Rotin_lecture 1_BCH444

Cargo concentration at coated pit bud formation and

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Unformatted text preview: ion at coated pit. Bud formation and vesicle scission. John Heuser Clathrin-mediated endocytosis Coated pit Dynamin ring (AP2) TD CLC=clathrin light chain CHC=clathrin heavy chain TD=terminal domain (Globular domain) Components of clathrin coated vesicles (CCVs): >30 proteins -clathrin -AP2 (binds clathrin, phospholipids, cargo and other components) -Dynamin (involved in vesicle fission) -Membrane -lipid binding proteins (AP180(in neurons)/CALM (in all cells), Amphiphysin) -Modifying enzymes (Synaptojanin, endophilin, amphiphysin) -Chaperons (Hsc70, Auxilin)-involved in disassembly of clathrin/AP2 complex -Endocytic kinases (AAK1 (adaptor-associate kinase1), GAK/auxilin2) -specialized cargo proteins (Dab2, ARH, epsin/eps15) -Scaffolding proteins (intersectin: binds clathrin, AP2, dynamin, Eps15,synaptojanin,actin, MAPK components, synaptosome components) Adaptors and linker proteins possess a Clathrin binding sequence: Some of the above proteins are necessary for vesicle formation (eg. clathrin, AP2,dynamin) and some are accessory/regulatory proteins (eg eps15, endophilin, amphiphysin). Alpha-adaptin (N) beta2-adaptin mu2 Alpha-adaptin (C) sigma2 AP2 adaptor: Main component of clathrin coated vesicles Functions as cargo recognition module and connects clathrin assembly to plasma membrane AP2 loss lethal in multicellular organisms Binding to accessory Proteins (eg epsin, AP180, which possess DPF motif) Binding to clathrin Binding to Plasma Membrane (PIP2/PIP3) Stabilization and cargo recognition Cargo recognition & binding (Yxx ) (di-Leu?) Phosphorylated by AAK1 Adaptor proteins: Link clathrin coat to membrane and cargo Multiple adaptors bind different cargo proteins to provide specificity in cargo recruitment Modular composition of adaptors allows for simultaneous binding of PM (PtdIns(4,5)P2), AP-2 and Clathrin Short linear motifs are recognized by clathrin adaptors AP-2 mediated clathrin dependent endocytosis Clathrin Associated Sorting Protein (CLASP) mediated clathrin dependent endocytosis Canonical sorting motif recognition: A short linear peptide motif YxxØ in cargo is recognized by dedicated folded domain in adaptor of AP-2 AAK1 mediated phosphorylation opens AP-2 to allow cargo binding via μ2 Open conformation is further stabilized by membrane lipid interaction Coincidence detection and Cargo selection: AP2 binds cytosolic cargo sorting motif YxxØ at PM but not Golgi Additional AP-2 binding site provided by PtdIns(4,5)P2 at PM reinforces fidelity of cargo sorting process Self-assembly & stimulation of the GTPase activity Dynamin (shibire) Binds phospholipids (PIP2) and membranes Binding to endocytic components Lipid coated coated microspheres incubated with GTP and dynamin leads to vesicle formation in vitro Dynamin is a mechanochemical enzyme and drives membrane fission, but most likely has additional functions through multiple interactions with proteins involved in early and late steps of clathrin dependent endocytosis Temperature sensitive dynamin mutant shibire induces paralysis in fly at restrictive temperature due to defective synaptic vesicle endocytosis and recycling. Shibire WT Temporal assembly of components during clathrin-mediated endocytosis Endocytic routes Summary (Sorting endosomes) (MVBs)...
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