife 2019;8:e50003 doi: 10.7554/eLife.50003

Abstract

Endocytosis of transmembrane proteins is orchestrated by the AP2 clathrin adaptor complex. AP2 dwells in a closed, inactive state in the cytosol, but adopts an open, active conformation on the plasma membrane. Membrane-activated complexes are also phosphorylated, but the significance of this mark is debated. We recently proposed that NECAP negatively regulates AP2 by binding open and phosphorylated complexes (Beacham et al., 2018). Here, we report high-resolution cryo-EM structures of NECAP bound to phosphorylated AP2. The site of AP2 phosphorylation is directly coordinated by residues of the NECAP PHear domain that are predicted from genetic screens in C. elegans. Using membrane mimetics to generate conformationally open AP2, we find that a second domain of NECAP binds these complexes and cryo-EM reveals both domains of NECAP engaging closed, inactive AP2. Assays in vitro and in vivo confirm these domains cooperate to inactivate AP2. We propose that phosphorylation marks adaptors for inactivation.

Article and author information

Author details

  1. Edward A Partlow

    Department of Molecular Medicine, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5513-088X
  2. Richard W Baker

    Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, United States
    For correspondence
    ribaker@ucsd.edu
    Competing interests
    The authors declare that no competing interests exist.
  3. Gwendolyn M Beacham

    Department of Molecular Medicine, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Joshua S Chappie

    Department of Molecular Medicine, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Andres E Leschziner

    Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, United States
    For correspondence
    aleschziner@ucsd.edu
    Competing interests
    The authors declare that no competing interests exist.
  6. Gunther Hollopeter

    Department of Molecular Medicine, Cornell University, Ithaca, United States
    For correspondence
    gh383@cornell.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6409-0530

Funding

National Institute of General Medical Sciences (R01 GM127548-01A1)

  • Gunther Hollopeter

Damon Runyon Cancer Research Foundation (DRG-#2285-17)

  • Richard W Baker

National Science Foundation (DGE-1650441)

  • Gwendolyn M Beacham

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

Reviewing Editor

  1. Suzanne R Pfeffer, Stanford University School of Medicine, United States

Publication history

  1. Received: July 9, 2019
  2. Accepted: August 28, 2019
  3. Accepted Manuscript published: August 29, 2019 (version 1)
  4. Version of Record published: September 10, 2019 (version 2)

Copyright

© 2019, Partlow 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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