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.
Highlights
Clathrin-mediated vesicle formation is an essential step in the intracellular trafficking of the membrane compartment
Expressed CALM was able to induce the assembly of clathrin cage and bind to clathrin triskelia, like AP180 through C terminal domain
The results showed the glycosylated and phosporylated CALM retained its functional properties as AP in clathrin vesicle formation
Summary
Clathrin-mediated vesicle formation is an essential step in the intracellular trafficking of the membrane compartment. Expressed CALM was able to induce the assembly of clathrin cage and bind to clathrin triskelia, like AP180 through C terminal domain. GST-CALM could interact with SH3 domain, and be proteolysed by calpain and caspase (Kim et al, 2000b).
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
