TY - JOUR
T1 - Lipid requirements for reconstitution of the proton-translocating complex of clathrin-coated vesicles.
AU - Xie, X. S.
AU - Tsai, S. J.
AU - Stone, D. K.
PY - 1986/12
Y1 - 1986/12
N2 - We have recently reported the reconstitution of the clathrin-coated vesicle proton-translocating complex with liposomes prepared from ethanol-extracted crude bovine brain lipids. Reconstitution of proton pumping by the isolated proton ATPase has now been achieved with liposomes prepared from pure lipids. Optimal proton pumping, as assessed by ATP-generated acridine orange quenching and 32Pi-ATP exchange, is achieved with liposomes prepared from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cholesterol at a weight ratio of 40:26.5:7.5:26, and a lipid-to-protein weight ratio of 200:1. Under such conditions, the extent of decrease of ATP-generated acridine orange quenching is 150-fold greater than that of native clathrin-coated vesicles. Cholesterol is required to stabilize the proteoliposome. Phosphatidylserine, which is most effective in activating ATP-hydrolytic activity of the solubilized enzyme, is not obligatory for reconstitution of proton pumping.
AB - We have recently reported the reconstitution of the clathrin-coated vesicle proton-translocating complex with liposomes prepared from ethanol-extracted crude bovine brain lipids. Reconstitution of proton pumping by the isolated proton ATPase has now been achieved with liposomes prepared from pure lipids. Optimal proton pumping, as assessed by ATP-generated acridine orange quenching and 32Pi-ATP exchange, is achieved with liposomes prepared from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cholesterol at a weight ratio of 40:26.5:7.5:26, and a lipid-to-protein weight ratio of 200:1. Under such conditions, the extent of decrease of ATP-generated acridine orange quenching is 150-fold greater than that of native clathrin-coated vesicles. Cholesterol is required to stabilize the proteoliposome. Phosphatidylserine, which is most effective in activating ATP-hydrolytic activity of the solubilized enzyme, is not obligatory for reconstitution of proton pumping.
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U2 - 10.1073/pnas.83.23.8913
DO - 10.1073/pnas.83.23.8913
M3 - Article
C2 - 2878429
AN - SCOPUS:0022959246
SN - 0027-8424
VL - 83
SP - 8913
EP - 8917
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 23
ER -