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.

A multivariate proteomics approach identified numerous new clathrin-coated vesicle proteins as well as the first AP-4 accessory protein, and also revealed how auxilin depletion causes mitotic arrest through sequestration of spindle proteins in clathrin cages.

SummarySNAREs provide the specificity and energy for the fusion of vesicles with their target membrane, but how they are sorted into the appropriate vesicles on post-Golgi trafficking pathways is largely unknown. We demonstrate that the clathrin-mediated endocytosis of the SNARE VAMP7 is directly mediated by Hrb, a clathrin adaptor and ArfGAP. Hrb wraps 20 residues of its unstructured C-terminal tail around the folded VAMP7 longin domain, demonstrating that unstructured regions of clathrin adaptors can select cargo. Disrupting this interaction by mutation of the VAMP7 longin domain or depletion of Hrb causes VAMP7 to accumulate on the cell's surface. However, the SNARE helix of VAMP7 binds back onto its longin domain, outcompeting Hrb for binding to the same groove and suggesting that Hrb-mediated endocytosis of VAMP7 occurs only when VAMP7 is incorporated into a cis-SNARE complex. These results elucidate the mechanism of retrieval of a postfusion SNARE complex in clathrin-coated vesicles.