UBIQUITIN METABOLISM IN CYCLING XENOPUS EGG EXTRACTS

Xenopus egg extract is capable of supporting mitosis in vitro, which m akes it ideal for biochemical analysis of the cell cycle. Since severa l studies have implicated the ubiquitin system in cell cycle progressi on, we have measured ubiquitin conjugation rates, proteolysis of ubiqu itin-lysozyme conjugates, and rates of isopeptidase activity in cyclin g Xenopus egg extracts. Although ubiquitin conjugation in cytostatic f actor arrested extract was half that in activated extract, there were no changes in rates of ubiquitin conjugation during the cell cycle. Ub iquitin conjugates are degraded by a 26 S ATP-stimulated protease. The ability of the 26 S protease to degrade ubiquitin-lysozyme conjugates and a fluorigenic peptide also remained constant across the cell cycl e. In contrast to previously characterized systems, isopeptidase activ ity in Xenopus egg extract is energy-dependent. Glycerol gradient frac tionation of Xenopus egg extract separated two ATP-dependent isopeptid ases. One co-sedimented with the 26 S protease; the other sedimented s lower and was not associated with any additional proteolytic activity. As found for rates of Ub conjugation and conjugate proteolysis, there was little or no variation in isopeptidase activity during the cell c ycle.