Membrane Protein Trafficking in Photoreceptors

抽象的。 光感受器中的膜蛋白运输。感光细胞生物学的核心是理解 膜蛋白的分类和运输，包括整体和外周膜相关， 内部部分。整合膜蛋白由ER相关的核糖体合成，并输出到 高尔基体异戊烯化或酰化的外周膜蛋白在胞质溶胶中合成， 与ER相关。囊泡从高尔基体反式网络（TGN）中出现，并运输到基底膜。 与细胞膜融合的纤毛。最后，货物组装为鞭毛内运输。 基于我们以前的鸟苷酸环化酶敲除结果，我们假设一些外周血细胞中的 膜相关蛋白（PMPs）的光转导机制共运输与含GC 囊泡在GC 1/GC 2双敲除的杆状细胞中，PDE 6不能转运，而转导素（T）和视蛋白激酶（PKK）不能转运。 （GRK 1）不受影响。在GC 1-/-视锥细胞中，整个光传导机制都不存在于COS中 这表明大货物的形成需要GC 1，GC 1本身是一种完整的膜蛋白。基于 删除异戊二烯基结合蛋白PrBP/<$（Pde 6d），我们发现， PDE 6C和法尼基化GRK 1被阻断到锥体外节，而PDE 6和T杆被转运到锥体外节。 预期本提案的目的1将解决新的推定的 异戊烯基/酰基结合蛋白（UNC 119和UNC 119 B）通过基因敲除和敲除。目标2将检查 GC 1在高尔基体后转运视锥细胞膜蛋白中的作用，强调了GC 1的重要性 细胞外结构域和GC活性，以及驱动蛋白-II在鞭毛内运输（IFT）中的作用。目标3将 阐明Ca 2+结合蛋白，centrin-1和centrin-2，在鞭毛内运输中的作用，通过杆和锥， 特定的基因敲除这些研究将共同扩大我们对蛋白质的认识 参与感光细胞维持和更新的功能和相互作用。叙事 光感受器的膜蛋白运输 沃尔夫冈·贝尔 光感受器外节每十天更新一次。每日更新 约10%（约100个磁盘）的外部段磁盘堆栈需要连续 高生物合成率、可靠的转运和靶向替代途径 外节蛋白感光细胞生物学的一个中心问题是 膜相关蛋白的生物合成后转运， 瞄准外节进行椎间盘组装的机制-- 对细胞的维持和存活至关重要。换句话说，“如何 高度极化的光感受器建立一个外节？“拟议的研究 检查膜和外周膜蛋白的整合方面 从光感受器内节（生物合成位点）到外节的运输 片段（光转导位点）。在aim 1中，异戊烯基或酰基结合的作用 蛋白质将被研究。在目标2中，我们将探讨鸟苷酸的作用 环化酶1和驱动蛋白-II，参与鞭毛内运输的分子马达。 最后，在目标3中，研究了光受体特异性缺失中心蛋白- 1将被研究。膜蛋白的运输，特别是高尔基体后 通过纤毛的运输和鞭毛内运输是复杂的， 对疾病的病因学认识不足。

Abstract. Membrane Protein Trafficking in Photoreceptors. Central to photoreceptor cell biology is an understanding of the sorting and transport of membrane proteins, both integral and peripherally membrane-associated, within the inner segment. Integral membrane proteins are synthesized by ER-associated ribosomes and exported to the Golgi apparatus. Prenylated or acylated peripheral membrane proteins are synthesized in the cytosol and become ER-associated. Vesicles emerge from the trans-Golgi network (TGN) and transport to the base of the cilium where fusion with the cell membrane occurs. Finally, cargo is assembled for intraflagellar transport. Based on our previous results with guanylate cyclase knockouts, we postulate that some peripheral membrane-associated proteins (PMPs) of the phototransduction machinery cotransport with GC-containing vesicles. In GC1/GC2 double knockout rods, PDE6 failed to transport whereas transducin (T) and opsin kinase (GRK1) were unaffected. In GC1-/- cones, the entire phototransduction machinery was absent from the COS suggesting that formation of a large cargo requires GC1, itself an integral membrane protein. Based on deletion of a prenyl binding protein, PrBP/¿ (Pde6d), we discovered that transport of geranylgeranylated PDE6C and farnesylated GRK1 to cone outer segments was blocked while rod PDE6 and rod T transported as expected. Aim 1 of this proposal will address the identification and functional characterization of novel putative prenyl/acyl binding proteins (UNC119 and UNC119B) by gene knockdowns and knockouts. Aim 2 will examine the role of GC1 in post-Golgi transport of cone membrane proteins, emphasizing the importance of the GC1 extracellular domain and GC activity, as well as the role of kinesin-II in intraflagellar transport (IFT). Aim 3 will elucidate the roles of Ca2+-binding proteins, centrin-1 and centrin-2, in intraflagellar transport by rod- and cone- specific knockouts of their genes. These studies will collectively expand our knowledge regarding the protein functions and interactions that participate in photoreceptor maintenance and renewal. Narrative Membrane Protein Trafficking in Photoreceptors Wolfgang Baehr Photoreceptor outer segments are renewed every ten days. Daily renewal of ~10% (about 100 disks) of the outer segment disk stack requires a continuous high rate of biosynthesis, reliable transport, and targeting pathways to replace outer segment proteins. A central question in photoreceptor cell biology concerns post-biosynthetic transport of membrane-associated proteins and the mechanisms of targeting to the outer segments for disk assembly--pathways that are essential for cell maintenance and survival. Otherwise stated, "How does the highly polarized photoreceptor build an outer segment?" The proposed research examines aspects of integral membrane and peripheral membrane protein transport from photoreceptor inner segment (site of biosynthesis) to the outer segment (site of phototransduction). In aim 1, the role of prenyl- or acyl binding proteins will be investigated. In aim 2, we will explore the roles of guanylate cyclase 1 and kinesin-II, a molecular motor involved in intraflagellar transport. Finally in aim 3, the consequences of photoreceptor-specific deletions of centrin- 1 will be studied. Transport of membrane proteins, particularly post-Golgi transport and intraflagellar transport through the cilium, are complex and insufficiently understood in disease etiology.