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FUNCTIONAL ANALYSIS OF SMALL GTP-BINDING PROTEIN, RAB6, BY GENE TRANSFER

通过基因转移对小 GTP 结合蛋白 RAB6 进行功能分析

基本信息

批准号：
10670021
负责人：
IIDA Hiroshi
金额：
$ 2.05万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670021/
关键词：
Small GTP-binding protein Rab6 Vesicular transport Golgi apparatus Gene transfer

项目摘要

9. SUMMARY OF RESEARCH RESULTSThe Golgi complex is an exceptionally dynamic organelle whose structure is maintained by a balance of vesicles entering and exiting the organelle as well as a balance between anterograde and retrograde membrane traffic within the organelle. A small GTP-binding protein, rab6p, appeared to be involved in the intra-Golgi vesicular transport. We have examined the potential roles of rab6p in maintaining the structural integrity of the Golgi complex. Wild-type rab6, rab6 Q72L (a GTPase-deficient mutant), and rab6 N126I (a mutant with low affinity for both GTP and GDP) were expressed in NRK cells by transfection-mediated gene transfer, and the effects of these proteins on the structural integrity of the Golgi complex were monitored by confocal laser scanning microscopy using mannosidase II (Man II) as a Golgi marker. Expression of wild-type rab6 N126I did not bring about remarkable change of Man II immunostaining pattern, while expression of rab6 Q72L was found to induce disassembly of the Golgi complex into the fragmented elements. Electron microscopy revealed that expression of rab6 Q72L not only promoted swelling or vacuolation of the Golgi cisternae but also caused dilation of the rough endoplasmic reticulum, suggesting that the normal function of rab6p in control of vesicular traffic is essential for maintaining the normal structure of both the Golgi cisternae and the rough endoplasmic reticulum. We also provided morphological evidence that swelling of the Golgi cisternae predominantly occurred at the trans pole of the organelle, which might trigger vacuolation of the Golgi cisternae and subsequent collapse of the organelle. Our findings suggest that rab6 Q72L might disturb the anterograde vesicular traffic from the medial to the trans Golgi cisternae that is regulated by endogenous rab6p, by which the normal morphology of the Golgi complex is disrupted.

9.研究概述高尔基复合体是一种异常动态的细胞器，其结构由进入和离开细胞器的小泡的平衡以及细胞器内顺行和逆行的膜交通平衡维持。一个小的GTP结合蛋白Rab6p似乎参与了高尔基体内的囊泡运输。我们研究了rab6p在维持高尔基复合体结构完整性方面的潜在作用。以甘露糖苷酶II(Man II)为高尔基体标记物，通过转基因方法在NRK细胞中表达野生型Rab6、Rab6 Q72L(GTP缺失突变体)和Rab6 N126I(GTP和GDP低亲和力突变体)，并用激光共聚焦扫描显微镜观察这些蛋白对高尔基体结构完整性的影响。野生型Rab6N126I的表达没有引起Man II免疫染色模式的显著变化，而Rab6Q72L的表达则导致高尔基体复合体解体为碎裂的元件。电子显微镜显示，Rab6Q72L的表达不仅促进高尔基体池的肿胀或空泡化，而且使粗面内质网扩张，提示Rab6p控制囊泡运输的正常功能对于维持高尔基体池和粗面内质网的正常结构是必不可少的。我们还提供了高尔基体池的肿胀主要发生在细胞器的横极的形态证据，这可能引发高尔基体池的空泡化和随后细胞器的崩溃。我们的研究结果提示，Rab6Q72L可能干扰了由内源性Rab6p调控的内侧到跨高尔基体囊泡的顺行运输，从而破坏了高尔基复合体的正常形态。