Membrane Protein Trafficking in Photoreceptors

描述(由申请人提供)：光感受器细胞生物学的核心是对膜蛋白在内节内的分类和运输的理解，膜蛋白既有完整的膜相关的，也有外周的膜相关的。整膜蛋白是由内质网相关的核糖体合成的，并输出到高尔基体。胞浆中合成预酰化或酰化的外周膜蛋白，并成为内质网相关蛋白。囊泡从跨高尔基体网(TGN)中产生，并运输到纤毛底部，在那里与细胞膜融合。最后，货物被组装起来，以便在鞭毛内运输。根据我们先前对鸟苷环化酶基因敲除的结果，我们推测光转导机制中的一些外周膜相关蛋白(PMPs)与含GC的小泡共运输。在GC1/GC2双基因敲除棒中，PDE6不能转运，而转导蛋白(T)和视蛋白激酶(GRK1)不受影响。在Gc1-/-锥体中，COS中没有整个光转导机制，这表明形成一个大的货物需要Gc1，而Gc1本身是一个完整的膜蛋白。基于PrBP/d(Pde6d)的缺失，我们发现香叶基香叶酰化的PDE6C和法尼化的GRK1到锥体外节的转运被阻断，而杆状PDE6和杆状T的转运如期进行。本提案的目标1将通过基因敲除和敲除来鉴定和鉴定新的可能的戊烯基/酰基结合蛋白(UN119和UN119B)。目的2研究Gc1在高尔基体后锥膜蛋白转运中的作用，强调Gc1胞外区和GC活性的重要性，以及Kinesin-II在鞭毛内转运(IFT)中的作用。目的3阐明钙离子结合蛋白Centin-1和Centin-2在杆状和锥状敲除其基因的鞭毛内转运中的作用。这些研究将共同扩展我们关于参与光感受器维护和更新的蛋白质功能和相互作用的知识。与公共卫生相关：光感受器外节每十天更新一次。每天更新约10%(约100个磁盘)的外节段磁盘堆需要持续的高生物合成速度、可靠的运输和靶向途径来取代外节段蛋白。光感受器细胞生物学中的一个中心问题涉及膜相关蛋白的生物合成后运输以及靶向磁盘组装的外部片段的机制--这是细胞维持和生存所必需的途径。换句话说，“高度极化的光感受器是如何构建外部部分的？”这项拟议的研究考察了从感光细胞内段(生物合成部位)到外段(光转导部位)的完整膜和外周膜蛋白运输的各个方面。在目标1中，将研究戊烯基或酰基结合蛋白的作用。在目标2中，我们将探索鸟苷环化酶1和运动蛋白-II的作用，它是参与鞭毛内运输的分子马达。最后，在目标3中，将研究光感受器特异性缺失Centin-1的后果。膜蛋白的转运，特别是高尔基体后转运和鞭毛内转运通过纤毛是复杂的，在病因学上还不够清楚。

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.