In vitro fusion of rabbit liver Golgi membranes with liposomes.
Fusion of Golgi membranes isolated from rabbit liver with liposomes was studied by lipid mixing of fluorescent lipid analogues and internal content mixing and by electron microscopic observation of transfer of horseradish peroxidase from liposomes into Golgi membranes. A monoclonal antibody was used to confirm fusion of Golgi membranes but not other contaminating vesicles. Fusion was rapid and efficient, reaching about 20% of the maximum after a 5-min incubation using small or large unilamellar dioleoylphosphatidylcholine vesicles. The fusion was dependent on temperature, decreasing at lower temperatures, and becoming nearly zero below 10 degrees C. The addition of ATP, GTP, cytosolic factors, or N-ethylmaleimide did not affect fusion. Treatments of Golgi membranes with 0.1 M Na2CO3 or 1 M KCl did not cause any changes in fusion. However, treatment with proteases inhibited fusion. These results suggest that Golgi integral membrane protein(s) are involved in fusion. Changing the medium to an isoosmotic substance, sucrose, in place of KCl or NaCl inhibited fusion. The binding assay of fluorescent liposomes to Golgi membranes showed that lowering the temperature or replacing salts with sucrose did not affect binding. However, treatment of Golgi membranes with proteases inhibited binding. Addition of phosphatidylserine or phosphatidylethanolamine to dioleoylphosphatidylcholine liposomes caused a 2-fold increase in binding and fusion. Fusion between Golgi membranes by themselves did not occur. These results provide some information on the mechanism of intracellular vesicular transport.
Kagiwada
S
S
Department of Biophysics, Faculty of Science, Kyoto University, Japan.
Murata
M
M
Hishida
R
R
Tagaya
M
M
Yamashina
S
S
Ohnishi
S
S
D000818:Animals; D000911:Antibodies, Monoclonal; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D008081:Liposomes; D008099:Liver / Q000378:metabolism*; D008561:Membrane Fusion; D008854:Microscopy, Electron; D010713:Phosphatidylcholines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D011817:Rabbits
D000911:Antibodies, Monoclonal; D008081:Liposomes; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; C017251:1,2-oleoylphosphatidylcholine