SummaryThe AP2 adaptor complex (α, β2, σ2, and μ2 subunits) crosslinks the endocytic clathrin scaffold to PtdIns4,5P2-containing membranes and transmembrane protein cargo. In the “locked” cytosolic form, AP2's binding sites for the two endocytic motifs, YxxΦ on the C-terminal domain of μ2 (C-μ2) and [ED]xxxL[LI] on σ2, are blocked by parts of β2. Using protein crystallography, we show that AP2 undergoes a large conformational change in which C-μ2 relocates to an orthogonal face of the complex, simultaneously unblocking both cargo-binding sites; the previously unstructured μ2 linker becomes helical and binds back onto the complex. This structural rearrangement results in AP2's four PtdIns4,5P2- and two endocytic motif-binding sites becoming coplanar, facilitating their simultaneous interaction with PtdIns4,5P2/cargo-containing membranes. Using a range of biophysical techniques, we show that the endocytic cargo binding of AP2 is driven by its interaction with PtdIns4,5P2-containing membranes.

Clathrin-coated vesicles (CCVs) originating from the transGolgi network (TGN) provide a major transport pathway from the secretory system to endosomes/lysosomes. Herein we describe paralogous Sec14 domain-bearing proteins, clavesin 1/CRALBPL and clavesin 2, identified through a proteomic analysis of CCVs. Clavesins are enriched on CCVs and form a complex with clathrin heavy chain (CHC) and adaptor protein-1, major coat components of TGN-derived CCVs. The proteins co-localize with markers of endosomes and the TGN as well as with CHC and adaptor protein-1. A membrane mimic assay using the Sec14 domain of clavesin 1 reveals phosphatidylinositol 3,5-bisphosphate as a specific lipid partner. Phosphatidylinositol 3,5-bisphosphate is localized to late endosomes/lysosomes, and interestingly, isoform-specific knockdown of clavesins in neurons using lentiviral delivery of interfering RNA leads to enlargement of a lysosome-associated membrane protein 1-positive membrane compartment with no obvious influence on the CCV machinery at the TGN. Since clavesins are expressed exclusively in neurons, this new protein family appears to provide a unique neuron-specific regulation of late endosome/lysosome morphology.