Clathrin-coated vesicle formation

Schmid, S. L. Clathrin-coated vesicle formation and protein sorting an integrated process. Annu. Rev. Biochem. 66 511-548,1997. 

Clathrin-mediated endocytosis is a major vesicular transport mechanism in the neuron, which enables the internalization of plasma membrane-bound proteins, nutrients, hormones and other molecules associated with the plasma membrane into intracellular compartments. Clathrin and various adaptor and accessory proteins work in concert at different stages of clathrin coated vesicle formation and disassembly, and many of these proteins (such as clathrin light chain, AP-2, dynamin 1, synaptojanin 1, and the amphiphysins) are substrates for protein kinases (Korolchuk et al. 2003). In addition, it has been suggested that directing synaptotagmin 1 to the synaptic vesicle is dependent on the N-terminal glycosylation of this protein (Han et al. 2004). 

Clathrin-Coated Vesicle Formation from Isolated Plasma Membranes... 

Lipid monolayers have been used for many years as templates for the formation of two-dimensional crystals (Chiu et al, 1997), and for surface tension measurements (Stahelin et al., 2003). We have used them as mimics of the inner leaflet of the plasma membrane to reconstitute the early stages of clathrin-coated vesicle formation (Ford et al, 2001, 2002) and they can equally well be applied to other vesicle and tubule budding mechanisms. A lipid monolayer forms when a droplet of solvent-dissolved Upid is placed onto an aqueous droplet. As the solvent evaporates, lipid molecules become oriented at the air-water interface with head groups in contact... 

A pre-requisite for clathrin-coat assembly is the recruitment to the membrane of an adaptor complex. Similar to what has been observed for the recruitment of coatomer to Golgi membranes, adaptor binding is dependent on the presence of ARF-GTP. However, in contrast to COPI vesicle formation, ARF-GTP is suggested to act in a process before budding and not as a stoichiometric coat component. Other differences between COP-coated and clathrin-coated vesicles concern their uncoating mechanism. Disassembly of clathrin-coated vesicles is believed to depend on the chaperoneHSC 70 and on auxilin. 

Clathrin-coated vesicles mediate transport from the Golgi apparatus to endosomes, and from the plasma membrane to endosomes. A multi-subunit protein, clath-rin, constitutes the major protein of this vesicle type (see Ch. 2). Clathrin is composed of three large and three small polypeptide chains, which assemble to form a triskelion (Fig. 9-2). Regulatory mechanisms control the assembly and formation of a convex, polyhexa-pentagonal basketlike structure by these triskelions [5], This structure is responsible for the formation of coated pits on the cytosolic face of plasma membranes. 

In the classic model of synaptic vesicle recycling in nerve terminals, synaptic vesicles fuse completely with the plasma membrane and the integrated vesicle proteins move away from the active zone to adjacent membrane regions (Fig. 9-9A). In these regions, clathrin-mediated synaptic vesicle endocytosis takes place rapidly after neurotransmitter release (within seconds) [64]. The process starts with the formation of a clathrin-coated pit that invaginates toward the interior of the cell and pinches off to form a clathrin-coated vesicle [83]. Coated vesicles are transient organelles that rapidly shed their coats in an ATP/chaperone dependent process. Once uncoated, the recycled vesicle fuses with a local EE for reconstitution as a synaptic vesicle. Subsequently, the recycled synaptic vesicle is filled with neurotransmitter and it returns to the release site ready for use. This may be the normal pathway when neurotransmitter release rates are modest. Clathrin/ EE-based pathways become essential when synaptic proteins have been incorporated into the presynaptic plasma membrane. 

Brodsky FM, Chen CY, Knuehl C, et al. Biological basket weaving formation and function of clathrin-coated vesicles. Annu Rev Cell Dev Biol 2001 17 517-568. 

Both intracellular depletion of potassium and hypertonic treatment lead to disruption of clathrin from the inner side of the plasma membrane. Consequently, the formation of clathrin-coated pits and clathrin-coated vesicles is... 

For the formation of clathrin-coated vesicles, several proteins are required. Currently, there are several recombinant inhibitors available, which block different steps of coated pit/vesicle formation for example, amphiphysin (53), clathrin assembly protein AP180 (54), epsin (55), and clathrin mutant (56). 

Both endocytosis of material at the plasma membrane and exocytosis from the Golgi apparatus involve the formation of clathrin-coated pits and vesicles. On the cytosolic side of the membrane these structures have an electron-dense coat consisting mainly of the protein clathrin, the polypeptides of which form a three-legged structure known as a triskelion. The clathrin triskelions assemble into a basket-like convex framework that causes the membrane to invaginate at that point and eventually to pinch off and form a vesicle. In endocytosis these clathrin-coated vesicles migrate into the cell where the clathrin coats are lost before delivering their contents to the lysosomes. 

Acidifies the cytoplasm and freezes clathrin networks Blocks budding of clathrin-coated vesicles Inhibits macropinocytosis Inhibits v-ATPases raises endosomal pH Fluorescent marker of the golgi complex Inhibitor of protein transport in the golgi complex Weak base raises endosomal pH disrupts endosomes Inhibits clathrin lattice formation Disrupts clathrin lattices... 

Vesicular transport occurs when a membrane completely surrounds a compound, particle, or cell and encloses it into a vesicle. When the vesicle fuses with another membrane system, the entrapped compounds are released. Endocytosis refers to vesicular transport into the cell, and exocytosis to transport out of the cell. Endocytosis is further classified as phagocytosis if the vesicle forms around particulate matter (such as whole bacterial cells or metals and dyes from a tattoo), and pinocy-tosis if the vesicle forms around fluid containing dispersed molecules. Receptor-mediated endocytosis is the name given to the formation of clathrin-coated vesicles that mediate the internalization of membrane-bound receptors in vesicles coated on the intracellular side with subunits of the protein clathrin (Eig. 10.14). Potocytosis is the name given to endocytosis that occurs via caveolae (small invaginations or caves ), which are regions of the cell membrane with a unique lipid and protein composition (including the protein caveolin-1). 

F. 10.14. Formation of a clathrin-coated vesicle. Ligands entering the cell through receptor-mediated endocytosis bind to receptors that cluster in an area of the membrane. Adaptor proteins bind to the receptor tails and to the clathrin molecules to enclose the budding membrane in a cage-like clathrin coat. 

Receptor desensitization is commonly caused by receptor internalization, whereby the receptor is removed from its location on the membrane into the cytoplasm, and then either degraded or recycled back to the surface by two main mechanisms. The level of cell-surface expression of a chemokine receptor is a balance between the rate of internalization and the rate of recovery, which can occur via de novo receptor production or receptor recycling, and which in some cases is a constitutive process (Pelchen-Matthews et al., 1999). Internalization is dramatically increased in the presence of agonist, and it is an active process, as internalization is almost entirely prevented at temperatures below 16°C (von Zastrow and Kobilka, 1994). There appear to be two main mechanisms of chemokine receptor internalization 1) the classical recruitment of arrestin to the receptor, resulting in the formation of a clathrin-coated vesicle around the receptor and subsequent internalization, and 2) internalization via clathrin-independent pathways. 

The best-documented way of endocytosis is receptor-mediated uptake of ligands via clathrin-coated vesicles (reviewed in Schmid 1997). Receptors are recruited and concentrated into clathrin-coated pits at the plasma membrane. After coated vesicle formation, the clathrin coat is removed by the concerted action of auxihn and heat shock protein 70 (Ungewickell et al. 1995). The uncoated vesicles fuse with early endosomes in a rab5-regulated manner (Rubino et al. 2000). 

Endocytic clathrin-coated vesicle (CCV) formation is a complex process involving a large number of proteins and lipids. The minimum machinery and the hierarchy of the events involved in CCV formation have yet to be dehned. Here we describe an in vitro assay for CCV formation from highly purified rat liver plasma membranes. This rapid and easy assay can be used to quantitatively evaluate the different protein requirements for different endocytic receptors. 

Miwako, I., Schroter, T., and Schmid, S. L. (2003). Clathrin- and dynamin-dependent coated vesicle formation from isolated plasma membranes. Traffic 4, 376-389. 

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