Bavdek, Andrej Vazquez, Hector M. Conzelmann, Andreas 2015-08-29 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. application/pdf https://folia.unifr.ch/global/documents/304538 https://folia.unifr.ch/documents/304538/files/con_eca.pdf https://folia.unifr.ch/documents/304538/files/con_eca_sm.pdf eng info:eu-repo/semantics/altIdentifier/doi/10.1016/j.bbamem.2015.08.020 info:eu-repo/semantics/openAccess License undefined Biochimica et Biophysica Acta (BBA) - Biomembranes. - 2015, vol. 1848, no. 11, Part A, p. 2960–2966 info:eu-repo/classification/udc/57 Enzyme-coupled assays for flip-flop of acyl-Coenzyme A in liposomes http://purl.org/coar/resource_type/c_6501