Rodriguez de Turco E B, Deretic D, Bazan N G, Papermaster D S
LSU Neuroscience Center and Department of Ophthalmology, Louisiana State University Medical Center, School of Medicine, New Orleans, Louisiana 70112, USA.
J Biol Chem. 1997 Apr 18;272(16):10491-7. doi: 10.1074/jbc.272.16.10491.
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.
从反式高尔基体网络（TGN）出芽的高尔基体后囊泡参与视紫红质向光感受器视杆外段（ROS）的定向运输和递送。我们在此报告，新合成的二十二碳六烯酰（DHA）磷脂被含视紫红质的高尔基体后囊泡隔离并共同运输到ROS。在分离ROS并进行亚细胞分级之前，用[35S]甲硫氨酸/半胱氨酸和[3H]DHA对青蛙视网膜进行脉冲标记。脉冲1小时后，在所有富含高尔基体后囊泡、TGN、高尔基体和内质网（ER）膜的分级中观察到相对均匀的[3H]DHA-脂质标记（每分钟衰变数/微克蛋白质）。在随后的2小时追踪过程中，游离[3H]DHA从ROS向光感受器内段的转运导致脂质标记总体上进一步增加。脉冲和追踪后，富含ER的分级中的比活性（每分钟衰变数/纳摩尔DHA）与其他亚细胞分级中的相似或更高，表明大部分[3H]DHA-脂质是在ER中合成的。追踪后，观察到ER和高尔基体中脂质标记增加了2倍，在较轻的富含TGN的分级中增加了2.6倍。标记最高的是高尔基体后囊泡分级（增加了4倍），[3H]DHA-磷脂酰胆碱和[3H]DHA-磷脂酰乙醇胺增加最多。同时，新合成的[35S]视紫红质从ER和高尔基体向TGN和高尔基体后分级转移。因此，[35S]视紫红质和[3H]DHA-脂质在TGN膜结构域中的隔离和结合发生在它们离开并随后在高尔基体后囊泡上定向共同运输到ROS之前。ROS脂质的标记非常低，磷脂酰肌醇和二酰基甘油的标记最高。这表明绕过高尔基体的其他机制，即由脂质载体蛋白促进的机制，也可能有助于盘膜DHA-磷脂，特别是磷脂酰肌醇的分子替代。
