title: Cell-free expression and functional reconstitution of CALM in clathrin assembly.
abstract: Clathrin-mediated vesicle formation is an essential step in the intracellular trafficking of the protein and lipid. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). In order to better understand a possible role of post-translational modification of CALM (clathrin assembly protein lymphoid myeloid), the homologue of AP180, in the assembly of CCVs, CALM was expressed in the cell-free reticulocyte translation system that is capable of carrying out post-translational modification. The apparent molecular weight of the expressed recombinant CALM was estimated as 105 kD. Alkaline phosphatase treatment of CALM resulted in a mobility shift on SDS-PAGE. We found that CALM was associated with the proteins harboring SH3 domain, promote assembly of clathrin triskelia into clathrin cage and bound to the preformed clathrin cage. CALM was also proteolyzed by caspase 3 and calpain but not by caspase 8. These results indicated that the post-translationally modified CALM, expressed in the eukaryotic cell-free reticulocyte translation system was able to mediate the assembly of clathrin and the coated-vesicle formation.
authors.affiliation: Department of Biochemistry, Medical College, Ewha Womans University, Seoul, Korea.
authors.forename: J A
authors.initials: JA
authors.lastname: Kim
authors.forename: H L
authors.initials: HL
authors.lastname: Kim
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport; D000469:Alkaline Phosphatase / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000378:metabolism; D002154:Calpain / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry*; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002417:Cattle; D002474:Cell-Free System; D002966:Clathrin / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism; D008055:Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D034041:Monomeric Clathrin Assembly Proteins; D010766:Phosphorylation; D011485:Protein Binding; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D018909:src Homology Domains
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; D002966:Clathrin; D008055:Lipids; D008565:Membrane Proteins; D034041:Monomeric Clathrin Assembly Proteins; C439003:PICALM protein, human; D011994:Recombinant Proteins; C439023:clathrin assembly protein AP180; D005982:Glutathione Transferase; D000469:Alkaline Phosphatase; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D002154:Calpain; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases