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

Abstract

A temperature-sensitive growth mutant tsFS20 isolated from mouse FM3A cells was identified as a mutant with thermolabile ubiquitin-activating enzyme El by transfection with a full-length cDNA encoding the human El enzyme and cell-cell hybridization with an authentic El mutant ts85 previously isolated from FM3A cells. The resulting transformants produced thermoresistant El 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 Gl-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 El mutant, reached the G2-phase and were arrested. Thus, the El 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 wild-type level in the transformant cells. Relevance of the unusually high incidence of the temperature-sensitive El mutation was discussed in terms of the El as a determinant of heat tolerance of cells. © 1992, Japan Society for Cell Biology. All rights reserved.