The transbilayer distribution of phospholipids in disc membranes is a dynamic equilibrium - Evidence for rapid flip and flop movement

We studied the transbilayer redistribution of phospholipids in bovine rod o uter segment membranes on thoroughly washed, Ficoll-floated osmotically int act disc vesicles; freshly prepared membranes separated from the disc stack by osmotic shock; and intact disc stacks with a permeabilized plasma membr ane (A-discs, B-discs C-discs, respectively). In all cases, spin-labelled p hospholipid analogues (SL-PL) with choline, serine and ethanolamine head gr oups (PtdCho, PtdSer and PtdEtn, respectively) were taken up into the outer leaflet of the membranes by > 90% and within less than 30 s after SL-PL ad dition, as deduced from the disappearance of spin-label from the suspension medium and from the specific ESR spectrum of membrane-associated spin-labe l. Using BSA extraction, the amount of SL-PL in the outer leaflet of the bi layer was determined. It decreased with a mean half-time of < 5 min at 25 d egrees C, indicating rapid redistribution of all spin-labelled phospholipid s into the inner leaflet of the disc membranes. After 1 h, PtdCho and PtdEt n were distributed almost symmetrically, whereas PtdSer was 35 : 65% (in/ou t). Using subsequent incubation with BSA, the outward movement (flop) of th e analogues was observed directly, demonstrating that inward and outward mo vements proceed in thermodynamic equilibrium. No effect of N-ethylmaleimide or ATP on the redistribution could be measured, which makes it unlikely th at energy-consuming translocase or flippase processes are involved in the r edistribution in the dark. We reason that the solubilization zone around th e photoreceptor rhodopsin may be the locus of rapid redistribution of the h ighly unsaturated disc phospholipid.