Mechanism of protein-induced membrane fusion: fusion of phospholipid vesicles by clathrin associated with its membrane binding and conformational change.
The clathrin-induced fusion of liposome membranes, the membrane binding of clathrin, and the conformational states of clathrin were investigated over a wide pH range using large unilamellar and multilamellar vesicles composed of phosphatidylserine (PS), phosphatidylcholine (PC), PS/PC (2:1), PS/PC (1:1), or PS/PC (1:2). The pH profiles of clathrin-induced fusion of all types of liposomes containing PS showed biphasic patterns. Their pH thresholds were found in the pH range of 5-6 and shifted to lower pH values with decrease in the PS content. Similar shifts were observed in the pH range of 5-6 and shifted to lower pH values with decrease in the PS content. Similar shifts were observed in the pH profiles of clathrin binding to these vesicles, but the pH profiles of binding were different from the biphasic fusion patterns. With PC vesicles, only small degrees of fusion and clathrin binding were observed at pH 2-4. The pH dependences of the conformation and hydrophobicity of clathrin were determined by measuring the extent of the blue shift of the fluorescence maximum of 1-anilinonaphthalene-8-sulfonate in the presence of the protein, the fluorescence intensity of N-(1-anilinonaphthyl-4)maleimide bound to the clathrin molecule, the resonance energy transfer from its tryptophan to anilinonaphthyl residues, the partitioning of the protein in Triton X-114 solution, and the hydrophobicity index of clathrin using cis-parinaric acid. These measurements indicated that conformational change and exposure of hydrophobic regions occur below pH 6 and suggested that clathrin may adopt different conformational states in the pH region where it induced membrane fusion.(ABSTRACT TRUNCATED AT 250 WORDS)
Maezawa
S
S
Institute for Enzyme Research, UNivrsity of Tokushima, Japan.
Yoshimura
T
T
Hong
K
K
Düzgüneş
N
N
Papahadjopoulos
D
D
D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes; D008561:Membrane Fusion; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D002966:Clathrin; D008081:Liposomes; D010743:Phospholipids