title: Membrane-spanning peptides induce phospholipid flop: a model for phospholipid translocation across the inner membrane of E. coli.
abstract: The mechanism by which phospholipids translocate (flop) across the E. coli inner membrane remains to be elucidated. We tested the hypothesis that the membrane-spanning domains of proteins catalyze phospholipid flop by their mere presence in the membrane. As a model, peptides mimicking the transmembrane stretches of proteins, with the amino acid sequence GXXL(AL)(n)XXA (with X = K, H, or W and n = 8 or 12), were incorporated in large unilamellar vesicles composed of E. coli phospholipids. Phospholipid flop was measured by assaying the increase in accessibility to dithionite of a 2,6-(7-nitro-2,1,3-benzoxadiazol-4-yl)aminocaproyl (C(6)NBD)-labeled phospholipid analogue, initially exclusively present in the inner leaflet of the vesicle membrane. Fast flop of C(6)NBD-phosphatidylglycerol (C(6)NBD-PG) was observed in vesicles in which GKKL(AL)(12)KKA was incorporated, with the apparent first-order flop rate constant (K(flop)) linearly increasing with peptide:phospholipid molar ratios, reaching a translocation half-time of approximately 10 min at a 1:250 peptide:phospholipid molar ratio at 25 degrees C. The peptides of the series GXXL(AL)(8)XXA also induced flop of C(6)NBD-PG, supporting the hypothesis that transmembrane parts of proteins mediate phospholipid translocation. In this series, K(flop) decreased in the order X = K > H > W, indicating that peptide-lipid interactions in the interfacial region of the membrane modulate the efficiency of a peptide to cause flop. For the peptides tested, flop of C(6)NBD-phosphatidylethanolamine (C(6)NBD-PE) was substantially slower than that of C(6)NBD-PG. In vesicles without peptide, flop was negligible both for C(6)NBD-PG and for C(6)NBD-PE. A model for peptide-induced flop is proposed, which takes into account the observed peptide and lipid specificity.
authors.affiliation: Department Biochemistry of Membranes, Center for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. m.a.kol@chem.uu.nl
authors.forename: M A
authors.initials: MA
authors.lastname: Kol
authors.forename: A I
authors.initials: AI
authors.lastname: de Kroon
authors.forename: D T
authors.initials: DT
authors.lastname: Rijkers
authors.forename: J A
authors.initials: JA
authors.lastname: Killian
authors.forename: B
authors.initials: B
authors.lastname: de Kruijff
mesh_terms: D001681:Biological Assay; D001692:Biological Transport; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D004227:Dithionite / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005240:Feasibility Studies; D008081:Liposomes; D008565:Membrane Proteins / Q000378:metabolism*; D008954:Models, Biological; D010069:Oxadiazoles / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism*
chemical_list: C435383:C6NBD-phosphatidylglycerol; D008081:Liposomes; D008565:Membrane Proteins; D010069:Oxadiazoles; D010455:Peptides; D010715:Phosphatidylglycerols; D010743:Phospholipids; D004227:Dithionite