Intracellular localization of two betaine lipids by cell fractionation and immunomicroscopy

The cellular localization of the betaine lipids diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and diacylglycerylhydroxymethyl-N,N,N-trimethyl-beta-alanine (DGTA) was investigated by a) chemical analysis of subcellular fractions and b) immunochemical methods using specific antisera and either fluorescence microscopy or electron microscopy for detection of the label. A homogenate of Lycopodium annotinum (Pteridophyta) was fractionated by differential and density gradient centrifugation. The particulate fractions obtained were analyzed for chlorophyll, cyt c oxidase, NADH-cyt c reductase and DGTS. Non-plastidial fractions were enriched in DGTS and only minor amounts of this lipid could be attributed to chloroplasts. Anti-DGTS and anti-DGTA sera were produced by immunization of rabbits. The monospecificity of the antisera was examined with cells of Chlamydomonas reinhardtii (Chlorophyceae) containing DGTS, Pavlova lutheri (Haptophyceae) containing DGTA and Ochromonas danica (Chrysophyceae) containing both DGTS and DGTA. Euglena gracilis which is free of betaine lipids, was used as a control. For the test, a FITC-coupled goat anti-rabbit antibody was used and detected by fluorescence microscopy. Thin sections of Ochromonas and Pavlova were incubated first with the anti-lipid sera and subsequently with a gold-coupled anti-rabbit serum and then examined in the electron microscope. With Ochromonas, anti-DGTS as well as anti-DGTA sera gave an accumulation of gold label in the cytoplasmic space but not in the chloroplasts. Similar results were obtained with Pavlova using anti-DGTA serum. These results describe for the first time the cytochemical localization of DGTS and DGTA strongly suggesting both these lipids to be associated mainly with non-plastidial structures.