Enzyme-coupled assays for flip-flop of acyl-Coenzyme A in liposomes.
Acyl-Coenzyme A is made in the cytosol. Certain enzymes using acyl-CoA seem to operate in the lumen of the ER but no corresponding flippases for acyl-CoA or an activated acyl have been described. In order to test the ability of purified candidate flippases to operate the transport of acyl-CoA through lipid bilayers in vitro we developed three enzyme-coupled assays using large unilamellar vesicles (LUVs) obtained by detergent removal. The first assay uses liposomes encapsulating a water-soluble acyl-CoA:glycerol-3-phosphate acyl transferase plus glycerol-3-phosphate (G3P). It measures formation of [(3)H]lyso-phosphatidic acid inside liposomes after [(3)H]palmitoyl-CoA has been added from outside. Two other tests use empty liposomes containing [(3)H]palmitoyl-CoA in the inner membrane leaflet, to which either soluble acyl-CoA:glycerol-3-phosphate acyl transferase plus glycerol-3-phosphate or alkaline phosphatase are added from outside. Here one can follow the appearance of [(3)H]lyso-phosphatidic acid or of dephosphorylated [(3)H]acyl-CoA, respectively, both being made outside the liposomes. Although the liposomes may retain small amounts of detergent, all these tests show that palmitoyl-CoA crosses the lipid bilayer only very slowly and that the lipid composition of liposomes barely affects the flip-flop rate. Thus, palmitoyl-CoA cannot cross the membrane spontaneously implying that in vivo some transport mechanism is required.
Division of Biochemistry, Department of Biology, University of Fribourg, Chemin du Musée 10, Fribourg, CH-1700, Switzerland.
Andrej
A
Bavdek
Division of Biochemistry, Department of Biology, University of Fribourg, Chemin du Musée 10, Fribourg, CH-1700, Switzerland.
Hector M
HM
Vazquez
Division of Biochemistry, Department of Biology, University of Fribourg, Chemin du Musée 10, Fribourg, CH-1700, Switzerland. Electronic address: andreas.conzelmann@unifr.ch.
Andreas
A
Conzelmann
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000378:metabolism; D001692:Biological Transport; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000378:metabolism*; D005994:Glycerophosphates / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D053835:Unilamellar Liposomes / Q000737:chemistry / Q000378:metabolism
D000214:Acyl Coenzyme A; D005994:Glycerophosphates; D008051:Lipid Bilayers; D008081:Liposomes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D053835:Unilamellar Liposomes; C057561:1-palmitoyl-2-oleoylphosphatidylethanolamine; D012710:Serum Albumin, Bovine; D005992:Glycerol-3-Phosphate O-Acyltransferase; D000469:Alkaline Phosphatase; C017251:1,2-oleoylphosphatidylcholine; C028694:1-palmitoyl-2-oleoylphosphatidylcholine
Alkaline phosphatase; Elman's reagent; Squash glycerol-3-phosphate acyltransferase