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

为了研究功能蛋白在细胞中的原位转运途径，通过以细胞器特异性方式使用突变体Rab蛋白来中断蛋白质转运。克隆了10个果蝇Rab蛋白的全长cDNA并进行了序列测定。使用cDNA，在大肠杆菌细胞中合成相应的Rab蛋白，并注射到小鼠中以产生其抗血清。然后通过使用这些抗血清检查Rab蛋白的定位。每种Rab蛋白的大部分是以膜结合的形式存在，可能存在于特定的细胞器中。免疫组化结果表明，DRab1蛋白在感光细胞中的分布与高尔基体的分布相重叠，利用胚系转化技术构建了表达DRab1、DRab2和DRabRP4的转基因果蝇。在DRabRP4基因组中未发现突变体Drab表达的明显影响。在显性失活突变体中，大多数视紫红质分子正常成熟，但有一小部分在完全成熟前保持不变。这一结果表明，Rab2可能参与囊泡运输的后期阶段，在Dorabelila感光细胞。与此相反，在DRab1转基因突变体中观察到对视紫红质转运和细胞结构的显著影响。未成熟的视紫红质结合一个大的寡糖链在突变体中积累。在感光细胞中，高尔基体解体，大量的rER围绕着细胞核。这些结果表明，DRab1在rER和高尔基体之间的囊泡运输中起着重要作用。

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.