We have isolated a temperature-sensitive alanyl-tRNA synthetase mutant from hamster BHK21 cells, designated as ts ET12. It has a single nucleotide mutation, converting the 321st amino acid residue, 321Gly, to Arg. The mutation was localized between two RNA-binding domains of alanyl-tRNA synthetase. Thus far, we have isolated two temperature-sensitive aminoacyl-tRNA synthetase mutants from the BHK21 cell line: ts BN250 and ts BN269. They are defective in histidyl- and lysyl-tRNA synthetase respectively. Both mutants rapidly undergo apoptosis at the nonpermissive temperature, 39.5°C. ts ET12 cells, however, did not undergo apoptosis until 48 h after a temperature-shift to 39.5°C, while mutated alanyl-tRNA synthetase of ts ET12 cells was lost within 4 h. Loss of the mutated alanyl-tRNA synthetase was inhibited by a ubiquitin-dependent proteasome inhibitor, MG132, and by a protein-synthesis inhibitor, cycloheximide. Cell-cycle related proteins were also lost in ts ET12 cells at 39.5°C, as shown in ts BN250. In contrast, the mutated aminoacyl-tRNA synthetases of ts BN250 and ts BN269 were stable at 39.5°C. However, the defects of these mutants released EMAPII, an inducer of apoptosis at 39.5°C. No release of EMAPII occurred in ts ET12 cells at 39.5°C, consistent with the delay of apoptosis in these cells.
All Science Journal Classification (ASJC) codes
- Molecular Biology