Elucidation of novel molecular mechanisms in protein transportation from ER to Golgi apparatus and their roles in development

阐明蛋白质从内质网运输到高尔基体的新分子机制及其在发育中的作用

• 批准号: 21770066
• 负责人: KOTANI Tomoya
• 金额: $ 2.91万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Young Scientists (B)
• 财政年份: 2009
• 资助国家: 日本
• 起止时间: 2009 至 2010
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-21770066/
• 关键词: 蛋白質; 細胞・組織; 蛋白質相互作用; 小胞輸送; 遺伝子発現阻害; Mys蛋白質; 特異的抗体; 蛋白質間相互作用

We previously identified a novel gene misty somites (mys), which encodes Mys protein, and found that almost all vertebrate genomes contain the gene encoding Mys protein homolog. In this study, we newly produced antibodies recognizing carboxy-terminus of human Mys protein, a region highly conserved in vertebrate Mys protein homologs. Mys protein homologs were found to be expressed in human and mouse cultured cells and Xenopus embryos. We then examined the localization of Mys protein in human cultured cells using the newly produced antibody and found that Mys protein was localized in Golgi apparatus and vesicles distributed throughout cytoplasm. We isolated Sec23A protein as a protein interacting with Mys by performing a pull-down assay with Flag-tagged human Mys protein in human cultured cells followed by mass spectrometry analysis. Immunostaining using anti Mys antibody and anti-Sec23A antibody showed co-localization of Mys and Sec23A in Golgi apparatus. Immunoprecipitation using these two antibodies showed the interaction of endogenous Mys and Sec23A. These results suggest that Mys and Sec23A function in Golgi apparatus in cooperation with each other. We are currently performing knock-down experiences and will elucidate roles of Mys in protein transportation.

我们以前发现了一个新的基因misty somites（myss），它编码的Mys蛋白，并发现几乎所有的脊椎动物基因组中含有编码Mys蛋白同源物的基因。在这项研究中，我们新产生的抗体识别人类Mys蛋白的羧基末端，一个高度保守的脊椎动物Mys蛋白同源物的区域。发现Mys蛋白同源物在人类和小鼠培养细胞和爪蟾胚胎中表达。然后，我们使用新产生的抗体检测了Mys蛋白在人培养细胞中的定位，发现Mys蛋白定位于高尔基体和分布在整个细胞质中的囊泡。我们通过在人培养细胞中用Flag标记的人Mys蛋白进行下拉测定，随后进行质谱分析，分离出Sec23A蛋白作为与Mys相互作用的蛋白。用抗Mys抗体和抗Sec23A抗体进行免疫组化染色，结果显示Mys和Sec23A共定位于高尔基体。使用这两种抗体的免疫沉淀显示内源性Mys和Sec23A的相互作用。这些结果表明，Mys和Sec23A在高尔基体中的功能相互合作。我们目前正在进行敲除实验，并将阐明Mys在蛋白质转运中的作用。

项目成果

蛋白質間相互作用による新規プロテインキナーゼA活性化機構

蛋白质-蛋白质相互作用的新型蛋白激酶 A 激活机制

• 发表时间: 2009
• 作者: 小谷友也;家村俊一郎;夏目徹;川上浩一;山下正兼
• 通讯作者: 山下正兼

Mys protein antagonizes Hedgehog signaling through activation of PKA during embryonic development

Mys 蛋白在胚胎发育过程中通过激活 PKA 来拮抗 Hedgehog 信号传导

• 发表时间: 2009
• 作者: Tomoya Kotani;Shun-ichiro Iemura;Tohru Natsume;Koichi Kawakami;Masakane Yamashita
• 通讯作者: Masakane Yamashita

Mys Protein Regulates Protein Kinase A Activity by Interacting with Regulatory Type IαSubunit during Vertebrate Development.

Mys 蛋白在脊椎动物发育过程中通过与调节型 Iα 亚基相互作用来调节蛋白激酶 A 活性。

• 发表时间: 2010
• 期刊: The Journal of Biological Chemistry 285(in press)
• 作者: Kotani;T.;Iemura;S.I.;Natsume;T.;Kawakami;K.;Yamashita;M.
• 通讯作者: M.