Abstract
Post-Golgi vesicles budding from the trans-Golgi network (TGN) are involved in the vectorial transport and delivery of rhodopsin to photoreceptor 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 pulse-labeled with [35S]methionine/cysteine and [3H]DHA prior to ROS isolation and subcellular fractionation. After a 1-h pulse, relatively uniform [3H]DHA-lipid labeling (DPM/microg protein) was observed in all fractions enriched in post-Golgi vesicles, TGN, Golgi, and endoplasmic reticulum (ER) membranes. During the subsequent 2-h chase translocation of free [3H]DHA from ROS to the photoreceptor inner segment contributed to an additional overall increase in labeling of lipids. The specific activity (dpm/nmol DHA) in ER-enriched fraction was similar or higher than in other subcellular fractions after both the pulse and the chase, indicating that the bulk of [3H]DHA-lipids was synthesized in the ER. After the chase a 2-fold increase in labeling of lipids in the ER and Golgi and a 2.6-fold in lighter TGN-enriched fractions was observed. The highest labeling was in the post-Golgi vesicle fraction (4-fold increase), with [3H]DHA-phosphatidylcholine and [3H]DHA-phosphatidylethanolamine showing the greatest increase. At the same time, newly synthesized [35S]rhodopsin shifted from the ER and Golgi toward TGN and post-Golgi fractions. Therefore, sequestration and association of [35S]rhodopsin and [3H]DHA-lipids in a TGN membrane domain occurs prior to their exit and subsequent vectorial cotransport on post-Golgi vesicles to ROS. Labeling of ROS lipids was very low, with phosphatidylinositol 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 of disc membrane DHA-phospholipids, particularly phosphatidylinositol.
Highlights
Vertebrate photoreceptors are polarized and compartmentalized cells, with a photosensitive outer segment and a synaptic terminal domain at opposite ends of the cell
Post-Golgi Vesicles Are Enriched with Newly Synthesized [35S]Rhodopsin and [3H]DHA-Lipids—To determine whether newly synthesized [3H]DHA-PLs and [35S]rhodopsin are transported together in the same population of post-Golgi vesicles recovered in fraction 5 of the sucrose gradient, retinas were pulse-labeled for 1 h in the presence of both precursors and further incubated for 2 h in cold buffer prior to subcellular fractionation
This study provides the first available information about the closely coordinated trafficking, sorting, and association of newly synthesized [3H]DHA-PLs with [35S]rhodopsin in frog photoreceptors as the two major membrane components initiate their journey from the rough endoplasmic reticulum (ER), where they are synthesized, move through the Golgi, and leave the trans-Golgi network (TGN) on post-Golgi vesicles vectorially driven to rod outer segments (ROS) for the assembly of new disc membranes
Summary
Vertebrate photoreceptors are polarized and compartmentalized cells, with a photosensitive outer segment and a synaptic terminal domain at opposite ends of the cell. ROS lack the capacity for de novo synthesis of PLs [4, 9] They depend entirely on an external supply of PLs from the inner segment where they are synthesized mainly in the rough endoplasmic reticulum (ER) [10]. How these highly unsaturated lipids become components of ROS membranes and at which stage of membrane biosynthesis and disc morphogenesis they become associated with rhodopsin is not yet clear. Synthesized rhodopsin is vectorially transported from its site of synthesis in the rough ER to ROS by vesicles that bud from the trans-Golgi network (TGN), cluster beneath the connecting cilium, and fuse with the inner segment plasma membrane within the periciliary ridge complex [11, 12]. Phospholipid renewal of ROS membranes involves both membrane replacement (as new disc membranes are assembled at the base of the ROS) and molecular replacement
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