The mammalian GTP-binding protein GSPT, whose carboxyl-terminal sequence is homologous to the eukaryotic elongation factor EF1␣, binds to the polypeptide chain releasing factor eRF1 to function as eRF3 in the translation termination. The amino-terminal domain of GSPT was, however, not required for the binding. Search for other GSPT-binding proteins in yeast two-hybrid screening system resulted in the identification of a cDNA encoding polyadenylate-binding protein (PABP), whose amino terminus is associating with the poly(A) tail of mRNAs presumably for their stabilization. The interaction appeared to be mediated through the carboxyl-terminal domain of PABP and the aminoterminal region of GSPT. Interestingly, multimerization of PABP with poly(A), which is ascribed to the action of its carboxyl-terminal domain, was completely inhibited by the interaction with the amino-terminal domain of GSPT. These results indicate that GSPT/eRF3 may play important roles not only in the termination of protein synthesis but also in the regulation of mRNA stability. Thus, the present study is the first report showing that GSPT/eRF3 carries the translation termination signal to 3-poly(A) tail ubiquitously present in eukaryotic mRNAs.The yeast GSPT gene, whose product is a GTP-binding protein structurally related to the translation elongation factor EF1␣, was first isolated based on the ability to complement a temperature-sensitive gst1 mutation of Saccharomyces cerevisiae (1). Because DNA synthesis was substantially arrested at the nonpermissive temperature in this mutant, the GSPT gene product appears to play an essential role at the G 1 to S phase transition in the yeast cell cycle. On the other hand, SUP35 was cloned by another group investigating omnipotent suppressor mutant of S. cerevisiae, and the gene turned out to be identical to GSPT (2). Omnipotent suppressor is a class of nonsense suppressors that is recessive and effective toward three types of nonsense codons (3). Mutations in the GSPT/ SUP35 gene were shown to increase the level of translational ambiguity (4, 5), suggesting that this gene product may also function as a positive regulator of translational accuracy in yeast.In eukaryotic protein synthesis, all three types of termination codons are directly recognized by a polypeptide chain releasing factor, eRF1, to release synthesized polypeptide chain from ribosome, and another releasing factor, eRF3, appears to be essentially required for the ribosomal binding of eRF1. Recently, it was reported in S. cerevisiae (6) and Xenopus laevis (7) that the product of the GSPT/SUP35 gene forms a complex with eRF1 to function as eRF3. We previously cloned a human homologue of the yeast GSPT gene (8) and more recently isolated two mouse GSPT genes, the counterpart of human GSPT1 and a novel member of the GSPT gene family, GSPT2 (9). The mammalian GSPT1 and GSPT2 could also associate with eRF1 to function as eRF3, although the two GSPTs were clearly distinct from each other in terms of their amino-terminal sequences, the expression...

Yeast GST1 gene, whose product is a GTP-binding protein structurally related to polypeptide chain elongation factor-1␣ (EF1␣), was first described to be essential for the G 1 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-1␣-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.

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