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的分类和运输的理解，包括整体和外周膜相关的，在内段内。整体膜蛋白由内质网相关核糖体合成并输出到高尔基体。烯丙基化或酰基化的外周膜蛋白在细胞质中合成并与er相关。囊泡从反式高尔基网络（TGN）产生并运输到纤毛基部，在那里与细胞膜发生融合。最后，货物被组装起来进行鞭毛内运输。基于我们之前鸟苷酸环化酶敲除的结果，我们假设光导机制的一些外周膜相关蛋白（pmp）与含gc的囊泡共同运输。在GC1/GC2双敲除棒中，PDE6运输失败，而转导蛋白(T)和视蛋白激酶（GRK1）未受影响。在GC1-/-视锥细胞中，COS中缺少整个光导机制，这表明大货的形成需要GC1，而GC1本身是一个完整的膜蛋白。基于戊烯基结合蛋白PrBP/d （Pde6d）的缺失，我们发现香叶基基化PDE6C和香叶基化GRK1向锥形外段的运输被阻断，而杆状体PDE6和杆状体T如期运输。本提案的目标1将通过基因敲除和敲除解决新的假定的戊烯基/酰基结合蛋白（UNC119和UNC119B）的鉴定和功能表征。目的2将研究GC1在锥体膜蛋白高尔基转运中的作用，强调GC1胞外结构域和GC活性的重要性，以及肌动素- ii在鞭毛内转运（IFT）中的作用。目的3将阐明Ca2+结合蛋白，中心蛋白-1和中心蛋白-2的作用，在鞭毛内运输通过杆和锥特异性敲除他们的基因。这些研究将共同扩大我们对参与光感受器维护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个磁盘）的外段磁盘堆栈需要持续的高生物合成速率，可靠的运输和靶向途径来替代外段蛋白质。光感受器细胞生物学的一个核心问题是膜相关蛋白的生物合成后转运和靶向到外节段的机制，这是细胞维持和生存所必需的途径。换句话说，“高度偏振光感受器是如何构建外部片段的？”拟议的研究考察了整体膜和外周膜蛋白从光感受器内段（生物合成位点）到外段（光转导位点）的运输方面。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，我们将探讨鸟苷酸环化酶1和肌动蛋白ii的作用，肌动蛋白ii是一种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中心蛋白- 1缺失的后果。膜蛋白的转运，特别是经过纤毛的高尔基转运和鞭毛内转运，是复杂的，在疾病的病因学中还没有得到充分的了解。

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.