Membrane Protein Trafficking in Photoreceptors

光感受器中的膜蛋白运输

• 批准号: 8389899
• 负责人: WOLFGANG BAEHR
• 金额: $ 33.97万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8389899
• 关键词: Abbreviations; Address; Affect; Anabolism; Binding; Binding Proteins; Caenorhabditis elegans; Calmodulin-Binding Proteins; Cell Maintenance; Cell Survival; 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; abstracting; base; guanylate cyclase 1; immunocytochemistry; kinetosome; mutant; novel; peripherin; phosphodiesterase 6; prenyl; prevent; protein function; recombinase; research study; retinal rods; trans-Golgi Network

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.

抽象的。 光感受器中的膜蛋白运输。光感受器细胞生物学的核心是对 膜蛋白的分选和运输，包括完整的和外周与膜相关的，在 内线段。完整的膜蛋白由ER相关的核糖体合成，并输出到 高尔基仪器。胞浆中合成预酰化或酰化的外周膜蛋白 成为与ER相关的。囊泡从跨高尔基体网络(TGN)中出现，并运输到 纤毛，与细胞膜融合的地方。最后，货物被组装起来，以便在鞭毛内运输。 根据我们之前对鸟苷环化酶基因敲除的结果，我们假设一些外围设备 光信号转导机制的膜相关蛋白与GC共转运 水泡。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)未受影响。在Gc1-/-锥体中，COS中没有整个光转导机制 这表明大货物的形成需要Gc1，而Gc1本身是一种完整的膜蛋白。基于 PrBP/β(Pde6d)的缺失，我们发现香叶基香叶的转运 PDE6C和法尼化GRK1到锥体外段被阻断，而杆状PDE6和杆状T作为 预期中。本提案的目标1将解决新的推定的识别和功能特征 异丙烯基/酰基结合蛋白(UN119和UNC119B)通过基因敲除和敲除。Aim 2将检查 Gc1在锥膜蛋白高尔基体后转运中的作用，强调Gc1的重要性 胞外区和GC活性，以及Kinesin-II在鞭毛内运输(IFT)中的作用。目标3将 阐明钙离子结合蛋白Centin-1和Centin-2在杆状和锥状细胞鞭毛内转运中的作用 他们基因的特定敲除。这些研究将共同扩展我们对蛋白质的了解 参与光感受器维护和更新的功能和相互作用。叙述性 光感受器中的膜蛋白运输 沃尔夫冈·贝尔 光感受器外节每十天更新一次。每日续费 ~10%(约100个磁盘)的外段磁盘堆需要连续 高生物合成率、可靠的运输和可替代的靶向途径 外段蛋白。感光细胞生物学中的一个中心问题是 膜相关蛋白的生物合成后转运 靶向磁盘组件外部节段的机制--路径 对于细胞的维持和生存是必不可少的。另一种说法是，“如何 高度偏振的光感受器建立外节？“这项研究建议 整体膜蛋白和外周膜蛋白的研究进展 光感受器内段(生物合成部位)向外的运输 片段(光转导部位)。在目标1中，戊烯基或酰基结合的作用 将对蛋白质进行研究。在目标2中，我们将探索鸟苷的作用 Cyclase 1和Kinesin-II，参与鞭毛内运输的分子马达。 最后，在目标3中，光感受器特异性缺失中央蛋白的后果- 1将进行研究。膜蛋白的运输，特别是高尔基体后 纤毛的运输和鞭毛内的运输是复杂的和 在疾病病因学方面理解不足。