Structure, Regulatory and Functional Mechanisms, and Physiological Roles of Peptide Transporter

肽转运蛋白的结构、调节和功能机制以及生理作用

• 批准号: 13142206
• 负责人: MAEDA Masatomo
• 金额: $ 46.66万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research on Priority Areas
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13142206/
• 关键词: Proteome; Peptide; Transporter; TAPL; MAT-8; Nano-Bio; Pharmaceutical Sciences; ABC輸送体; ABCB9; トランスポーター; Mat-8; FXYDファミリー

1. Human ABC transporter TAPL was stably expressed in the cultured cells, and its intracellular localization was determined. The TAPL was localized on the lysosomal membrane. Furthermore, TAPL formed a homo-dimer as determined by the expression of TAPL tagged with different fluorescent proteins. The amino terminal 4 trans-membrane segments were enough for TAPL to be localized to lysosome. Mutation introduced into a residue important for ATP binding of TAPL abolished the high sensitivity of the yeast to an antibiotic.2. TAPL had carboxyl-terminal splicing isoforms (3 and 4 in human and rat, respectively), which would produce functional diversity of TAPL.3. We also demonstrated that two TAPL homologues of C. elegance are expressed on the granule membranes in intestinal cells. The deletion of the genes for these homologues further indicated that they are important for formation and/or maintenance of such intestinal intracellular granules.4. Mat-8, a member of FXYD family, co-localized with Nat, K+-ATPase on the plasma membrane of colorectal cancer cells, suggesting that Mat-8 could be a regulatory component of a membrane transport nano-machine. When the mutation was introduced into the conserved residue in its transmembrane domain, Mat-8 was expressed rather on the intracellular membranes instead of plasma membrane.5. ln this project, we constructed various expression systems for membrane proteins TAPL and Mat-8 together with those tagged with fluorescent proteins. These systems were adopted for visualization of transporters and their regulators under fluorescent microscope. Further construction of mutants and their expression will give us important information on the association and dissociation of the membrane transporter complexes. Taken together, these findings would be valuable and become basic knowledge for future studies on function and structure of membrane transport nano-machines.

1.人ABC转运蛋白TAPL在培养细胞中稳定表达，并确定其在细胞内的定位。TAPL定位于溶酶体膜上。此外，通过标记不同荧光蛋白的TAPL的表达，TAPL形成了一种同源二聚体。氨基末端的4个跨膜片段足以使TAPL定位于溶酶体。在TAPL结合ATP的重要残基中引入突变，消除了酵母菌对抗生素的高度敏感性。TAPL具有羧基末端剪接异构体(在人和大鼠中分别为3和4)，这将产生TAPL.3的功能多样性。我们还证明了优雅线虫的两个TAPL同源物在肠道细胞的颗粒膜上表达。这些同源基因的缺失进一步表明，它们对于这种肠道细胞内颗粒的形成和/或维持是重要的。Mat-8是FXYD家族的一员，与NAT，K+-ATPase共定位于结直肠癌细胞的质膜上，提示Mat-8可能是膜转运纳米机器的调控元件。当突变被引入到其跨膜区的保守残基中时，Mat-8更多地在细胞膜上表达，而不是在质膜上表达。在本项目中，我们构建了不同的膜蛋白TAPL和Mat-8的表达系统，以及那些被荧光蛋白标记的表达系统。这些系统被用来在荧光显微镜下对转运蛋白及其调节子进行可视化。突变体的进一步构建及其表达将为我们提供关于膜转运蛋白复合体的结合和解离的重要信息。综上所述，这些发现将是有价值的，并成为未来膜传输纳米机器功能和结构研究的基础知识。

项目成果

GATA DNA-binding protein expressed in mouse I-10 Leydig testicular tumor cells.

GATA DNA 结合蛋白在小鼠 I-10 Leydig 睾丸肿瘤细胞中表达。

• 发表时间: 2001
• 期刊: Biochem.Biophys.Res.Commun. 283
• 作者: Katanosaka;Y.;Iwata;Y.;Kobayashi;Y.;Shibasaki;F.;Wakabayashi;S.;Shigekawa;M.;Reiko Nakagawa
• 通讯作者: Reiko Nakagawa

Membrane localization of transporter associated with antigen processing (TAP)-like (ABCB9) visualized in vivo with a fluorescence protein-fusion technique

• DOI: 10.1248/bpb.27.1916
• 发表时间: 2004-12-01
• 期刊: BIOLOGICAL & PHARMACEUTICAL BULLETIN
• 影响因子: 2
• 作者: Kobayashi, A;Maeda, T;Maeda, M
• 通讯作者: Maeda, M

Proteolytic activation of SREBPs during adipocyte differentiation

• DOI: 10.1006/bbrc.2001.4915
• 发表时间: 2001-05-25
• 期刊: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
• 影响因子: 3.1
• 作者: Inoue, J;Kumagai, H;Sato, R
• 通讯作者: Sato, R

H^+/K^+-トランスポーテイングATPase広川タンパク質化学 第4巻 酵素4.3ヒドロラーゼ[III](分担執筆)

H^+/K^+-转运 ATP 酶 Hirokawa Protein Chemistry 第 4 卷酶 4.3 水解酶 [III]（贡献者）

• 发表时间: 2002
• 作者: Matsumoto K;Inoue H;Nakano T;Tsuda M;Yoshiura Y;Fukuyama S;Tsushima F;Hoshino T;Aizawa H;Akiba h;Pardoll D;Yagita H;Azuma M;Hara N.;前田正知
• 通讯作者: 前田正知

Na^+/K^+-トランスポーテイングATPase広川タンパク質化学 第4巻 酵素4.3ヒドロラーゼ[III](分担執筆)

Na^+/K^+-转运 ATP 酶 Hirokawa Protein Chemistry Volume 4 Enzyme 4.3 Hydrolase [III]（贡献者）

• 发表时间: 2002
• 作者: Kadota;K. et al.;小林綾子
• 通讯作者: 小林綾子