INTRACELLULAR-LOCALIZATION OF 2 BETAINE LIPIDS BY CELL FRACTIONATION AND IMMUNOMICROSCOPY

The cellular localization of the betaine lipids diacylglyceryl-N,N,N-t rimethylhomoserine (DGTS) and glycerylhydroxymethyl-N,N,N-trimethyl-be ta-alanine (DGTA) was investigated by a) chemical analysis of subcellu lar fractions and b) immunochemical methods using specific antisera an d 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 ox idase, NADH-cyt c reductase and DGTS. Non-plastidial fractions were en riched in DGTS and only minor amounts of this lipid could be attribute d to chloroplasts. Anti-DGTS and anti-DGTA sera were produced by immun ization of rabbits. The monospecificity of the antisera was examined w ith 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 FIT C-coupled goat anti-rabbit antibody was used and detected by fluoresce nce microscopy. Thin sections of Ochromonas and Pavlova were incubated first with the anti-lipid sera and subsequently with a gold-coupled a nti-rabbit serum and then examined in the electron microscope. With Oc hromonas, anti-DGTS as well as anti-DGTA sera gave an accumulation of gold label in the cytoplasmic space but not in the chloroplasts. Simil ar results were obtained with Pavlova using anti-DGTA serum. These res ults describe for the first time the cytochemical localization of DGTS and DGTA strongly suggesting both these lipids to be associated mainl y with non-plastidial structures.