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

我们先前发现了一个新的编码Mys蛋白的基因Mys，并且发现几乎所有脊椎动物的基因组中都含有编码Mys蛋白同源基因。在这项研究中，我们制造了识别人类Mys蛋白的羧基末端的抗体，这是脊椎动物Mys蛋白同源物中高度保守的区域。Mys蛋白同源物在人、小鼠培养细胞和非洲爪哇胚胎中均有表达。然后，我们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中，小泡分布在细胞质中。我们通过对人培养细胞进行Flag标记的人Mys蛋白的下拉分析和质谱分析，分离出了与Mys相互作用的Sec23A蛋白。抗Mys抗体和抗Sec23A抗体免疫染色显示Mys和Sec23A在高尔基体共定位。用这两种抗体进行免疫沉淀表明内源性Mys与Sec23A之间存在相互作用。这些结果表明，Mys和Sec23A在高尔基体中的作用是相互协同的。我们目前正在进行击倒实验，并将阐明Mys在蛋白质运输中的作用。

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.