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-nitro2,1,3-benzoxadiazol-4-yl)aminocaproyl (C6NBD)-labeled phospholipid analogue, initially exclusively present in the inner leaflet of the vesicle membrane. Fast flop of C6NBD-phosphatidylglycerol (C6NBD-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 similar to 10 min at a 1:250 peptide:phospholipid molar ratio at 25 degreesC. The peptides of the series GXXL(AL)(8)XXA also induced flop Of C6NBD-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 C6NBD-phosphatidylethanolamine (C6NBD-PE) was substantially slower than that of C6NBD-PG. In vesicles without peptide, flop was negligible both for C6BD-PG and for C6NBD-PE. A model for peptide-induced flop is proposed, which takes into account the observed peptide and lipid specificity.