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Mechanism of selective transport and localization of functional proteins in the neuronal cells

神经元细胞中功能蛋白的选择性运输和定位机制

基本信息

批准号：
07804060
负责人：
OZAKI Koichi
金额：
$ 1.47万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

In order to investigate the transport pathway of functional proteins in the cells in situ, protein transport was interrupted by the use of mutant Rab proteins in the organellaspecific manner. Full-length cDNAs of 10 Drosophila Rab proteins were cloned and sequenced. Using the cDNAs, corresponding Rab proteins were synthesized in E.coli cells, and injected into the mice to produce their antisera. Localization of Rab proteins were then examined by the use of these antisera. Major fraction of each Rab proteins was in the membrane-bound form, which would probably resides in a particular cell organella. Immunohistological investigation of DRab1 protein revealed that its distribution overlaps that of the Golgi body in the photoreceptor cells.Transgenic flies expressing the dominant-negative versions of DRab1, DRab2 and DRabRP4 were constructed with the germ-line transformation technique. No apparent affects of the mutant Drab expression were found in the DRabRP4 transformant. In the dominant negative mutant of DRab2, most rhodopsin molecules were matured normally but a small fraction of them stayd just before full-mature form. This result suggests that Rab2 may be involved in a late stage of vesicle transport in Dorosophila photoreceptor cells. In contrast, remarkable affects on rhodopsin transport and cell structure were observed in the DRab1 transgenic mutant. Immature rhodopsin binding a large oligosaccharide chain was accumulated in the mutant. In the photoreceptor cells, Golgi bodies were disassembled and a numerous rER surrounded a nucleus. These results indicates that DRab1 plays an essential role in the vesicle transport between rER and Golgi body.

为了研究功能蛋白在原位细胞中的转运途径，利用突变的Rab蛋白以细胞器特异性的方式中断了蛋白质的转运。克隆了10个果蝇Rab蛋白的全长cdna并进行了测序。利用这些cdna，在大肠杆菌细胞中合成相应的Rab蛋白，并将其注射到小鼠体内以产生抗血清。然后用这些抗血清检测Rab蛋白的定位。每个Rab蛋白的主要部分以膜结合形式存在，这可能存在于特定的细胞器中。对DRab1蛋白的免疫组织学研究显示其在光感受器细胞中的分布与高尔基体重叠。利用种系转化技术构建了表达DRab1、DRab2和DRabRP4显性阴性版本的转基因果蝇。在DRabRP4的转化中，没有发现突变体Drab表达的明显影响。在DRab2显性阴性突变体中，大部分视紫红质分子正常成熟，但有一小部分在完全成熟之前停留。这一结果表明Rab2可能参与了嗜Dorosophila光感受器细胞囊泡运输的后期。相比之下，DRab1转基因突变体对视紫红质转运和细胞结构有显著影响。在突变体中积累了结合大寡糖链的未成熟视紫红质。在感光细胞中，高尔基体被分解，大量的内质网包围着细胞核。这些结果表明，DRab1在内质网和高尔基体之间的囊泡运输中起重要作用。