Recently, the gene encoding clathrin assembly protein of lymphoid myeloid leukemia (CALM), which is homologous to the AP180, was cloned from rat brain, and its expression differential to AP180 was reported (Kim and Lee, 1999). This gene product promotes the polymerization of clathrin into clathrin cage and found to be a regulator in membrane trafficking between intracellular compartments in eukaryotic cells (Kim et al., 2000). In this study, we have purified the CALM protein from clathrin-coated vesicles of rat liver using the monoclonal antibody against the recombinant N-terminal region of the CALM. The coated proteins extracted from the coated vesicle fraction was further purified by multi-step procedures involving gel-filtration and ion-exchange chromatography and SDS-PAGE. The purified protein with an apparent molecular weight of 100 kD promoted the assembly of clathrin triskelia into clathrin cage. In this respect the CALM protein bears a functional resemblance to the AP180 that has been previously described.
Animal ; Clathrin/*metabolism ; Clathrin-Coated Vesicles/*chemistry ; Liver/*chemistry ; Nerve Tissue Proteins/*isolation & purification ; Phosphoproteins/*isolation & purification ; Rats

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.
Animal ; Antibodies, Monoclonal ; Calpain/chemistry ; Caspases/chemistry ; Clathrin-Coated Vesicles/metabolism* ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli/metabolism ; Escherichia coli/genetics ; Female ; Glutathione Transferase/genetics* ; Mice ; Mice, Inbred BALB C ; Nerve Tissue Proteins/metabolism ; Nerve Tissue Proteins/metabolism ; Nerve Tissue Proteins/chemistry* ; Phosphoproteins/metabolism ; Phosphoproteins/genetics ; Phosphoproteins/chemistry* ; Protein Binding ; Rabbits ; Recombinant Fusion Proteins/metabolism ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/chemistry* ; src Homology Domains