Abstract
Clathrin-coated vesicles (CCVs) are involved in protein and lipid trafficking between intracellular compartments in eukaryotic cells. CCVs are composed of clathrin and assembly proteins. The clathrin assembly protein lymphoid myeloid leukemia (CALM) gene, encodes a homologoue of the neuronal clathrin assembly protein AP180. In this study, we characterized the properties of the CALM expressed in E. coli. The molecular weight of bacterially expressed GST-CALM fusion protein was approximately 105 kD on SDS-PAGE. The CALM protein could promote clathrin triskelia into clathrin cages and could bind the preformed clathrin cage. However, 33 kD N-terminal domain of CALM could not bind pre-assembled clathrin cages, but assemble clathrin triskelia into clathrin cages. The CALM protein was bound to SH3 domain through N-terminal domain1, in vitro. The CALM protein is proteolyzed by caspase 3, caspase 8 and calpain through C-terminal domain.
Highlights
Clathrin-mediated vesicle formation is an essential step in the intracellular trafficking of the protein and lipid
When clathrin triskelia were sedimented following dialysis in the presence of glutathion sulfur transferase (GST)-N33 kD, GST-N33 kD remained in the pellet with the clathrin cages, but GST was not cosedimented with clathrin cages (Figure 4, lanes 3-6)
We found that clathrin assembly protein lymphoid myeloid leukemia (CALM) bounds to SH3 domain of PLCγ1 in vitro using a recombinant fusion protein of glutathione-S-transferase with the PLCγ1 SH3 domain (GST-SH3)
Summary
Clathrin-mediated vesicle formation is an essential step in the intracellular trafficking of the protein and lipid. The native AP180 was shown to interact with clathrin triskelia by one to one stoichiometry and thereby induced clathrin assembly into a uniformly sized of 60-70 nm coat structures (Kondury and Roland, 1988). These appear to be somewhat smaller and to be sediment considerably more slowly that than those containing AP-2 (80 nm) (Ye and Lafer 1995). We expressed the GST-fusion protein encoding cDNA of the CALM in bacterial system and characterized it’s properties in order to understand a possibile role of CALM protein in clathrin coated vesicle modulating function
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