Purification of a new clathrin assembly protein from bovine brain coated vesicles and its identification as myelin basic protein.
The multimeric clathrin assembly proteins AP-1 and AP-2 with molecular masses of approximately 270 kDa and the monomeric clathrin assembly proteins AP180 and auxilin with molecular masses of approximately 90 kDa catalyze the assembly of clathrin into artificial clathrin baskets under physiological conditions. We have now identified a much smaller approximately 20-kDa clathrin assembly protein in 0.5 M Tris, pH 7.0, extracts of bovine-brain coated vesicles and purified it to near homogeneity. A polyclonal antibody against this protein did not cross-react with any of the other assembly proteins, and sequencing data suggest that this new protein is similar or identical to myelin basic protein (MBP). At a molar ratio of 3 molecules per clathrin triskelion, MBP catalyzes polymerization of clathrin into artificial baskets that appear structurally similar to the baskets assembled by the other assembly proteins. In addition, like the other baskets, the clathrin-MBP baskets are uncoated by hsp70. MBP represents a significant fraction of the total assembly protein activity present in 0.5 M Tris, pH 7.0, extracts of coated vesicles. It is not clear if it acts as an assembly protein in vivo, but because it is well characterized and easily available, MBP will be a useful protein to investigate the mechanism of clathrin assembly and disassembly in vitro.
Prasad
K
K
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-0301, USA.
Barouch
W
W
Martin
B M
BM
Greene
L E
LE
Eisenberg
E
E
D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002850:Chromatography, Gel; D002966:Clathrin / Q000378:metabolism / Q000648:ultrastructure; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D007700:Kinetics; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D034041:Monomeric Clathrin Assembly Proteins; D004676:Myelin Basic Protein / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010750:Phosphoproteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
D033942:Adaptor Proteins, Vesicular Transport; D000906:Antibodies; D002966:Clathrin; D018840:HSP70 Heat-Shock Proteins; D034041:Monomeric Clathrin Assembly Proteins; D004676:Myelin Basic Protein; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; C439023:clathrin assembly protein AP180