Exposure of hydrophobic domains of clathrin in its membrane fusion-inducible pH region.

Clathrin induces fusion of phospholipid membranes containing phosphatidylserine when the pH of the medium is reduced below about 6. The hydrophobicity of clathrin in the membrane fusion-inducible pH region was examined. In the presence of clathrin, the fluorescence maximum of 1-anilinonaphthalene-8-sulfonate was shifted to shorter wavelengths and its fluorescence intensity increased at pH values below about 6. Steep increase of the fluorescence intensity of the fluorescent probe, N-(1-anilinonaphthyl-4)maleimide covatently bound to clathrin was observed in a similar pH region. The efficiency of resonance energy transfer from tryptophan to anilinonaphthyl residues in the clathrin molecule showed similar pH dependency. When Triton X-114 solutions containing clathrin were subjected to phase separation by raising the temperature from 0 degree to 30 degrees C at different pH values, clathrin remained in the aqueous phase above pH 6, whereas the protein was partitioned between the aqueous and detergent phases at pH 5-6, and was present only in the detergent phase below pH 5. The effective hydrophobicity of clathrin determined by the fluorescence method using cis-parinaric acid also increased at pH values below 6. Moreover, clathrin was aggregated by lowering the pH below 6. These results show that exposure of hydrophobic domains of clathrin through conformational change occurs in its membrane fusion-inducible pH region, suggesting the crucial role of protein hydrophobicity in the initiation of membrane fusion.