Exp Mol Med.  2001 Jun;33(2):89-94.

Cell-free expression and functional reconstitution of CALM in clathrin assembly

• Kim JA
• Kim HL

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.

Keyword

expression; clathrin-coated vesicle; CALM; SH3 domain; cleavage

MeSH Terms

Alkaline Phosphatase/pharmacology
Animal
Brain/metabolism
Calpain/metabolism
Carrier Proteins/*chemistry
Caspases/metabolism
Cattle
Cell-Free System
Clathrin/*chemistry
Electrophoresis, Polyacrylamide Gel
Glutathione Transferase/metabolism
Lipids/chemistry
Membrane Proteins/*chemistry
Phosphorylation
Protein Binding
Protein Processing, Post-Translational
Protein Structure, Tertiary
Protein Transport
Recombinant Proteins/chemistry/metabolism
Reticulocytes/metabolism
Support, Non-U.S. Gov't
Translation, Genetic
src Homology Domains