Membrane Protein Trafficking in Photoreceptors

描述（由申请人提供）：感光细胞生物学的核心是了解内节内膜蛋白的分选和转运，包括整体和外周膜相关蛋白。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并成为ER相关的。囊泡从高尔基体网络（trans-Golgi network，TGN）中出现，并运输到纤毛的基部，在那里与细胞膜融合。最后，货物组装为鞭毛内运输。基于我们以前的结果与鸟苷酸环化酶敲除，我们假设，一些外周膜相关蛋白（PMPs）的光转导机制cotransport与含GC囊泡。在GC 1/GC 2双敲除棒中，PDE 6不能转运，而转导素（T）和视蛋白激酶（GRK 1）不受影响。在GC 1-/-视锥细胞中，整个光转导机制不存在于COS中，这表明大货物的形成需要GC 1，其本身是一种整合的膜蛋白。基于异戊烯基结合蛋白PrBP/d（Pde 6d）的缺失，我们发现香叶基香叶基化的PDE 6C和法尼基化的GRK 1转运到视锥外节被阻断，而视杆PDE 6和视杆T转运如预期。本提案的目的1将通过基因敲除和敲除来解决新型推定的异戊二烯基/酰基结合蛋白（UNC 119和UNC 119 B）的鉴定和功能表征。目的2将研究GC 1在锥膜蛋白的高尔基体后转运中的作用，强调GC 1胞外结构域和GC活性的重要性，以及驱动蛋白II在鞭毛内转运（IFT）中的作用。目的3通过敲除视杆细胞和视锥细胞的钙结合蛋白centrin-1和centrin-2基因，阐明其在鞭毛内转运中的作用。这些研究将共同扩大我们对参与感光细胞维持和更新的蛋白质功能和相互作用的认识。公共卫生相关性：感光器外节每十天更新一次。每天更新约10%（约100个盘）的外节盘堆叠需要连续的高速率生物合成、可靠的运输和靶向途径来替换外节蛋白。光感受器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以及靶向外节进行盘组装的机制-这是细胞维持和存活所必需的途径。换句话说，“高度极化的感光细胞如何建立一个外节？“拟议的研究检查了从光感受器内段（生物合成位点）到外段（光转导位点）的完整膜和外周膜蛋白质转运的各个方面。在目标1中，异戊二烯基或酰基结合蛋白的作用将被调查。在目标2中，我们将探讨鸟苷酸环化酶1和驱动蛋白-II的作用，驱动蛋白-II是参与鞭毛内运输的分子马达。最后，在目标3中，将研究中心蛋白-1的光受体特异性缺失的后果。膜蛋白的运输，特别是高尔基体后运输和鞭毛内运输通过纤毛，是复杂的，在疾病的病因学还不充分了解。

DESCRIPTION (provided by applicant): 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/d (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. PUBLIC HEALTH RELEVANCE: 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.