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The NECAP PHear domain increases clathrin accessory protein binding potential Brigitte Ritter1,3 , Alexei Yu Denisov2,3 , Jacynthe Philie1 , Patrick D Allaire1 , Valerie Legendre-Guillemin1,4 , Peter Zylbergold1 , Kalle Gehring2, * and Peter S McPherson1, *

1 Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada and

2 Department of Biochemistry, McGill University, Montreal, Quebec, Canada AP-2 is a key regulator of the endocytic protein machinery driving clathrin-coated vesicle (CCV) formation.

One cri- tical function, mediated primarily by the AP-2 a-ear, is the recruitment of accessory proteins. NECAPs are a-ear-bind- ing 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 bear- ing FxDxF motifs, which were previously thought to bind exclusively to the AP-2 a-ear. Structural analysis of the PHear domain reveals the molecular surface for FxDxF motif binding, which was con?rmed by site-directed muta- genesis. The reciprocal analysis of the FxDxF motif in amphiphysin I identi?ed distinct binding requirements for binding to the a-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 con- vergence of two evolutionarily and structurally distinct modules to recognize a common peptide motif and pro- mote ef?cient endocytosis. The EMBO Journal (2007) 26, 4066C4077. doi:10.1038/ sj.emboj.7601836;

Published online

30 August

2007 Subject Categories: membranes &

transport;

structural biology Keywords: AP-2;

clathrin;

endocytosis;

NMR;

PH domain Introduction The formation of endocytic clathrin-coated vesicles (CCVs) is a complex multistep process requiring coordination of a low- af?nity interaction network based on proteinCprotein and proteinClipid interplay (Ritter and McPherson, 2004;

Traub, 2005). These interactions are mediated through protein modules that speci?cally recognize lipid head groups and/ or peptide motifs in target molecules (McPherson and Ritter, 2005;

Traub, 2005). One hub for the organization of this machinery is the a-ear of the heterotetrameric clathrin adap- tor AP-2 (Owen et al, 2004;

Honing et al, 2005). The a-ear, which is connected to the AP-2 trunk by a ?exible linker, extends into the surrounding cytosol to recruit endocytic accessory proteins through N-terminal platform and C-term- inal sandwich subdomains that each present a conserved protein-binding interface. Through these interactions, the a- ear functions as a major organizer for numerous endocytic accessory proteins including Eps15, epsins, amphiphysin I and II, AP180, connecdenn, NECAP

1 and 2, synaptojanin, and AAK1 (McPherson and Ritter, 2005;

Allaire et al, 2006). The platform subdomain utilizes overlapping binding sites to interact with DPF/W, FxDxF, and FxxFxxL peptide motifs (Owen et al, 1999;

Traub et al, 1999;

Brett et al, 2002;

Praefcke et al, 2004). Recently, we and others identi?ed a spatially distinct binding site on the sandwich subdomain that recruits proteins through WxxF-acidic motifs (Ritter et al, 2003;

Jha et al, 2004;

Mishra et al, 2004;

Praefcke et al, 2004;

Ritter and McPherson, 2004;

Walther et al, 2004). The NECAPs are a family of endocytic proteins that we identi?ed through a proteomic analysis of CCVs isolated from rat brain (Wasiak et al, 2002;