Coated vesicles were prepared from rat liver homogenates by sucrose density gradient centrifugation and the clathrin coat, dissociated by mild treatment with Tris-Cl, was characterized by sodium dodecyl sulfate gel electrophoresis. At pH 6.5 and below, the soluble clathrin preparations induced a voltage-dependent increase in ion conductance across a black lipid membrane of oxidized cholesterol under conditions where the intact coated vesicles were inert. Further evidence for the interaction of clathrin with the lipid bilayer was provided by the demonstration that the fluorescent dye, carboxyfluorescein, was released from the internal aqueous space of small unilamellar dioleoyl and dipalmitoyl phosphatidylcholine vesicles upon the addition of the soluble protein. Dye release was shown to be a function of both calcium and pH. In the presence of calcium, release occurred at or above pH 8.0; in the absence of calcium, release was most prominent at pH 5.0-6.5. Formation of stable protein-phospholipid complexes was shown by KBr density gradient centrifugation. Stability of the complexes in high salt concentration suggested that electrostatic interactions were not essential to maintain the recombinants. Here, as with the black lipid membrane, incubation of the phospholipid vesicles with intact coated vesicles failed to induce release of the dye at pH 6.5 or below. Interaction of the clathrin coat protein with unilamellar vesicles at low pH resulted in a change in vesicle size and morphology as shown by electron microscopy.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - Jul 25 1982|