RESCUE OF THE COMPLEX TEMPERATURE-SENSITIVE PHENOTYPE OF CHINESE-HAMSTER OVARY E36TS20 CELLS BY EXPRESSION OF THE HUMAN UBIQUITIN-ACTIVATING ENZYME CDNA

The ubiquitin conjugation system is a multi-step pathway in which ubiq uitin is activated and conjugated to acceptor proteins, one function o f which is to target acceptor proteins for rapid degradation within th e cell. The conjugation system is involved in many aspects of cellular functions, including the cell cycle. Several cell-cycle arrest mutant cell lines have been characterized and appear to harbour a mutant ubi quitin-activating enzyme, E1, as their primary defect. One such cell l ine is ts20, which is derived from Chinese hamster ovary E36 cells. Th is cell line has been used to characterize some of the potential funct ions of the ubiquitin conjugation system in vivo, such as its involvem ent in the maturation of autophagic vacuoles. The present study descri bes the complete rescue of the complex ts20 phenotype following the ex pression of the cDNA for human E1. Stable transfectants expressing the human E1 cDNA in the CMVneo expression vector were measured for ubiqu itin-conjugation activity, protein degradation and growth in culture a t the nonpermissive temperature. This rescue confirms that the phenoty pe observed in the ts20 cells is due to a defect in the E1 enzyme. Thu s, the ts20 cell line will serve as a useful tool to delineate the fun ctions of the ubiquitin system in vivo.