Phosphoinositides, ezrin/moesin, and rac1 regulate fusion of rhodopsin transport carriers in retinal photoreceptors
Phosphoinositides, ezrin/moesin, and rac1 regulate fusion of rhodopsin transport carriers in retinal photoreceptors
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DOI：
10.1091/mbc.e03-04-0203
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发表时间：
2004-01-01
影响因子：
3.3
通讯作者：
Ransom, N
中科院分区：
文献类型：
--
作者：
Deretic, D;Traverso, V;Ransom, N
视紫红质在感光细胞中的高尔基体后运输是由视紫红质承载运输载体（RTCs）介导的，并由小GTdR ab 8调节。在这项工作中，我们采取了结合药理学-蛋白质组学的方法来揭示RTC向专门的光敏细胞器，视杆细胞外节（ROS）运输的新调节因子。我们通过用1-磷酸鞘氨醇（SIP）激活磷脂酶D或用普萘洛尔（Pp 1）抑制磷脂酸磷酸水解酶来干扰磷脂合成。SIP刺激膜向ROS运输的总体速率。Pp 1刺激RTCs的出芽，但阻断膜向ROS的传递。Pp 1引起的RTCs在融合位点附近的积累，表明拴系缺陷，类似于先前描述的rab 8 T22 N突变体的表型。Pp 1处理后积累的RTCs的蛋白质组学分析显示磷脂酰肌醇-4，5-二磷酸结合蛋白ezrin和/或膜突蛋白显著减少。Ppl诱导膜突蛋白、肌动蛋白和小的GT-rac 1从RTCs重新分布到胞质溶胶中。通过共聚焦显微镜，ezrin/膜突蛋白和rac 1共定位与rab 8的RTC在其与质膜融合的网站，然而，这种分布后PP 1治疗丢失。我们的数据表明，在光感受器磷脂酰肌醇-4，5-二磷酸，膜突蛋白，肌动蛋白和rac 1的行为与rab 8协调一致，以调节系留和融合的RTCs。因此，他们是必要的视紫红质负载膜交付的ROS，从而控制的光检测细胞器的生物发生的关键步骤。
The post-Golgi trafficking of rhodopsin in photoreceptor cells is mediated by rhodopsin-bearing transport carriers (RTCs) and regulated by the small GTPase rab8. In this work, we took a combined pharmacological-proteomic approach to uncover new regulators of RTC trafficking toward the specialized light-sensitive organelle, the rod outer segment (ROS). We perturbed phospholipid synthesis by activating phospholipase D with sphingosine 1-phosphate (SIP) or inhibiting phosphatidic acid phosphohydrolase by propranolol (Pp1). SIP stimulated the overall rate of membrane trafficking toward the ROS. Pp1 stimulated budding of RTCs, but blocked membrane delivery to the ROS. Pp1 caused accumulation of RTCs in the vicinity of the fusion sites, suggesting a defect in tethering, similar to the previously described phenotype of the rab8T22N mutant. Proteomic analysis of RTCs accumulated upon Pp1 treatment showed a significant decrease in phosphatidylinositol-4,5-bisphosphate- binding proteins ezrin and/or moesin. Ppl induced redistribution of moesin, actin and the small GTPase rac1 from RTCs into the cytosol. By confocal microscopy, ezrin/moesin and rac1 colocalized with rab8 on RTCs at the sites of their fusion with the plasma membrane; however, this distribution was lost upon Pp1 treatment. Our data suggest that in photoreceptors phosphatidylinositol-4,5-bisphosphate, moesin, actin, and rac1 act in concert with rab8 to regulate tethering and fusion of RTCs. Consequentially, they are necessary for rhodopsin-laden membrane delivery to the ROS, thus controlling the critical steps in the biogenesis of the light-detecting organelle.