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
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29.08.2015
Published in:
- 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
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- English
- Classification
- Biological sciences
- License
- License undefined
- Identifiers
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- RERO DOC 257827
- DOI 10.1016/j.bbamem.2015.08.020
- Persistent URL
- https://folia.unifr.ch/unifr/documents/304538
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