POST-GOLGI VESICLES COTRANSPORT DOCOSAHEXAENOYL-PHOSPHOLIPIDS AND RHODOPSIN DURING FROG PHOTORECEPTOR MEMBRANE BIOGENESIS

Post-Golgi vesicles budding from the trans-Golgi network (TGN) are inv olved in the vectorial transport and delivery of rhodopsin to photorec eptor rod outer segments (ROS). We report here that newly synthesized docosahexaenoyl (DHA) phospholipids are sequestered and cotransported by rhodopsin-bearing post-Golgi vesicles to ROS, Frog retinas were pul se-labeled with [S-35]methionine/cysteine and [H-3]DHA prior to ROS is olation and subcellular fractionation. After a 1-h pulse, relatively u niform [H-3]DHA-lipid labeling (DPM/mu g protein) was observed in all fractions enriched in post-Golgi vesicles, TGN, Golgi, and endoplasmic reticulum (ER) membranes. During the subsequent 2-h chase translocati on of free [H-3]DHA from ROS to the photoreceptor inner segment contri buted to an additional overall increase in labeling of lipids. The spe cific activity (dpm/nmol DHA) in ER-enriched fraction was similar or h igher than in other subcellular fractions after both the pulse and the chase, indicating that the bulk of [H-3]DHA-lipids was synthesized in the ER. After the chase a a-fold increase in labeling of lipids in th e ER and Golgi and a 2.6-fold in lighter TGN-enriched fractions was ob served. The highest labeling was in the post-Golgi vesicle fraction (4 -fold increase), with [H-3]DHA-phosphatidylcholine and [H-3]DHA-phosph atidylethanolamine showing the greatest increase. At the same time, ne wly synthesized [S-35]rhodopsin shifted from the ER and Golgi toward T GN and post-Golgi fractions. Therefore, sequestration and association of [S-35]rhodopsin and [H-3]DHA-lipids in a TGN membrane domain occurs prior to their exit and subsequent vectorial cotransport on post-Golg i vesicles to ROS. Labeling of ROS lipids was very low, with phosphati dylinositol and diacylglycerols displaying the highest labeling, This indicates that other mechanisms by-passing Golgi, i.e. facilitated by lipid carrier proteins, may also contribute to molecular replacement o f disc membrane DHA-phospholipids, particularly phosphatidylinositol.