Phosphatidylethanolamine Synthesis by Castor Bean Endosperm

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Abstract
The bilayer distribution of phosphatidylethanolamine (PtdEtn) in the endoplasmic reticulum of castor bean (Ricinus communis L. var Hale) endosperm following synthesis by both the CDP-ethanolamine: 1,2-diacylglycerol ethanolaminephosphotransferase and ethanolamine base exchange reactions have been studied. Two chemical probes, 1-fluoro-2,4-dinitrobenzene (FDNB) and 2,4,6-trinitrobenzenesulfonic acid (TNBS), which covalently bind to the free amino groups, were utilized. The endoplasmic reticulum membranes were impermeable to TNBS at 4 and 25°C, but were permeable to FDNB at both temperatures. FDNB treatment of the PtdEtn from the base exchange reacted with 92% of the PtdEtn, while 80% of the lipid reacted with TNBS. Thus, at least 80% of the PtdEtn synthesized by the base exchange reaction was localized in the outer leaflet of the membrane, with about 12% occurring on the inner leaflet and about 8% being inaccessible. For PtdEtn formed by the CDP-ethanolamine pathway, 85% reacted with FDNB and 70% with TNBS, indicating that at least 70% was produced to the cytoplasmic face of the ER and 15% to the lumen side. The remainder was inaccessible to the probes. The sensitivity to trypsin of the two reactions also was tested. The ethanolamine base exchange enzyme, as well as that for l-serine exchange, retained activity following exposure to trypsin, but the activity of ethanolaminephosphotransferase disappeared after such treatment. This indicates that both base exchange enzymes are exposed to the lumenal side of the ER, while CDP-ethanolamine: 1,2-diacylglycerol ethanolaminephosphotransferase is exposed on the outer or cytoplasmic side. These results are discussed with respect to the final phospholipid distributions and the presumed sources of the water-soluble substrates.