Structure, function and regulation of Na^+/H^+ antiporters and intracellular localization mechanism.

Na^/H^反向转运蛋白的结构、功能和调控以及细胞内定位机制。

• 批准号: 13680689
• 负责人: KANAZAWA Hiroshi
• 金额: $ 2.24万
• 依托单位: Department of Biological Sciences, Graduate School of Science, 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: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680689/
• 关键词: Na^+; H^+ antiporter; Ion transport; Salt tolerance; Active transport; Genetic approach; Intracellular localization; Regulatory protein; 膜タンパク質; 膜タンパクの細胞内局在化; 膜蛋白質の精製と再構成; イオン輸送蛋白質の構想と機能; 能動輸送の分子機構; 新規カルシューム結合性蛋白質; キメラ蛋白質の合成; イオン輸送分子機構; H^

Maintaining homeostasis of intracellular ion concentrations is the most basic requirement for every living cells. This mechanism is supported by various ion transpoting proteins located in cellular membranes. Na^+/H^+ antiporters among the ion-transporting proteins play a central role to maintain intracellular Na^+ and H^+ concentrations and named Nha or NHE, for bacteria or mammalian cells, respectively. In the present study, we set a final goal of this project to clarify the molecular basis of adaptation mechanism of living cells to various ion environments, especially Na^+ and H^+, by analyzing structure, function and regulation of Na^2/H^+ antiporters from bacteria, yeast and mammalian cells. During 2 years project term, we successfully obtained several new information of Na^+/H^2 antiporters as follows: (1) We clarified transmembrane domains essential for ion transport as well as domains required for pH sensing. We showed that H. pylori antiporter have a specific structure for pH … More sensing which is different from E. coli antiproter. We also successfully purified H. pylon antiporters and reconstituted the antiporter activity in vitro. (2) We showed that various different yeast species have similar Na^+/H^+ (Nhalp) by cloning the genes. We also found that Nhalp has a two-domain structure, comprised of hydrophobic membrane region, and hydrophilic cytoplasmic region which overall structure is close to mammalian NHE. Within this cytoplasmic region, we revealed that domains important for antiport and also intracellular localization exist. Further we identified that a novel membrane protein binds to the domain and enhance the antiporter activity. (3) We identified a novel Ca^<2+> protein (CHP) capable of binding to NHE previously. In this project we identified a new protein kinase and a new kinesin as the binding target of CHP. We studied functional significance of CHP in vitro and in vivo and discussed the multifunction of CHP. Further study is required for understanding intracellular regulatory function of CHP in the future study.The several different lines of result in terms of Na^+/H^+ antiporters in this project opened a new future study. The results were published as 6 independent full papers. Less

维持细胞内离子浓度的稳态是每个活细胞最基本的要求。这种机制得到了位于细胞膜上的各种离子转运蛋白的支持。离子转运蛋白中的 Na^+/H^+ 反向转运蛋白在维持细胞内 Na^+ 和 H^+ 浓度方面发挥着核心作用，对于细菌或哺乳动物细胞分别称为 Nha 或 NHE。在本研究中，我们的最终目标是通过分析细菌、酵母和哺乳动物细胞的Na^2/H^+反向转运蛋白的结构、功能和调控，阐明活细胞对各种离子环境，特别是Na^+和H^+的适应机制的分子基础。在两年的项目期间，我们成功获得了Na^+/H^2反向转运蛋白的一些新信息：（1）我们阐明了离子转运必需的跨膜结构域以及pH传感所需的结构域。我们发现，幽门螺杆菌反向转运蛋白具有与大肠杆菌反向转运蛋白不同的 pH 传感特定结构。我们还成功纯化了 H. pylon 反向转运蛋白并在体外重建了反向转运蛋白活性。 (2)我们通过克隆基因证明了各种不同的酵母物种具有相似的Na^+/H^+ (Nhalp)。我们还发现Nhalp具有双结构域结构，由疏水性膜区和亲水性细胞质区组成，整体结构与哺乳动物NHE接近。在这个细胞质区域内，我们发现存在对反向端口和细胞内定位重要的结构域。此外，我们还发现一种新型膜蛋白与该结构域结合并增强逆向转运蛋白活性。 (3)我们先前鉴定了一种能够与NHE结合的新型Ca^2+蛋白(CHP)。在这个项目中，我们确定了一种新的蛋白激酶和一种新的驱动蛋白作为 CHP 的结合靶点。我们研究了CHP的体外和体内功能意义，并讨论了CHP的多功能性。在未来的研究中，需要进一步研究了解CHP的细胞内调节功能。本项目中Na^+/H^+反向转运蛋白的几个不同的结果开辟了新的未来研究。结果以6篇独立完整论文的形式发表。较少的

项目成果

Matsumoto, M., Miyake, Y., Nagita, M., Inoue, H., Shitakubo, D., Takemoto, K., Ohtsuka, C., Nakamura, N., Kanazawa, H.: "A serine/threonine kinase which causes apoptosis like cell death interacts with a calcineurin B like protein capable of binding Na^+ /

Matsumoto, M.、Miyake, Y.、Nagita, M.、Inoue, H.、Shitakubo, D.、Takemoto, K.、Ohtsuka, C.、Nakamura, N.、Kanazawa, H.：“丝氨酸/苏氨酸

Keiko Hayami, Takato Noumi, Hiroki Inoue, Ge-Hong Sijn-Wada, Takao Yoshimizu and Hiroshi Kanazawa: "The murine genome contains one functional gene and two pseudogenes coding for the 16 kDa proteolipid subunit of vacuolar H^+-ATPase"Gene. 273. 199-206 (200

Keiko Hayami、Takato Noumi、Hiroki Inoue、Ge-Hong Sijn-Wada、Takao Yoshimizu 和 Hiroshi Kanazawa：“小鼠基因组包含一个功能基因和两个编码液泡 H^-ATP 酶 16 kDa 蛋白脂质亚基的基因”。

U.Tokumoto, S.Nomura, Y.Minami, H.Mihara, S.Kato, T.Kurihara, N.Esaki, H.Kanazawa, H.Matsuhara., Y.Takahashi: "Network of Protein Protein Interactions among Iron-Sulfur Cluster Assembly Proteins in Escherichia coli"J. Biochem.. 131. 713-719 (2002)

U.Tokumoto、S.Nomura、Y.Minami、H.Mihara、S.Kato、T.Kurihara、N.Esaki、H.Kanazawa、H.Matsuhara.、Y.Takahashi：“铁-蛋白质之间的蛋白质相互作用网络

Norihiro Nakamura, Y.Miyake, M.Matsushita, S.Tanaka, H.Inoue, H.Kanazawa: "KIF1Bb2, capable of interacting with CHP, is localized to Synaptic vesicles"J. Biochem.. 132. 483-491 (2002)

Norihiro Nakamura、Y.Miyake、M.Matsushita、S.Tanaka、H.Inoue、H.Kanazawa：“能够与 CHP 相互作用的 KIF1Bb2 定位于突触小泡”J.

Yumi Tsuboi, Hiroki Inoue, Norihiro Nakamura, Hiroshi Kanazawa: "Identification of membrane domains of Na^+/H^+ antiporter (NhaA) from Helicobacter pylori required for ion transport and pH sensing"J. Biol. Chem.. June issue(in press). (2003)

Yumi Tsuboi、Hiroki Inoue、Norihiro Nakamura、Hiroshi Kanazawa：“幽门螺杆菌中离子转运和 pH 传感所需的 Na^ /H^ 逆向转运蛋白 (NhaA) 的膜域的鉴定”J.