学 位 記 番 号 理工博 第

氏 名 高橋

俊樹

所 属 理工学研究科 生命科学専攻 学 位 の 種 類 博士（理学）

学 位 記 番 号 理工博 第

号 学位授与の日付 平成

年

月

日 課程・論文の別 学位規則第４条第１項該当

学 位 論 文 題 名

小胞輸送を制御する

タンパク質の分解機構とその意義（英文）

論 文 審 査 委 員 主査 教 授 川原 裕之 委員 教 授 岡本 龍史 委員 准教授 安藤 香奈絵

【論文の内容の要旨】

Cells are composed of various organelles such as endoplasmic reticulum, Golgi apparatus, endosome, and the like. Organelle transports information and proteins to each other and maintains its homeostasis using membrane vesicles network "membrane traffic". The Rab family protein is a monomeric GTPase that controls membrane traffic.

The structure and function of Rab proteins are changed by cycling betI en GTP-bound type and GDP-bound type. The Rab protein bound to GTP becomes active form and is anchored to the membrane to promote vesicular transport. On the other hand, the Rab protein bound to GDP becomes inactive and localizes in the cytoplasm away from the membrane vesicles. Recently, structural analysis of Rab protein revealed that GDP type Rab protein exposes a hydrophobic region (Switch-I). The exposure of the hydrophobic region in the cytoplasm can cause protein aggregation. Therefore, the mechanism including a chaperone that recognizes a hydrophobic region is crucial, but the mechanism has not yet been sufficiently clarified. Therefore, I started this research to elucidate the mechanism of protecting and controlling GDP-type Rab protein. I identified BAG6 as a novel chaperone that recognizes the hydrophobic region of the protein and found that its function was involved in intracellular membrane protein transport. Therefore, I focused on BAG6 as a chaperone candidate for Rab protein. First, I investigate the possibility that BAG6 functions as a hydrophobic recognition chaperone of GDP-bound. Co-immunoprecipitation revealed that BAG6 associated with Rab8a depending on GDP-bound. In addition, BAG6-Rab8a interaction was abolished

by deletion of BAG6-N465 region that was important to recognize hydrophobicity substrate to proteasome. Next, in order to clarify the possibility that the Switch-I region is involved in Rab8a recognition by BAG6, I examined whether BAG6 recognize Rab8a mutant that the hydrophobic residue in the Switch-I region was substituted with a hydrophilic group. As a result, BAG6 was not able to recognize the Mutant-Rab8a with reduced hydrophobicity of Switch-I. From the above results, it was shown that BAG6 recognizes Rab8a via Switch-I exposed upon binding of GDP. BAG6 is known to induce ubiquitination and degradation of the target substrate. So I examined whether GDP-bound Rab8a might be unstable and ubiquitinated. As a result, it was shown for the first time that GDP-bound Rab8a undergoes ubiquitination modification and is rapidly degraded. Furthermore, it was found that degradation of GDP-bound Rab8a was inhibited under BAG6 knockdown condition. These results indicate that BAG6 controls GDP-bound Rab8a specific proteasome-dependent degradation. Finally, in order to investigate the influence of Rab proteolysis by BAG6 on membrane traffic, the effect of BAG6 knockdown on the localization of transferrin receptor (Tfn-R) known as the substrate of Rab8a. I found that endosomal localization of Tfn-R changed by BAG6 knockdown. Since BAG6 interacts to multiple Rab family proteins including Rab8a, I suggest possibility in this study that BAG6 broadly control the stability of GDP-bound Rab protein and influence Rab-dependent membrane traffic.