Kwok Michael C M, Holopainen Juha M, Molday Laurie L, Foster Leonard J, Molday Robert S
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Mol Cell Proteomics. 2008 Jun;7(6):1053-66. doi: 10.1074/mcp.M700571-MCP200. Epub 2008 Jan 31.
The outer segment is a specialized compartment of vertebrate rod and cone photoreceptor cells where phototransduction takes place. In rod cells it consists of an organized stack of disks enclosed by a separate plasma membrane. Although most proteins involved in phototransduction have been identified and characterized, little is known about the proteins that are responsible for outer segment structure and renewal. In this study we used a tandem mass spectrometry-based proteomics approach to identify proteins in rod outer segment preparations as an initial step in defining their roles in photoreceptor structure, function, renewal, and degeneration. Five hundred and sixteen proteins were identified including 41 proteins that function in rod and cone phototransduction and the visual cycle and most proteins previously shown to be involved in outer segment structure and metabolic pathways. In addition, numerous proteins were detected that have not been previously reported to be present in outer segments including a subset of Rab and SNARE proteins implicated in vesicle trafficking and membrane fusion. Western blotting and immunofluorescence microscopy confirmed the presence of Rab 11b, Rab 18, Rab 1b, and Rab GDP dissociation inhibitor in outer segments. The SNARE proteins, VAMP2/3, syntaxin 3, N-ethylmaleimide-sensitive factor, and Munc 18 detected in outer segment preparations by mass spectrometry and Western blotting were also observed in outer segments by immunofluorescence microscopy. Syntaxin 3 and N-ethylmaleimide- sensitive factor had a restricted localization at the base of the outer segments, whereas VAMP2/3 and Munc 18 were distributed throughout the outer segments. These results suggest that Rab and SNARE proteins play a role in vesicle trafficking and membrane fusion as part of the outer segment renewal process. The data set generated in this study is a valuable resource for further analysis of photoreceptor outer segment structure and function.
外段是脊椎动物视杆和视锥光感受器细胞的一个特殊区域,光转导在此发生。在视杆细胞中,它由一堆由独立质膜包裹的有序排列的圆盘组成。尽管大多数参与光转导的蛋白质已被鉴定和表征,但对于负责外段结构和更新的蛋白质却知之甚少。在本研究中,我们使用基于串联质谱的蛋白质组学方法来鉴定视杆外段制剂中的蛋白质,作为确定它们在光感受器结构、功能、更新和退化中作用的第一步。共鉴定出516种蛋白质,其中包括41种在视杆和视锥光转导以及视觉循环中起作用的蛋白质,以及大多数先前已证明参与外段结构和代谢途径的蛋白质。此外,还检测到许多以前未报道在外段中存在的蛋白质,包括与囊泡运输和膜融合有关的Rab和SNARE蛋白亚群。蛋白质免疫印迹法和免疫荧光显微镜检查证实了外段中存在Rab 11b、Rab 18、Rab 1b和Rab GDP解离抑制剂。通过质谱和蛋白质免疫印迹法在外段制剂中检测到的SNARE蛋白VAMP2/3、 syntaxin 3、N - 乙基马来酰亚胺敏感因子和Munc 18,也通过免疫荧光显微镜在外段中观察到。Syntaxin 3和N - 乙基马来酰亚胺敏感因子在外段基部有局限定位,而VAMP2/3和Munc 18分布在外段各处。这些结果表明,Rab和SNARE蛋白作为外段更新过程的一部分,在囊泡运输和膜融合中发挥作用。本研究中生成的数据集是进一步分析光感受器外段结构和功能的宝贵资源。