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 m ere presence in the membrane. As a model, peptides mimicking the transmembr ane stretches of proteins, with the amino acid sequence GXXL(AL)(n)XXA (wit h X = K, H, or W and n = 8 or 12), were incorporated in large unilamellar v esicles composed of E. coli phospholipids. Phospholipid flop was measured b y assaying the increase in accessibility to dithionite of a 2,6-(7-nitro2,1 ,3-benzoxadiazol-4-yl)aminocaproyl (C6NBD)-labeled phospholipid analogue, i nitially 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 f lop rate constant (K-flop) linearly increasing with peptide:phospholipid mo lar 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 se ries GXXL(AL)(8)XXA also induced flop Of C6NBD-PG, supporting the hypothesi s that transmembrane parts of proteins mediate phospholipid translocation. In this series, K-flop decreased in the order X = K > H > W. indicating tha t peptide-lipid interactions in the interfacial region of the membrane modu late the efficiency of a peptide to cause flop. For the peptides tested, fl op Of C6NBD-phosphatidylethanolamine (C6NBD-PE) was substantially slower th an 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.