Laboratory of Toxicology, Course of Veterinary Science, Graduate School of Life and Environmental Biosciences, Osaka Prefecture University, Osaka, Japan.
J Cell Biochem. 2011 Sep;112(9):2250-6. doi: 10.1002/jcb.23142.
In our previous study, fluoride (AlF(4) ) disturbed ER-to-Golgi transport through the activation of ER-resident heterotrimeric G protein (ER-G protein). Therefore, ER-G protein may be implicated in ER-to-Golgi transport at the early stage prior to coat protein assembly. Sar1 translocation onto the endoplasmic reticulum (ER) membrane is suppressed by non-selective protein kinase inhibitor H89, suggesting the participation of H89-sensitive kinase in this process. To investigate the involvement of ER-G protein in ER-to-Golgi transport, the effect of G(i) protein activator (mastoparan 7) was examined on Sar1 translocation onto the ER in a cell-free system consisting of microsome membrane and cytosol. Sar1 translocation onto the microsome membrane was induced by addition of GTPγS in the cell-free system. Translocation of Sar1 by GTPγS was suppressed significantly by both H89 and mastoparan 7. Mastoparan 7 suppressed the translocation of Sar1 onto the microsome membrane with dosage dependency, but mastoparan 17, the inactive analog of mastoparan 7, had no effect on Sar1 translocation. The suppressive effect of mastoparan 7 was recovered by treatment with pertussis toxin (IAP). Moreover, G(i2) protein was detected on the microsome membrane by western blotting for heterotrimeric G(i) proteins. These results indicate that ER-G(i2) protein modulated Sar1 translocation onto the ER, suggesting that ER-resident G(i2) protein is an important negative regulator of vesicular transport at the early stage of vesicle formation before coat protein assembly on the ER.
在我们之前的研究中，氟化物 (AlF(4) ) 通过激活内质网驻留的异三聚体 G 蛋白（ER-G 蛋白）干扰内质网到高尔基体的运输。因此，ER-G 蛋白可能在内质网到高尔基体的运输的早期阶段，即在衣壳蛋白组装之前参与其中。非选择性蛋白激酶抑制剂 H89 抑制 Sar1 转位到内质网膜上，表明 H89 敏感激酶参与了这个过程。为了研究 ER-G 蛋白在 ER 到高尔基体运输中的作用，我们在由微粒体膜和细胞质组成的无细胞系统中检查了 G(i) 蛋白激活剂（蜂毒素 7）对 Sar1 转位到内质网的影响。无细胞系统中加入 GTPγS 可诱导 Sar1 转位到微粒体膜上。H89 和蜂毒素 7 均可显著抑制 GTPγS 诱导的 Sar1 转位。蜂毒素 7 以剂量依赖性方式抑制 Sar1 转位到微粒体膜上，但蜂毒素 17，蜂毒素 7 的非活性类似物，对 Sar1 转位没有影响。用百日咳毒素（IAP）处理可恢复蜂毒素 7 的抑制作用。此外，通过 Western blot 检测到异三聚体 G(i) 蛋白在微粒体膜上存在 G(i2) 蛋白。这些结果表明，内质网驻留的 G(i2) 蛋白调节 Sar1 转位到内质网，表明内质网驻留的 G(i2) 蛋白是在衣壳蛋白组装到内质网之前，囊泡形成的早期阶段，对囊泡运输的一个重要负调控因子。
