Hoshino S, Imai M, Mizutani M, Kikuchi Y, Hanaoka F, Ui M, Katada T
Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan.
J Biol Chem. 1998 Aug 28;273(35):22254-9. doi: 10.1074/jbc.273.35.22254.
Yeast GST1 gene, whose product is a GTP-binding protein structurally related to polypeptide chain elongation factor-1alpha (EF1alpha), was first described to be essential for the G1 to S phase transition (GSPT) of the cell cycle, and the product was recently reported to function as a polypeptide chain release factor 3 (eRF3) in yeast. Although we previously cloned a human homologue (renamed as GSPT1) of the yeast gene, it has remained to be determined whether GSPT1 also functions as eRF3 or if another GSPT may have such a function in mammalian cells. In the present study, we isolated two mouse GSPT genes, the counterpart of human GSPT1 and a novel member of the GSPT gene family, GSPT2. Both the mouse GSPTs had a two-domain structure characterized as an amino-terminal no-homologous region (approximately 200 amino acids) and a carboxyl-terminal conserved eukaryotic elongation factor-1alpha-like domain (428 amino acids). Messenger RNAs of the two GSPTs could be detected in all mouse tissues surveyed, although the level of GSPT2 message appeared to be relatively abundant in the brain. The mouse GSPT1 was expressed in a proliferation-dependent manner in Swiss 3T3 cells, whereas the expression of GSPT2 was constant during the cell-cycle progression. Immunoprecipitation assays in COS-7 cells expressing flag epitope-tagged proteins demonstrated that not only GSPT1 but also GSPT2 was capable of interacting with eRF1. Such interaction between GSPT2 and eRF1 was also confirmed by yeast two-hybrid analysis. Taken together, these data indicated that the novel GSPT2 may interact with eRF1 to function as eRF3 in mammalian cells.
酵母GST1基因的产物是一种与多肽链延伸因子-1α（EF1α）结构相关的GTP结合蛋白，最初被描述为对细胞周期的G1到S期转变（GSPT）至关重要，最近有报道称该产物在酵母中作为多肽链释放因子3（eRF3）发挥作用。尽管我们之前克隆了该酵母基因的人类同源物（重新命名为GSPT1），但GSPT1是否也作为eRF3发挥作用，或者是否有另一种GSPT在哺乳动物细胞中具有这种功能，仍有待确定。在本研究中，我们分离出了两个小鼠GSPT基因，即人类GSPT1的对应物以及GSPT基因家族的一个新成员GSPT2。两种小鼠GSPT均具有双结构域结构，其特征为氨基末端无同源区域（约200个氨基酸）和羧基末端保守的真核延伸因子-1α样结构域（428个氨基酸）。在所检测的所有小鼠组织中均可检测到两种GSPT的信使RNA，尽管GSPT2信使RNA的水平在脑中似乎相对丰富。小鼠GSPT1在瑞士3T3细胞中以增殖依赖的方式表达，而GSPT2的表达在细胞周期进程中保持恒定。在表达带有flag表位标签蛋白的COS-7细胞中进行的免疫沉淀分析表明，不仅GSPT1，而且GSPT2都能够与eRF1相互作用。酵母双杂交分析也证实了GSPT2与eRF1之间的这种相互作用。综上所述，这些数据表明新的GSPT2可能与eRF1相互作用，在哺乳动物细胞中作为eRF3发挥作用。
