Clathrin light chains

GFP-Clathrin Light Chain, Clathrin Light Chain-GFP... 

The plasmid-encoding human clathrin light chain A, with GFP attached at its NH2 terminus, can be used to label clathrin-coated invaginations at the plasma membrane (40,113). With this technique, it should be possible to monitor the first step of this pathway—the binding and concentration at the plasma membrane. 

Acton, S. L.,and Brodsky, F. M. (1990). Predominance of clathrin light chain LCb correlates with the presence of a regulated secretory pathway.y. Cell Biol. Ill, 1419-1426. 

DeLuca-Flaherty, C., McKay, D. B., Parham, P., and Hill, B. L. (1990). Uncoating protein (hsc70) binds a conformationally labile domain of clathrin light chain LC to stimulate ATP hydrolysis. Cell Cambridge, Mass.) 62, 875-887. 

Schmid, S. L., Braell, W. A., Schlossman, D. M., and Rothman, J. E. (1984). A role for clathrin light chains in the recognition of clathrin cages by uncoating ATPase. Nature (London) 311, 228-231. 

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). 

A protein with a similar dumbell shape and structure is troponin C of skeletal muscles. Troponin C binds to a complex of proteins that assemble on the thin actin filaments of muscle fibers and control con-trachon in response to changes in the calcium ion con-centrahon (Chapter 19). Other proteins that contain EF-hand mohfs and are therefore responsive to Ca + include spectrin of cell membranes, clathrin light chains from coated vesicles, the extracellular osteonectin of bones and teeth, ° and a birch pollen anhgen. 2 Another group of 17 or more small SlOO EF-hand proteins play a variety of other roles. One of these, which has a high affinity for Zn +, has been named psoriasin because of its 5-fold or greater... 

The best-characterized vesicles that bud from the trans-Golgi network (TGN) have a two-layered coat an outer layer composed of the fibrous protein clathrin and an inner layer composed of adapter protein (AP) complexes. Purified clathrin molecules, which have a three-limbed shape, are called triskelions from the Greek for three-legged (Figure 17-19a). Each limb contains one clathrin heavy chain (180,000 MW) and one clathrin light chain... 

Clathrin is a protein complex composed of three heavy and three light chains, which assemble in a so-called triskelion. Clathrin is a major constituent of endocytic vesicles. 

Biochemical characterization of clathrin-coated vesicles revealed that their major coat components are clathrin and various types of adaptor complexes. Clathrin assembles in triskelions that consist of three heavy chains of approximately 190 kDa and three light chains of 30 40 kDa. Four types of adaptor complexes have been identified to date, AP-1, AP-2, AP-3 and AP-4 (AP for adaptor protein). Whereas AP-1, AP-3 and AP-4 mediate sorting events at the TGN and/or endosomes, AP-2 is involved in endocytosis at the plasma membrane. Each adaptor complex is a hetero-tetrameric protein complex, and the term adaptin was extended to all subunits of these complexes. One complex is composed of two large adaptins (one each of y/a/S/s and [31-4, respectively, 90-130 kDa), one medium adaptin (pi -4, <50 kDa), and one small adaptin (ol-4, <20 kDa). In contrast to AP-1, AP-2 and AP-3, which interact directly with clathrin and are part of the clathrin-coated vesicles, AP-4 seems to be involved in budding of a certain type of non-clathrin-coated vesicles at the TGN. 

Clathrins, first observed by Pearse, are structures with pentagonal and hexagonal faces that can form three dimensional structures with the same truncated icosahedral morphology as a C6o molecule or a soccer ball [1], The vertices of the clathrin coating are formed by three intertwined proteins composed of heavy and light chains that emanate from a central hub in a structure known as a triskelion, shown in Fig. 3.1. 

In the context of the initial interaction of HSC70 with clathrin requiring the presence of light chains, a site of interaction of HSC70 with light chains, and hence by inference a candidate for the initial binding site on clathrin, has been characterized (DeLuca-Flaherty et ai, 1990). The... 

The question of how DnaJ proteins interact with substrates and mediate their transfer onto Hsp70 partner proteins is not answered for any of the three classes of DnaJ proteins. Some DnaJ homologs have broad substrate specificity, such as E. coli DnaJ and yeast Ydjl, while others have more restricted substrate spectra. In particular the DnaJ proteins of class III may either bind a restricted number of substrates, such as the clathrin-specific auxilin or the kinesin light chain, or they may not bind substrates themselves but rather are positioned in close proximity to substrates. The latter seems to be the case for Dj 1A in the plasma membrane of E. coli (Clarke et al, 1997 Kelley and Georgopoulos, 1997a), Sec63 at the translocation pore in the ER (Corsi and Schekman, 1996 Rapoport et al., 1996), and cysteine string proteins on the surface of neurosecretory vesicles (Buchner and Bundersen, 1997). 

Electron micrograph of clathrin cages, like those that surround clathrin-coated transport vesicles, formed by the in vitro polymerization of clathrin heavy and light chains. [John Heuser,... 

Wakeham, D. E., L. Abi-Rached, M. C. Towler, J. D. Wilbur, P. Parham, and F. M. Brodsky. (2005). Clathrin heavy and light chain isoforms originated by independent mechanisms of gene duplication during chordate evolution. Pfoc Natl Acad Sci USA, Vol.102, pp.7209-7214.. 

In the Chinese hamster ovary cell line ts20, containing a thermosensitive ubiquitin-activating enzyme, El, Sachse et al. (2002) showed that this coat is predominantly present on early endosomes and has a characteristic bilayered appearance in the electron microscope. The coat contains clathrin heavy as well as light chain, but lacks the adaptor complexes API, AP2, and AP3, by which it differs from clathrin coats on endocytic vesicles and recycling endosomes. The coat is insensitive to short incubations with brefeldin A, but disappears in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin. 

Clathrin is a major coat protein of coated pits and coated vesicles formed in receptor-mediated endocytosis and intracellular vesicle-mediated transport and has a unique pinwheel-like structure in solution termed a triskelion composed of three heavy chains and three light chains [9]. Previously, we found that below pH 6, clathrin forms self-aggregates in the absence of liposomes and fusogenic assemblies in their presence [10, 11]. 

F1GURE 27-40 Clathrin. (a) Three light (L) chains (Mr 35,000) and three heavy (H) chains (Mr 180,000) of the (HL)3 clathrin unit, organized as a three-legged structure called a triskelion. (b) Triskelions... 

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