Ubiquitin metabolism in cycling Xenopus egg extracts

Xenopus egg extract is capable of supporting mitosis in vitro, which makes it ideal for biochemical analysis of the cell cycle. Since several studies have implicated the ubiquitin system in cell cycle progression, we have measured ubiquitin conjugation rates, proteolysis of ubiquitin-lysozyme conjugates, and rates of isopeptidase activity in cycling Xenopus egg extracts. Although ubiquitin conjugation in cytostatic factor arrested extract was half that in activated extract, there were no changes in rates of ubiquitin conjugation during the cell cycle. Ubiquitin 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 cycle. In contrast to previously characterized systems, isopeptidase activity in Xenopus egg extract is energy-dependent. Glycerol gradient fractionation of Xenopus egg extract separated two ATP-dependent isopeptidases. On co-sedimented with the 26 S protease; the other sedimented slower 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 cycle.