Enzyme-coupled assays for flip-flop of acyl-Coenzyme A in liposomes

Bavdek, Andrej; Vazquez, Hector M.; Conzelmann, Andreas

doi:10.1016/j.bbamem.2015.08.020

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Enzyme-coupled assays for flip-flop of acyl-Coenzyme A in liposomes

Bavdek, Andrej Division of Biochemistry, Department of Biology, University of Fribourg, Switzerland
Vazquez, Hector M. Division of Biochemistry, Department of Biology, University of Fribourg, Switzerland
Conzelmann, Andreas Division of Biochemistry, Department of Biology, University of Fribourg, Switzerland

29.08.2015

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Biochimica et Biophysica Acta (BBA) - Biomembranes. - 2015, vol. 1848, no. 11, Part A, p. 2960–2966

English 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 [3H]lyso-phosphatidic acid inside liposomes after [3H]palmitoyl-CoA has been added from outside. Two other tests use empty liposomes containing [3H]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 [3H]lyso-phosphatidic acid or of dephosphorylated [3H]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.

Faculty

Faculté des sciences et de médecine

Department

Département de Biologie

Language