Membrane-spanning peptides induce phospholipid flop: a model for phospholipid translocation across the inner membrane of E. coli.
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.
Kol
M A
MA
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
de Kroon
A I
AI
Rijkers
D T
DT
Killian
J A
JA
de Kruijff
B
B
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*
C435383:C6NBD-phosphatidylglycerol; D008081:Liposomes; D008565:Membrane Proteins; D010069:Oxadiazoles; D010455:Peptides; D010715:Phosphatidylglycerols; D010743:Phospholipids; D004227:Dithionite