Clathrin-coated vesicles, endocytic protein

Le Borgne, R., and Hoflack, B. (1998a). Mechanisms of protein sorting and coat assembly insights from the clathrin-coated vesicle pathway. Curr. Opin. Cell Biol. 10, 499-503. Le Borgne, R., and Hoflack, B. (1998b). Protein transport from the secretory to the endocytic pathway in mammalian cells. Biochim. Biophys. Acta 1404, 195-209. 

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. 

Specialized regions of internalization from the plasma membrane, coated with a polyhedral lattice of the protein clathrin. It is in these regions that the first step of the process of endocytosis takes place, with the formation of clathrin-coated endocytic vesicles. 

Shortly after formation of coated vesicles, the clathrin coat is removed and the vesicles are referred to as endosomes. Endosomes are roughly 300 400 nm in diameter when fully mature. Antibodies to earlier and late endosomes are available from antibodies-online GmbH, Aachen, Germany (http //www.antikoerper-online. de/). Early endosomal antigen 1 (EEA1) is a 162 kDa membrane-bound protein component specific to the early endosomes and is essential for their fusion with early endocytic vesicles for subsequent redistribution of extracellular compounds to... 

Fig. 5 Synaptic vesicle recycling in the synapse. For synaptic vesicle recycling, several endocytic mechanisms appear to co-exist in synaptic nerve terminals. In the case of fast kiss-and-ran exo-cytosis/endocytosis, the fused vesicle does not collapse into the membrane but is retrieved directly by a fast process. The molecular machinery underlying this pathway is unknown. Vesicles that have fully collapsed into the membrane are recycled by clathrin-mediated endocytosis. Clathrin, along with other proteins, is involved in membrane invagination (see figure and text) and leads finally to the formation of a constricted pit. The GTPase dynamin (black ring) mediates membrane scission of the constricted pit. After removal of the clathrin coat, two pathways are possible (direct recycling and recycling via the early endosome). In all cases, before fusion the recycled vesicles have to be loaded with neurotransmitters (NT).

In the kiss-and-run mode exocytosis and endocytosis are directly coupled to each other, while in the case of classical complete vesicle fusion, exocytosis and slow clathrin-mediated endocytosis are timely and spatially separated. However, it appears that also in the latter case exocytosis and endocytosis occur coordinated, as both are stimulated by an increase of the cytoplasmic calcium concentration. It has been shown that after calcium entry the enzyme phospho-inositol-5 kinase Iy, which is enriched in the synapse, catalyzes the synthesis of phosphatidylinos-itol (4,5)-bisphosphate and that this mechanism is important for synaptic vesicle trafficking (Di Paolo et al. 2004). As many proteins involved in clathrin-mediated endocytosis are recruited to the plasma membrane by binding to phosphatidylinosi-tol (4,5)-bisphosphate (e.g., amphiphysin, dynamin, epsin, AP-180, and AP-2) it is attractive to speculate that elevated levels of calcium mediate the recruitment of en-docytic proteins to the plasma membrane by this mechanism. The increased level of phosphatidylinositol (4,5)-bisphosphate could be in part degraded by synaptojanin that thereby initiates the disassembly of the clathrin coat. Hence, calcium-induced transient increases in the level of phosphatidylinositol (4,5)-bisphosphate appear to play a central role for coupling exocytosis to clathrin-mediated endocytosis. In addition, it has been demonstrated that calcium also leads to the dephosphorylation of endocytic proteins as amphiphysin, dynamin, and synaptojanin, which in vitro is important for efficient coat assembly (Cousin and Robinson 2001). 

LDL binds specifically to lipoprotein receptors on the cell surface. The resulting complexes become clustered in regions of the plasma membrane called coated pits. Endocytosis follows (see Fig. 13-3). The clathrin coat dissociates from the endocytic vesicles, which may recycle the receptors to the plasma membrane or fuse with lysosomes. The lysosomal proteases and lipases then catalyze the hydrolysis of the LDL-receptor complexes the protein is degraded completely to amino acids, and cholesteryl esters are hydrolyzed to free cholesterol and fatty acid. New LDL receptors are synthesized on the endoplasmic reticulum (ER) membrane and are subsequently reintroduced into the plasma membrane. The cholesterol is incorporated in small amounts into the endoplasmic reticulum membrane or may be stored after esterification as cholesteryl ester in the cytosol this occurs if the supply of cholesterol exceeds its utilization in membranes. Normally, only very small amounts of cholesteryl ester reside inside cells, and the majority of the free cholesterol is in the plasma membrane. 

Synaptic vesicles are formed primarily by endocytic budding from the plasma membrane of axon terminals. Endocytosis usually involves clathrin-coated pits and is quite specific, in that several membrane proteins unique to the synaptic vesicles (e.g., neurotransmitter transporters) are specifically incorporated into the endocytosed vesicles. In this way, synaptic-vesicle membrane proteins can be reused and the recycled vesicles refilled with neurotransmitter (see Figure 17-36). 

Pearse and Bretscher (1981) have discussed the role of coated vesicles in membrane synthesis and function. Eukaryotic cells are able to specifically take up macromolecules by absorptive endocytosis. The macromolecules are usually transferred to lyso-somes where they may be degraded. The first stage of the process involves the binding of macromolecules to receptors which are localized in coated pits. The latter are indented sites on the plasma membrane and the coated pit buds into the cytoplasm to form a coated vesicle in which lie the endocytosed macromolecules. The coated vesicle sheds its coat rapidly and the endocytic vesicles fuse with each other. This allows receptors to be returned to the plasma membrane while the contents are transferred to the lyso-somes. In order to explain how lysosomal and plasma membranes remain different, it was suggested that the coated pits are able to accept certain macromolecules while excluding others. The accepted proteins enter the coated pit and were presumed to bind directly or indirectly to clathrin. Clathrin, a 180000-dalton protein on the cytoplasmic face of coated pits, provides the polyhedron skeleton for the coated vesicles. Examples of the use of coated vesicles for mediated endocytosis are in the uptake of low-density lipoprotein from the blood and in humans for the transport of immunoglobulins from the mother to the child. For other mammals such as the rat the antibodies are selectively absorbed from the mother s milk by the intestinal epithelium. Coated vesicles also provide a mechanism for virus transport into cells. 

I. Clathrin-eoated vesicle a transport vesicle, diameter about 800 A, formed in the process of endocy-tosis (pinocytosis), and found in vinually all eukaryotic cells. A C-c.v. is therefore also an endocytic or pi-nocytic vesicle. A C-c. v. is formed by invagination of a coated pit in the plasma membrane. A coated pit is a highly specialized region of the plasma membrane carrying cell surface receptors for a variety of ligands that are normally taken up by the cell, e.g. serum proteins, insulin, lipoproteins, etc. The receptors become concentrated in the pits either before or after association with their ligands. The process of C-c.v. formation from the plasma membrane is therefore known as receptor-mediated endocytosis... 

Many vesicles that bud from the frans-Golgi network as well as endocytic vesicles bear a coat composed of AP (adapter protein) complexes and clathrin (see Figure 17-19). 

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