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.
Subject(s)
Cattle , Alkaline Phosphatase/pharmacology , Animals , Brain/metabolism , Calpain/metabolism , Carrier Proteins/chemistry , Caspases/metabolism , 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 , Reticulocytes/metabolism , Protein Biosynthesis , src Homology DomainsABSTRACT
The most efficient means of protein internalization from the membrane are through clathrin-coated pits, which concentrate protein interactions with the clathrin-associated assembly protein complex AP-2 and internalization signals in the cytoplasmic domain of transmembrane proteins. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). Due to a difficulty of isolating clathrin molecules from their complex or assembly state in the cells, most of the studies were carried out with recombinant clathrin proteins, which may present different conformation and structural variation. In this study, we have developed an efficient method of isolating the native clathrin assembly protein lymphoid myeloid (CALM) from the bovine brain that is enriched with clathrin and clathrin associated proteins and characterized by their sensitivity to proteases and it's ability to form CCV. The purified CALM has molecular weight of approximately 100,000 dalton on SDS-PAGE, which is consistent with the result of in vitro translation. The purified CALM protein could promote the assembly of clathrin triskelia into clathrin cage, and cleaved CALM proteolysed by caspase 3 and calpain could not promote them. In this respect, our data support a model in which CALM functions like AP180 as a monomeric clathrin assembly protein and might take part in apoptotic process in neuronal cells.
Subject(s)
Cattle , Adaptor Proteins, Vesicular Transport , Animals , Brain Chemistry , Calpain/metabolism , Carrier Proteins , Caspases/metabolism , Clathrin/metabolism , Coated Pits, Cell-Membrane/metabolism , Hydrolysis , Membrane Proteins , Molecular Weight , Nerve Tissue Proteins/chemistry , Neurons/chemistry , Protein Binding , Protein Conformation , Recombinant Proteins/chemistryABSTRACT
The most efficient means of protein internalization from the membrane are through clathrin-coated pits, which concentrate protein interactions with the clathrin-associated assembly protein complex AP-2 and internalization signals in the cytoplasmic domain of transmembrane proteins. Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicles (CCVs). Due to a difficulty of isolating clathrin molecules from their complex or assembly state in the cells, most of the studies were carried out with recombinant clathrin proteins, which may present different conformation and structural variation. In this study, we have developed an efficient method of isolating the native clathrin assembly protein lymphoid myeloid (CALM) from the bovine brain that is enriched with clathrin and clathrin associated proteins and characterized by their sensitivity to proteases and it's ability to form CCV. The purified CALM has molecular weight of approximately 100,000 dalton on SDS-PAGE, which is consistent with the result of in vitro translation. The purified CALM protein could promote the assembly of clathrin triskelia into clathrin cage, and cleaved CALM proteolysed by caspase 3 and calpain could not promote them. In this respect, our data support a model in which CALM functions like AP180 as a monomeric clathrin assembly protein and might take part in apoptotic process in neuronal cells.
Subject(s)
Cattle , Adaptor Proteins, Vesicular Transport , Animals , Brain Chemistry , Calpain/metabolism , Carrier Proteins , Caspases/metabolism , Clathrin/metabolism , Coated Pits, Cell-Membrane/metabolism , Hydrolysis , Membrane Proteins , Molecular Weight , Nerve Tissue Proteins/chemistry , Neurons/chemistry , Protein Binding , Protein Conformation , Recombinant Proteins/chemistryABSTRACT
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.
Subject(s)
Female , Mice , Rabbits , Animals , Antibodies, Monoclonal , Calpain/chemistry , Caspases/chemistry , Clathrin-Coated Vesicles/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Escherichia coli/genetics , Glutathione Transferase/genetics , Mice, Inbred BALB C , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/chemistry , Phosphoproteins/metabolism , Phosphoproteins/genetics , Phosphoproteins/chemistry , Protein Binding , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/chemistry , src Homology DomainsABSTRACT
Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicle (CCV) in neurons. The clathrin assembly protein gene (rCALM) was cloned from rat brain cDNA library. rCALM deduced 69 kD molecule has overall 73% amino acid homology compared with that of AP180 protein. The N-terminal domain, where amino acid sequences are very similar with AP180, harbours binding sites for clathrin and inositides, as well as possible phosphorylation sites, but the proline rich C-terminal domain is different from that of AP180. The mRNA expression of rCALM and AP180 by in situ hybridization histochemistry revealed that the rCALM mRNA was more intensely expressed than that of AP180, and the distribution patterns were different from each other. These results suggest that the rCALM mediates the assembly of clathrin in neural and supporting cells of brain, and regulates the clathrin coated-vesicle formation through phosphorylation and inositide metabolism. Copyright 2000 Academic Press.