A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex
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...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2019-08-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/50003 |
| _version_ | 1811202753023705088 |
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| author | Edward A Partlow Richard W Baker Gwendolyn M Beacham Joshua S Chappie Andres E Leschziner Gunther Hollopeter |
| author_facet | Edward A Partlow Richard W Baker Gwendolyn M Beacham Joshua S Chappie Andres E Leschziner Gunther Hollopeter |
| author_sort | Edward A Partlow |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-12T02:43:48Z |
| format | Article |
| id | doaj.art-094aeadc1c9548f2b101e3cf7d38a0e8 |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-12T02:43:48Z |
| publishDate | 2019-08-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-094aeadc1c9548f2b101e3cf7d38a0e82022-12-22T03:51:13ZengeLife Sciences Publications LtdeLife2050-084X2019-08-01810.7554/eLife.50003A structural mechanism for phosphorylation-dependent inactivation of the AP2 complexEdward A Partlow0https://orcid.org/0000-0001-5513-088XRichard W Baker1https://orcid.org/0000-0003-1136-6000Gwendolyn M Beacham2https://orcid.org/0000-0001-7158-6887Joshua S Chappie3https://orcid.org/0000-0002-5733-7275Andres E Leschziner4Gunther Hollopeter5https://orcid.org/0000-0002-6409-0530Department of Molecular Medicine, Cornell University, New York, United StatesDepartment of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, United StatesDepartment of Molecular Medicine, Cornell University, New York, United StatesDepartment of Molecular Medicine, Cornell University, New York, United StatesDepartment of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, United States; Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, United StatesDepartment of Molecular Medicine, Cornell University, New York, United StatesEndocytosis 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.https://elifesciences.org/articles/50003endocytosisclathrin adaptorphosphorylationNECAPcryo-EMgenetic screen |
| spellingShingle | Edward A Partlow Richard W Baker Gwendolyn M Beacham Joshua S Chappie Andres E Leschziner Gunther Hollopeter A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex eLife endocytosis clathrin adaptor phosphorylation NECAP cryo-EM genetic screen |
| title | A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex |
| title_full | A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex |
| title_fullStr | A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex |
| title_full_unstemmed | A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex |
| title_short | A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex |
| title_sort | structural mechanism for phosphorylation dependent inactivation of the ap2 complex |
| topic | endocytosis clathrin adaptor phosphorylation NECAP cryo-EM genetic screen |
| url | https://elifesciences.org/articles/50003 |
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