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

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.

Metrics

  • 4,436
    views
  • 586
    downloads
  • 18
    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. 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

Further reading

    1. Cell Biology
    Qi Zeng, Chen Yao ... Shuai Chen
    Research Article

    Mounting evidence has demonstrated the genetic association of ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3) gene polymorphisms with bronchial asthma and a diverse set of inflammatory disorders. However, its role in type I interferon (type I IFN) signaling remains poorly defined. Herein, we report that ORMDL3 is a negative modulator of the type I IFN signaling by interacting with mitochondrial antiviral signaling protein (MAVS) and subsequently promoting the proteasome-mediated degradation of retinoic acid-inducible gene I (RIG-I). Immunoprecipitation coupled with mass spectrometry (IP-MS) assays uncovered that ORMDL3 binds to ubiquitin-specific protease 10 (USP10), which forms a complex with and stabilizes RIG-I through decreasing its K48-linked ubiquitination. ORMDL3 thus disrupts the interaction between USP10 and RIG-I, thereby promoting RIG-I degradation. Additionally, subcutaneous syngeneic tumor models in C57BL/6 mice revealed that inhibition of ORMDL3 enhances anti-tumor efficacy by augmenting the proportion of cytotoxic CD8 positive T cells and IFN production in the tumor microenvironment (TME). Collectively, our findings reveal the pivotal roles of ORMDL3 in maintaining antiviral innate immune responses and anti-tumor immunity.

    1. Cell Biology
    Swastika Sur, Maggie Kerwin ... Minnie M Sarwal
    Research Article

    Understanding the unique susceptibility of the human kidney to pH dysfunction and injury in cystinosis is paramount to developing new therapies to preserve renal function. Renal proximal tubular epithelial cells (RPTECs) and fibroblasts isolated from patients with cystinosis were transcriptionally profiled. Lysosomal fractionation, immunoblotting, confocal microscopy, intracellular pH, TEM, and mitochondrial stress test were performed for validation. CRISPR, CTNS -/- RPTECs were generated. Alterations in cell stress, pH, autophagic turnover, and mitochondrial energetics highlighted key changes in the V-ATPases in patient-derived and CTNS-/- RPTECs. ATP6V0A1 was significantly downregulated in cystinosis and highly co-regulated with loss of CTNS. Correction of ATP6V0A1 rescued cell stress and mitochondrial function. Treatment of CTNS -/- RPTECs with antioxidants ATX induced ATP6V0A1 expression and improved autophagosome turnover and mitochondrial integrity. Our exploratory transcriptional and in vitro cellular and functional studies confirm that loss of Cystinosin in RPTECs, results in a reduction in ATP6V0A1 expression, with changes in intracellular pH, mitochondrial integrity, mitochondrial function, and autophagosome-lysosome clearance. The novel findings are ATP6V0A1’s role in cystinosis-associated renal pathology and among other antioxidants, ATX specifically upregulated ATP6V0A1, improved autophagosome turnover or reduced autophagy and mitochondrial integrity. This is a pilot study highlighting a novel mechanism of tubular injury in cystinosis.