Membrane Protein Trafficking in Photoreceptors

光感受器中的膜蛋白运输

基本信息

批准号：
7564341
负责人：
WOLFGANG BAEHR
金额：
$ 37.63万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-12-01 至 2013-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7564341
关键词：
Abbreviations Address Affect Anabolism Binding Binding Proteins Caenorhabditis elegans Calmodulin-Binding Proteins Cell Maintenance Cell membrane Cellular biology Cilia Co-Immunoprecipitations Complex Cyclic GMP Cytosol Disease Electron Microscopy Endoplasmic Reticulum Etiology Extracellular Domain Filament G protein coupled receptor kinase GRK1 gene Genes Golgi Apparatus Guanylate Cyclase Integral Membrane Protein KRP protein Knock-out Knowledge Light Maintenance Membrane Membrane Protein Traffic Membrane Proteins Membrane Transport Proteins Microtubules Molecular Motors Motor Mus Opsin Partner in relationship Pathway interactions Peripheral Phenotype Phosphotransferases Photoreceptors Phototransduction Physiology Process Proteins Research Retina Retinal Cone Rhodopsin Ribosomes Role Sequence Homology Side Site Sorting - Cell Movement Structure Testing Transducin Transgenes Transgenic Mice Trimethoprim-Sulfamethoxazole Vertebrate Photoreceptors Vesicle base guanylate cyclase 1 immunocytochemistry kinetosome mutant novel peripherin phosphodiesterase 6 prenyl prevent protein function public health relevance recombinase research study retinal rods trans-Golgi Network

项目摘要

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.

描述（由申请人提供）：光感受器细胞生物学的中心是对内部段内膜蛋白的分类和转运的理解，无论是整合性和周围膜相关的。整合性膜蛋白通过ER相关核糖体合成并导出到高尔基体。在细胞质中合成前或酰化的周围膜蛋白并与ER相关。囊泡从反式高尔基网络（TGN）出现，并转移到纤毛的底部，其中与细胞膜融合发生。最后，将货物组装用于flagellar内运输。基于我们先前使用鸟苷酸环化酶敲除的结果，我们假设光转传机械共转运的某些外周膜相关蛋白（PMP）含有含GC的囊泡。在GC1/GC2双基因敲除杆中，PDE6未能运输，而透明蛋白（T）和Opsin激酶（GRK1）不受影响。在GC1 - / - 锥中，整个光转导机器都是从COS中提出的，这表明大型货物的形成需要GC1，这本身就是整体膜蛋白。基于删除蛋白质的蛋白质PRBP/d（PDE6D），我们发现黄烷基凝聚酰化的PDE6C和Farnesylated GRK1的转运被阻断，而ROD PDE6和ROD t却被预期运输。该提案的目标1将通过基因敲除和敲除来探讨新型假定的前蛋白/酰基结合蛋白（UNC119和UNC119B）的鉴定和功能表征。 AIM 2将检查GC1在锥膜蛋白的高尔基后运输中的作用，强调GC1细胞外结构域和GC活性的重要性，以及驱动蛋白II在Flagellar内转运（IFT）中的作用。 AIM 3将阐明Ca2+结合蛋白，Centrin-1和Centrin-2在其基因的杆内运输中的Ca2+结合蛋白，Centrin-1和Centrin-2。这些研究将共同扩大我们有关参与光感受器维持和更新的蛋白质功能和相互作用的知识。公共卫生相关性：光感受器的外部细分市场每十天更新一次。每日续订约10％（约100个磁盘）外部段磁盘堆栈需要连续高的生物合成率，可靠的运输和靶向替代外部段蛋白的靶向途径。光感受器细胞生物学的一个核心问题涉及膜相关蛋白的生物合成后转运以及靶向外部段的磁盘组装的机制 - 对于细胞维持和生存至关重要的途径。否则说：“高度极化的光感受器如何构建外部段？”拟议的研究研究了整合膜和周围膜蛋白从感光细胞内部段（生物合成位点）转移到外部段（光转移位点）的各个方面。在AIM 1中，将研究蛋白基或酰基结合蛋白的作用。在AIM 2中，我们将探索鸟烯基环化酶1和驱动蛋白II的作用，这是一种参与氟法内转运的分子运动。最后，将研究中心蛋白1的光感受器特异性缺失的后果。膜蛋白的运输，尤其是高尔基后运输和通过纤毛的氟法运输，在疾病病因学中是复杂的，并且不足以理解。