Clathrin Polymerization Research Articles

Polymerization of clathrin protomers into basket structures.

The effects of pH, ionic strength, temperature, and protein concentration on the rate of clathrin (8 S) polymerization to form coat (or basket) structures (approximately 300 S) have been measured by turbidity. The extent of polymerization has also been evaluated under the same experimental conditions by analytical centrifugation. The characteristic polygonal structure of the re-formed coat was confirmed by electron microscopy. The rate of polymerization is sensitive to all the variables investigated. The reaction is very slow at pH approximately 7 and becomes very rapid by pH approximately 6. The polymerization is readily reversed by increasing the pH slightly. The time dependence of the polymerization does not conform to either a first- or a second-order reaction but to a higher order. Increasing temperature increases the rate but decreases the extent of reaction. Increasing the salt concentration decreases the rate. The effects of several salts on the rate follow the Hofmeister ranking, with the exception of sulfate.

Molecular properties of the reassembled coat protein of coated vesicles.

Clathrin has been prepared from human and bovine brains by a rapid technique which does not require sucrose gradient centrifugation. The promoter molecule which is obtained has the ability to polymerize and form protein coats, i.e., so-called cages or baskets, which resemble the structures observed in coated vesicles. The polymerization of clathrin to form cage structures in 0.2 M ammonium acetate, pH 6.8, results in two distributions of sedimenting particles in the ultracentrifuge, one centered near 300S and the other near 150S. Equilibrium sedimentation gives molecular weights of the 150S and 300S particles near 25 million and 100 million, respectively. The turbidities of the two species have been measured during centrifugation in the ultracentrifuge. When the turbidity values are combined with the molecular weight values, the radii of the 150S and 300S species can be obtained, assuming a hollow sphere as a model for the clathrin polyhedral molecules.