Purification and properties of 100-kd proteins from coated vesicles and their reconstitution with clathrin.
Bullock brain coated vesicles contain a family of at least six 100-kd polypeptides which have the property of promoting clathrin assembly. These proteins have been purified from Triton X-100-extracted coated vesicles by a combination of gel filtration and chromatography on hydroxylapatite and DE-52 cellulose. Three major 100-kd species occur as complexes with a stoichiometric amount of a 50-kd polypeptide. On cross-linking these complexes, the chief products appear to contain two polypeptides of 100 kd and two of 50 kd. These 100-kd/50-kd complexes will polymerise with low concentrations of clathrin to give a relatively homogeneous population of coats predominantly of the 'barrel' size. In contrast, three other polypeptides of 100 kd lack the 50-kd protein but polymerise with clathrin under the same conditions to yield coats of a wide range of sizes including 'barrels', truncated icosahedra and particles of greater than 100 nm diameter. When clathrin cages are reassembled with a saturating amount of 100-kd/50-kd complexes and studied by electron microscopy, the additional proteins appear to follow the underlying geometry of the clathrin polyhedra, partially filling in the polygonal faces of the cage structures. Saturation appears to require approximately 3 molecules of 100-kd polypeptide per clathrin trimer.
D000818:Animals; D001921:Brain / Q000378:metabolism* / Q000648:ultrastructure; D002417:Cattle; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000648:ultrastructure*; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D011992:Endosomes / Q000648:ultrastructure*; D007700:Kinetics; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008961:Models, Structural; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification* / Q000378:metabolism; D011487:Protein Conformation; D013388:Succinimides
D002966:Clathrin; D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D013388:Succinimides; C035759:3,3'-dithiobis(sulfosuccinimidyl propionate)