1. Cell Biology
  2. Structural Biology and Molecular Biophysics

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
https://doi.org/10.7554/eLife.50003
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  1. Edward A Partlow
  2. Richard W Baker
  3. Gwendolyn M Beacham
  4. Joshua S Chappie
  5. Andres E Leschziner
  6. Gunther Hollopeter
(2019)
A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex
eLife 8:e50003.
https://doi.org/10.7554/eLife.50003
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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.

Data availability

The density maps generated during this study are available at the Electron Microscopy Data Bank (EMD-20215, unclamped and EMD-20220, clamped). The atomic structures generated during this study are available at the Protein Data Bank (PDB 6OWO, unclamped and 6OXL, clamped).

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.

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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  1. Edward A Partlow
  2. Richard W Baker
  3. Gwendolyn M Beacham
  4. Joshua S Chappie
  5. Andres E Leschziner
  6. Gunther Hollopeter
(2019)
A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex
eLife 8:e50003.
https://doi.org/10.7554/eLife.50003

Share this article

https://doi.org/10.7554/eLife.50003

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Further reading

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    NECAPs are negative regulators of the AP2 clathrin adaptor complex

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    Eukaryotic cells internalize transmembrane receptors via clathrin-mediated endocytosis, but it remains unclear how the machinery underpinning this process is regulated. We recently discovered that membrane-associated muniscin proteins such as FCHo and SGIP initiate endocytosis by converting the AP2 clathrin adaptor complex to an open, active conformation that is then phosphorylated (Hollopeter et al., 2014). Here we report that loss of ncap-1, the sole C. elegans gene encoding an adaptiN Ear-binding Coat-Associated Protein (NECAP), bypasses the requirement for FCHO-1. Biochemical analyses reveal AP2 accumulates in an open, phosphorylated state in ncap-1 mutant worms, suggesting NECAPs promote the closed, inactive conformation of AP2. Consistent with this model, NECAPs preferentially bind open and phosphorylated forms of AP2 in vitro and localize with constitutively open AP2 mutants in vivo. NECAPs do not associate with phosphorylation-defective AP2 mutants, implying that phosphorylation precedes NECAP recruitment. We propose NECAPs function late in endocytosis to inactivate AP2.

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