Abstract
Adaptor protein complex 2 α and β-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of β-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the β-appendage (the “top” and “side” sites) that bind motifs distinct from those previously identified on the α-appendage. We solved the structure of the β-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor β-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the β-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability (“matricity”). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as β-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.
Highlights
Clathrin-mediated endocytosis is the process whereby cargo molecules are selected for incorporation into vesicles surrounded by a coat protein, clathrin. ‘‘Accessory proteins’’ organise the process of coat assembly and disassembly as well as the process of membrane budding and fission [1,2,3,4,5]
Ligands of the b-Appendage The adaptor protein complex 2 (AP2) adaptor complex with its two appendages acts as an interaction hub for numerous accessory proteins, whether these be accessory cargo adaptors or accessory proteins involved in the mechanics of Clathrin-coated vesicle (CCV) formation
We have extended this using glutathione S-transferase (GST) a and b2 appendages coupled to agarose beads in HeLa, brain and liver extracts (Figure 1C and Figure S1), and analyse the protein interactors with LC-MS/MS
Summary
Clathrin-mediated endocytosis is the process whereby cargo molecules are selected for incorporation into vesicles surrounded by a coat protein, clathrin. ‘‘Accessory proteins’’ organise the process of coat assembly and disassembly as well as the process of membrane budding and fission [1,2,3,4,5]. ‘‘Accessory proteins’’ organise the process of coat assembly and disassembly as well as the process of membrane budding and fission [1,2,3,4,5]. Some of these accessory proteins act as ‘‘cargo adaptors’’ and help select the cargo content of the nascent vesicle. In synaptic vesicles there are over ten different transmembrane cargo components, each in their constant stoichiometries and packaged in vesicles of uniform size to ensure reliable synaptic transmission [6,7] This is a remarkable achievement of the clathrin-adaptor machinery. The tetrameric adaptor protein complex 2 (AP2) and clathrin are examples of such hubs in this network
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