Characterization of the charged components and their topology on the surface of plant seed oil bodies

Abstract

Oil bodies of plant seeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with alkaline proteins called oleosins. Oil bodies isolated from maize (Zea mays L.) in a medium of pH 7.2 maintained their entities but aggregated when the pH was lowered to 6.8 and 6.2. Aggregation did not lead to coalescence and was reversible with an elevation of the pH. Further decrease of the pH from 6.2 to 5.0 retarded the aggregation. Aggregation at pH 7.2 was induced with 2 mM CaCl2 or MgCl2 but not with NaCl. Aggregation at pH 6.8 was prevented by 10 microM sodium dodecyl sulfate but not with NaCl. We conclude that oil bodies have a negatively charged surface at pH 7.2 and an isoelectric point of about 6.0. This conclusion is supported by isoelectrofocusing results and by theoretical calculation of the positive charges in the oleosins and the negative charges in phosphatidylserine, phosphatidylinositol, and free fatty acids. Apparently, lowering of the pH from 7.2 to 6.2 protonates the histidine residues in the oleosins, and neutralizes the oil bodies. Further decrease of the pH to 5.0 likely protonates the free fatty acids and produces positively charged organelles. Similar charge properties were observed in the oil bodies isolated from rape, flax, and sesame seeds. An analysis of the oleosin secondary structures reveals an N-terminal amphipathic domain, a central hydrophobic anti-parallel beta-strand domain (not found in any other known protein), and a C-terminal amphipathic alpha-helical domain. In the two amphipathic domains, the positively charged residues are orientated toward the interior facing the negative charged lipids, whereas the negatively charged residues are exposed to the exterior. The negatively charged surface is a major factor in maintaining the oil bodies as stable individual entities.