Determination of the regions of the clathrin molecule inducing membrane fusion.
Clathrin induces fusion of liposome membranes containing phosphatidylserine at acidic pH [Maezawa, S., Yoshimura, T., Hong, K., Düzgüneş, N., & Papahadjopoulos, D. (1989) Biochemistry 28, 1422-1428]. The regions of the clathrin molecule inducing membrane fusion were determined by examining the fusion abilities of clathrin fragments obtained by limited proteolysis of clathrin cages with thermolysin. Membrane fusion was assessed by resonance energy transfer assay in terms of the dilution of fluorescent phospholipids in liposome membranes. Proteolysis of clathrin decreased the fusion rate and the amount of protein but did not affect the specific fusion rate (i.e., the fusion rate per unit of protein), indicating that clathrin fragments retain the ability to induce fusion. Of the two proteolytic fragments of the clathrin heavy chain, the terminal domain and the residual proximal part, which were separated by ultracentrifugation or gel chromatography, only the proximal part showed fusion activity. Light chains seemed to have no role in membrane fusion, since they are susceptible to proteolytic digestion. The terminal domain induced reversible liposome membrane aggregation, which was also induced by the residual proximal part of the heavy chain and the whole molecule of clathrin. These results suggest that the terminal domain and the proximal portion of clathrin have critical roles in the steps of close apposition and fusion of membranes, respectively.
Maezawa
S
S
Institute for Enzyme Research, University of Tokushima, Japan.
Yoshimura
T
T
D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002966:Clathrin / Q000302:isolation & purification / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes; D008561:Membrane Fusion; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D013820:Thermolysin
D002966:Clathrin; D008081:Liposomes; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D013820:Thermolysin