A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex
- Cornell University, United States
- 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
Gunther Hollopeter
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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A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex
eLife 8:e50003.
https://doi.org/10.7554/eLife.50003
