Article
- The EMBO Journal (2007) 26, 4066 - 4077
- doi:10.1038/sj.emboj.7601836
Published online: 30 August 2007
Subject Categories:
The NECAP PHear domain increases clathrin accessory protein binding potential
Brigitte Ritter1,a, Alexei Yu Denisov2,a, Jacynthe Philie1, Patrick D Allaire1, Valerie Legendre-Guillemin1,b, Peter Zylbergold1, Kalle Gehring2 and Peter S McPherson1
- Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada
- Department of Biochemistry, McGill University, Montreal, Quebec, Canada
Correspondence to:
Kalle Gehring, Department of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montreal, Canada H3G 1Y6. Tel.: +514 398 7287; Fax: +514 847 0220; E-mail: kalle.gehring@mcgill.ca
Peter S McPherson, Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, 3801 University Street, Montreal, Canada H3A 2B4. Tel.: +514 398 7355; Fax: +514 398 8106; E-mail: peter.mcpherson@mcgill.ca
aThese authors contributed equally to this work
bPresent address: Département des Sciences Fondamentales, Université du Québec à Chicoutimi, Chicoutimi, G7H 2B1, Canada
Received 11 April 2007; Accepted 30 July 2007
Abstract
AP-2 is a key regulator of the endocytic protein machinery driving clathrin-coated vesicle (CCV) formation. One critical function, mediated primarily by the AP-2 
-ear, is the recruitment of accessory proteins. NECAPs are -ear-binding proteins that enrich on CCVs. Here, we have solved the structure of the conserved N-terminal region of NECAP 1, revealing a unique module in the pleckstrin homology (PH) domain superfamily, which we named the PHear domain. The PHear domain binds accessory proteins bearing FxDxF motifs, which were previously thought to bind exclusively to the AP-2 -ear. Structural analysis of the PHear domain reveals the molecular surface for FxDxF motif binding, which was confirmed by site-directed mutagenesis. The reciprocal analysis of the FxDxF motif in amphiphysin I identified distinct binding requirements for binding to the -ear and PHear domain. We show that NECAP knockdown compromises transferrin uptake and establish a functional role for NECAPs in clathrin-mediated endocytosis. Our data uncover a striking convergence of two evolutionarily and structurally distinct modules to recognize a common peptide motif and promote efficient endocytosis.
Keywords:
- AP-2,
- clathrin,
- endocytosis,
- NMR,
- PH domain
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