Abstract
The translocation of apocytochrome c (apocyt.c) across large unilamellar vesicles (LUVs) constructed from mixtures of anionic and zwitterionic phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), has been studied. It was shown that the import ratio of horse heart apocyt.c in LUVs composed of phosphatidic acid (PA) combined with PE and PC (62±10%) was much higher than that in LUVs made of PE and PC plus any other acidic phospholipid species (20±5%). This feature was shared by tuna heart and chicken heart apocyt.c. In addition, the greater efficiency of the PA/PE/PC system versus others in facilitating apocyt.c translocation was maintained using synthetic anionic phospholipids with the same acyl chains. Besides, apocyt.c induces more leakage of entrapped fluorescein sulphonate (FS) from the interior of PA/PC/PE vesicles compared with phosphatidylglycerol (PG)/PC/PE ones. By measuring the intrinsic fluorescence emission spectrum and the accessibility of the preprotein to the fluorescence quencher, acrylamide, differences could be detected in the conformational changes of apocyt.c as a consequence of its interaction with PA/PE/PC and PG/PE/PC vesicles, respectively. Particularly notable is that PE is indispensable for the PA/PE/PC system to most efficiently facilitate apocyt.c translocation across the model membranes. With the fraction of PE increasing from 0 to 30 mol%, the translocation efficiency of apocyt.c as well as its ability to induce FS efflux was significantly enhanced in PA-containing LUVs, whereas this was not observed in the case of replacement of PA by PG or phosphatidylserine. It is also interesting to note that in LUVs containing PA, dioleoyl-PE, but not dielaidoyl-PE, can exert such influences, indicative of the role of non-bilayer formation propensity. On the basis of these results it is postulated that PA might increase the bilayer-destabilizing effects of PE, and hence increase the translocation efficiency of apocyt.c and its leakage-induction ability.
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