AP2 controls clathrin polymerization with a membrane-activated switch

Clathrin-mediated endocytosis (CME) is vital for the internalization of most cell-surface proteins. In CME, plasma membrane-binding clathrin adaptors recruit and polymerize clathrin to form clathrin-coated pits into which cargo is sorted. Assembly polypeptide 2 (AP2) is the most abundant adaptor and is pivotal to CME. Here, we determined a structure of AP2 that includes the clathrin-binding beta 2 hinge and developed an AP2-dependent budding assay. Our findings suggest that an autoinhibitory mechanism prevents clathrin recruitment by cytosolic AP2. A large-scale conformational change driven by the plasma membrane phosphoinositide phosphatidylinositol 4,5-bisphosphate and cargo relieves this autoinhibition, triggering clathrin recruitment and hence clathrin-coated bud formation. This molecular switching mechanism can couple AP2's membrane recruitment to its key functions of cargo and clathrin binding

New tools for “hot-wiring” clathrin-mediated endocytosis with temporal and spatial precision

Clathrin-mediated endocytosis (CME) is the major route of receptor internalization at the plasma membrane. Analysis of constitutive CME is difficult because the initiation of endocytic events is unpredictable. When and where a clathrin-coated pit will form and what cargo it will contain are difficult to foresee. Here we describe a series of genetically encoded reporters that allow the initiation of CME on demand. A clathrin-binding protein fragment (“hook”) is inducibly attached to an “anchor” protein at the plasma membrane, which triggers the formation of new clathrin-coated vesicles. Our design incorporates temporal and spatial control by the use of chemical and optogenetic methods for inducing hook–anchor attachment. Moreover, the cargo is defined. Because several steps in vesicle creation are bypassed, we term it “hot-wiring.” We use hot-wired endocytosis to describe the functional interactions between clathrin and AP2. Two distinct sites on the β2 subunit, one on the hinge and the other on the appendage, are necessary and sufficient for functional clathrin engagement

From polymer chemistry to structural biology: The development of SMA and related amphipathic polymers for membrane protein extraction and solubilisation

Nanoparticles assembled with poly(styrene-maleic acid) copolymers, identified in the literature as Lipodisq, SMALPs or Native Nanodisc, are routinely used as membrane mimetics to stabilise protein structures in their native conformation. To date, transmembrane proteins of varying complexity (up to 8 beta strands or 48 alpha helices) and of a range of molecular weights (from 27 kDa up to 500 kDa) have been incorporated into this particle system for structural and functional studies. SMA and related amphipathic polymers have become versatile components of the biochemist's tool kit for the stabilisation, extraction and structural characterization of membrane proteins by techniques including cryo-EM and X-ray crystallography. Lipodisq formation does not require the use of conventional detergents and thus avoids their associated detrimental consequences. Here the development of this technology, from its fundamental concept and design to the diverse range of experimental methodologies to which it can now be applied, will be reviewed