No abstract

The local generation of phosphatidic acid plays a key role in the regulation of intracellular membrane transport through mechanisms which are largely unknown. Phosphatidic acid may recruit and activate downstream effectors, or change the biophysical properties of the membrane and directly induce membrane bending and/or destabilization. To evaluate these possibilities, we determined the phase properties of phosphatidic acid and lysophosphatidic acid at physiological conditions of pH and ion concentrations. In single-lipid systems, unsaturated phosphatidic acid behaved as a cylindrical, bilayer-preferring lipid at cytosolic conditions (37°C, pH 7.2, 0.5 mM free Mg 2+ ), but acquired a type-II shape at typical intra-Golgi conditions, a mildly acidic pH and submillimolar free Ca 2+ (pH 6.6±5.9, 0.3 mM Ca 2+ ). Lysophosphatidic acid formed type-I lipid micelles in the absence of divalent cations, but anhydrous cationlysophosphatidic acid bilayer complexes in their presence. These data suggest a similar molecular shape for phosphatidic acid and lysophosphatidic acid at cytosolic conditions; however, experiments in mixed-lipid systems indicate that their shape is not identical. Lysophosphatidic acid stabilized the bilayer phase of unsaturated phosphatidylethanolamine, while the opposite effect was observed in the presence of phosphatidic acid. These results support the hypothesis that a conversion of lysophosphatidic acid into phosphatidic acid by endophilin or BARS (50 kDa brefeldin A ribosylated substrate) may induce negative spontaneous monolayer curvature and regulate endocytic and Golgi membrane fission. Alternative models for the regulation of membrane fission based on the strong dependence of the molecular shape of (lyso)phosphatidic acid on pH and divalent cations are also discussed.

The formation of phosphatidic acid (PA) from lysophosphatidic acid (LPA), diacylglycerol, or phosphatidylcholine plays a key role in the regulation of intracellular membrane fission events, but the underlying molecular mechanism has not been resolved. A likely possibility is that PA affects local membrane curvature facilitating membrane bending and fission. To examine this possibility, we determined the spontaneous radius of curvature (R 0p ) of PA and LPA, carrying oleoyl fatty acids, using well-established X-ray diffraction methods. We found that, under physiological conditions of pH and salt concentration (pH 7.0, 150 mM NaCl), the R 0p values of PA and LPA were -46 Å and +20 Å, respectively. Thus PA has considerable negative spontaneous curvature while LPA has the most positive spontaneous curvature of any membrane lipid measured to date. The further addition of Ca 2+ did not significantly affect lipid spontaneous curvature; however, omitting NaCl from the hydration buffer greatly reduced the spontaneous curvature of PA, turning it into a cylindrically shaped lipid molecule (R 0p of -1.3 × 10 2 Å). Our quantitative data on the spontaneous radius of curvature of PA and LPA at a physiological pH and salt concentration will be instrumental in developing future models of biomembrane fission.