Clathrin-mediated endocytosis is the major process by which transmembrane proteins are internalized from the cell's limiting membrane into the first compartment of the endosomal system, the early endosome. From here, these transmembrane cargo proteins, which are of widely varying type and function, are trafficked to their required destination. Endocytosis plays, therefore, an important role in cell signalling, nutrient uptake, cellular homoeostasis and the interaction of the cell with its external environment. The formation of clathrin-coated endocytic vesicles requires the complex interplay of many proteins with each other and with the membrane itself. Their formation has served as a paradigm for formation of all types of transport vesicle, which move cargo between the various membrane-bound compartments of the cell. Clathrin-coated vesicles (CCVs) possess three layers: the inner membrane layer, in which the transmembrane cargo is embedded, linked to the outer clathrin lattice by a layer of cargo-binding adaptors and proteins that aid and regulate vesicle formation. Protein X-ray crystallography in combination with biochemical, biophysical and cell biological assays has been used to investigate the structure and function of some of the proteins that make up the middle layer of CCVs. These proteins are diverse in their functions, but are all modular in nature, consisting of folded domains joined by long unstructured linkers. Within these linkers are short motifs that interact with the folded domains of other components of the CCV formation machinery. Many of these folded domains also bind directly to the membrane. These interactions, whose molecular basis we have studied, have affinities in the low micromolar range, making them readily reversible and easily regulated. The mechanism of CCV formation is discussed in the light of this structural and biochemical data.

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