The N-End Rule Pathway inXenopusEgg Extracts

Abstract Ubiquitin-dependent degradation of intracellular proteins underlies a multitude of biological processes, including the cell cycle, cell differentiation, and responses to stress. One ubiquitin-dependent proteolytic system is the N-end rule pathway, whose targets include proteins that bear destabilizing N-terminal residues. This pathway, which has been characterized only in somatic cells, is shown here to be present also in germ line cells such as the eggs of the amphibian Xenopus laevis. We demonstrate that the set of destabilizing residues in the N-end rule pathway of Xenopus eggs is similar, if not identical, to that of somatic cells such as mammalian reticulocytes and fibroblasts. It is also shown that the degradation of engineered N-end rule substrates in egg extracts can be strongly and selectively inhibited by dipeptides bearing destabilizing N-terminal residues. This result allowed us to ask whether selective inhibition of the N-end rule pathway in egg extracts influences the apoptosis-like changes that are observed in these extracts. A dipeptide bearing a bulky hydrophobic (type 2) destabilizing N-terminal residue was found to delay the apoptotic changes in egg extracts, whereas dipeptides bearing basic (type 1) destabilizing N-terminal residues had no effect. High activity of the N-end rule pathway in egg extracts provides an alternative to reticulocyte extracts for the in vitro analyses of this pathway.