Complementation by a Cloned Human Ubiquitin-Activating Enzyme E1 of the S-Phase-Arrested Mouse FM3A Cell Mutant with Thermolabile E1

[摘要] References(49)Cited-By(7)A temperature-sensitive growth mutant tsFS20 isolated from mouse FM3A cells was identified as a mutant with thermolabile ubiquitin-activating enzyme E1 by transfection with a full-length cDNA encoding the human E1 enzyme and cell-cell hybridization with an authentic E1 mutant ts85 previously isolated from FM3A cells. The resulting transformants produced thermoresistant E1 activity. Upon shift-up of temperature, asynchronously growing tsFS20 cells showed multiple points of cell-cycle arrest. At the nonpermissive temperature, tsFS20 cells that had been synchronized at the G1-S-phase progressed and accumulated in the mid-S-phase, as evidenced by the absence of G2-specific cdc2 kinase activity, while ts85 mutant cells, the widely used E1 mutant, reached the G2-phase and were arrested. Thus, the E1 mutation seemed to be involved in progression in the S-phase as well as in the G2-phase in the cell cycle. Degradation of short-lived abnormal proteins in tsFS20 cells was decreased to about 50% at the nonpermissive temperature, while the block was fully restored to the wildtype level in the transformant cells. Relevance of the unusually high incidence of the temperature-sensitive E1 mutation was discussed in terms of the E1 as a determinant of heat tolerance of cells.

[学科分类] 分子生物学，细胞生物学和基因

[关键词] protein degradation;cdc2 kinase;temperature-sensitive mutant;DNA transfection;cell cycle