Soonthornsit Jeerawat, Sakai Noriko, Sasaki Yurika, Watanabe Ryota, Osako Shiho, Nakamura Nobuhiro
Division of Engineering, Graduate School, Kyoto Sangyo University, Motoyama, Kamigamo, Kita, Kyoto 603-8555, Japan; Department of Pre-clinic and Applied Animal Science, Faculty of Veterinary Science, Mahidol University, 999 Phutthamonthon Sai 4 Road Salaya, Phutthamonthon, Nakhon Pathom 73170 Thailand.
Graduate School of Natural Science and Technology and School of Pharmacy, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan.
Exp Cell Res. 2017 Apr 15;353(2):100-108. doi: 10.1016/j.yexcr.2017.03.011. Epub 2017 Mar 9.
In this study, we attempted to explore the function of three uncharacterized mammalian homologs of yeast Yip domain family proteins-YIPF6, a homolog of Yip1p, and YIPF1 and YIPF2, which are homologs of Yif1p. Immunofluorescence staining revealed that YIPF1, YIPF2, and YIPF6 mainly localize in the medial-/trans-Golgi and also partially in the trans-Golgi network (TGN). On treatment with brefeldin A (BFA), the homologs co-migrated partly with medial-/trans-Golgi markers and also with a TGN marker in earlier time point, but finally redistributed within cytoplasmic punctate structures that were distinct from medial-/trans-Golgi and the TGN markers. YIPF6 formed a stable complex separately with YIPF1 and YIPF2, and knockdown of YIPF6 reduced YIPF1 and YIPF2 levels. These results suggest that YIPF6 forms complexes with YIPF1 and YIPF2 for their stable expression and localization within the Golgi apparatus. Knockdown experiments showed that YIPF1 and YIPF2, by contrast, are not necessary for the expression and localization of YIPF6. The structure of the Golgi apparatus and its disassembly after BFA treatment were not significantly affected by the knockdown of YIPF1, YIPF2, or YIPF6. However, reassembly of the Golgi apparatus after the removal of BFA was markedly delayed by the knockdown of YIPF1 and YIPF2, but not by that of YIPF6. These results strongly suggest that free YIPF6 after disassociating with YIPF1 and YIPF2 interferes with the reassembly of the Golgi apparatus. Knockdown of YIPF1 and YIPF2, but not that of YIPF6, also reduced intracellular glycans in HT-29 cells. Thus, we confirmed that YIPF1, YIPF2, and YIPF6 play a significant role in supporting normal glycan synthesis.
在本研究中，我们试图探究酵母Yip结构域家族蛋白的三个未被表征的哺乳动物同源物——YIPF6（Yip1p的同源物）以及YIPF1和YIPF2（Yif1p的同源物）的功能。免疫荧光染色显示，YIPF1、YIPF2和YIPF6主要定位于高尔基体中间/反式面，也部分定位于反式高尔基体网络（TGN）。用布雷菲德菌素A（BFA）处理后，这些同源物在较早时间点部分与高尔基体中间/反式面标记物以及一个TGN标记物共迁移，但最终在与高尔基体中间/反式面和TGN标记物不同的细胞质点状结构中重新分布。YIPF6分别与YIPF1和YIPF2形成稳定复合物，敲低YIPF6会降低YIPF1和YIPF2的水平。这些结果表明，YIPF6与YIPF1和YIPF2形成复合物，以使其在高尔基体中稳定表达和定位。相反，敲低实验表明，YIPF1和YIPF2对于YIPF6的表达和定位并非必需。敲低YIPF1、YIPF2或YIPF6对高尔基体的结构及其在BFA处理后的解体没有显著影响。然而，去除BFA后高尔基体的重新组装在敲低YIPF1和YIPF2时明显延迟，但敲低YIPF6时则没有。这些结果强烈表明，YIPF6与YIPF1和YIPF2解离后的游离形式会干扰高尔基体的重新组装。敲低YIPF1和YIPF2而非YIPF6也会降低HT - 29细胞中的细胞内聚糖。因此，我们证实YIPF1、YIPF2和YIPF6在支持正常聚糖合成中发挥重要作用。
