Chain Clathrin-mediated endocytosis

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). 

Huang F, Khvorova A, Marshall W, Sorkin A. Analysis of clathrin-mediated endocytosis of epidermal growth factor receptor by RNA interference. J. Biol. Chem. 2004 279 16657-16661. Hinrichsen L, Harborth J, Andrees L, Weber K, Ungewickell EJ. Effect of clathrin heavy chain- and alpha-adaptin-specific small inhibitory RNAs on endocytic accessory proteins and receptor trafficking in HeLa cells. J. Biol. Chem. 2003 278 45160-45170. Wu AM, Senter PD. Arming antibodies prospects and challenges for immunoconjugates. Nat. Biotechnol. 2005 23 1137-1146. 

This conclusion is based on immunogold localization of PrP in these organelles by electron microscopy inhibition of PrP internalization by incubation of cells in hypertonic sucrose, which disrupts clathrin lattices and detection of PrP in purified preparations of coated vesicles from brain. The N-terminal half of the PrP polypeptide chain is essential for efficient clathrin-mediated endocytosis, because... 

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]. 

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. 

The work reviewed here has begun to provide a detailed picture of the biosynthesis and cellular processing of PrP and PrP. PrP is synthesized and matures along the secretory pathway much like other membrane glycoproteins. One important feature is the addition of a GPI anchor that serves to attach the polypeptide chain to the lipid bilayer without the necessity for a transmembrane domain. Once it reaches the plasma membrane, PrP is constitutively endocytosed via clathrin-coated vesicles, and a portion of the molecules is proteolyti-cally cleaved in a highly conserved domain before recycling to the cell surface. Efficient endocytosis depends on structural features in the N-terminal half of the PrP molecule and may be mediated by a transmembrane PrP receptor . 

---