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Volume 272, Number 16, Issue of April 18, 1997 pp. 10491-10497
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Post-Golgi Vesicles Cotransport Docosahexaenoyl-Phospholipids and Rhodopsin during Frog Photoreceptor Membrane Biogenesis

(Received for publication, September 19, 1996, and in revised form, December 30, 1996)

Elena B. Rodriguez de Turco Dagger , Dusanka Deretic § , Nicolas G. Bazan Dagger and David S. Papermaster par

From the Dagger  LSU Neuroscience Center and Department of Ophthalmology, Louisiana State University Medical Center, School of Medicine, New Orleans, Louisiana 70112, the § University of Michigan, Departments of Ophthalmology and Anatomy and Cell Biology, Ann Arbor, Michigan 48105, and par  Univeristy of Texas, Health Science Center, Department of Pathology, San Antonio, Texas 78284-7750

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/µg 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.


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