Abstract
The most efficient means of protein internalization from the membrane are through clathrin-coated pits, which concentrate protein interactions with the clathrin-associated assembly protein complex AP-2 and internalization signals in the cytoplasmic domain of transmembrane proteins. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). Due to a difficulty of isolating clathrin molecules from their complex or assembly state in the cells, most of the studies were carried out with recombinant clathrin proteins, which may present different conformation and structural variation. In this study, we have developed an efficient method of isolating the native clathrin assembly protein lymphoid myeloid (CALM) from the bovine brain that is enriched with clathrin and clathrin associated proteins and characterized by their sensitivity to proteases and it's ability to form CCV. The purified CALM has molecular weight of approximately 100,000 dalton on SDS-PAGE, which is consistent with the result of in vitro translation. The purified CALM protein could promote the assembly of clathrin triskelia into clathrin cage, and cleaved CALM proteolysed by caspase 3 and calpain could not promote them. In this respect, our data support a model in which CALM functions like AP180 as a monomeric clathrin assembly protein and might take part in apoptotic process in neuronal cells.
Highlights
Clathrin-mediated vesicle formation is an essential step in the intracellular trafficking of the protein and lipid
All buffers used in the protein work, even though it is not explicitly indicated in the citation, contained 0.1 mM phenylmethylsulfonyl fluoride (PMSF)
Proteolysis by caspase One microgram of purified clathrin assembly protein lymphoid myeloid (CALM) protein was incubated with caspase 3 and control bacterial lysate in 20 μl of reaction buffer (16 mM HEPES, 8 mM NaCl, and 0.004% IGEPAL)
Summary
Clathrin-mediated vesicle formation is an essential step in the intracellular trafficking of the protein and lipid. AP-1 and AP-2 were first characterized (Ahle and Ungewickell, 1986) as the major clathrin coated vesicle adaptor proteins. To understand the characteristics of CALM protein in mammalian cells, we have purified the CALM protein from bovine brain, through CCV preparation, Sepharose CL-4B gel-filtration chromatography, Mono S and Superose 6 FPLC.
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