MOLECULAR-CLONING OF A NOVEL MEMBER OF THE EUKARYOTIC POLYPEPTIDE CHAIN-RELEASING FACTORS (ERF) - ITS IDENTIFICATION AS ERF3 INTERACTING WITH ERF1

Yeast GST1 gene, whose product is a GTP-binding protein structurally r elated to polypeptide chain elongation factor-1 alpha (EF1 alpha), was first described to be essential for the (G) under bar(1) to (S) under bar phase transition (GSPT) of the cell cycle, and the product was re cently reported to function as a polypeptide chain release factor 3 (e RF3) in yeast. Although we previously cloned a human homologue (rename d as GSPT1) of the yeast gene, it has remained to be determined whethe r GSPT1 also functions as eRF3 or if another GSPT may have such a func tion in mammalian cells. In the present study, we isolated two mouse G SPT genes, the counterpart of human GSPT1 and a novel member of the GS PT gene family, GSPT2. Both the mouse GSPTs had a two domain structure characterized as an amino-terminal no-homologous region (approximatel y 200 amino acids) and a carboxyl-terminal conserved eukaryotic elonga tion factor-1 alpha-like domain (428 amino acids), Messenger RNAs of t he 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 mann er in Swiss 3T3 cells, whereas the expression of GSPT2 was constant du ring the cell-cycle progression. Immunoprecipitation assays in COS-7 c ells expressing flag epitope-tagged proteins demonstrated that not onl y GSPT1 but also GSPT2 was capable of interacting with eRF1. Such inte raction between GSPT2 and eRF1 was also confirmed by yeast two-hybrid analysis. Taken together, these data indicated that the novel GSPT2 ma y interact with eRF1 to function as eRF3 in mammalian cells.