1 Center for Regenerative and Developmental Biology, Department of Biology, Tufts University, Medford, MA, USA, 2The Future of Research (www.futureofresearch.org), Abington, MA, 3Manylabs (www.manylabs.org), San Francisco, CA, USA, 4 Department of Oncology, MRC/Hutchison Research Centre, University of Cambridge, Cambridge Biomedical Campus, Cambridge, UK and 5 Wellcome Trust – Medical Research Council, Cambridge Stem Cell Institute, Cambridge, UK
The small protein modifier, ubiquitin, can be covalently attached to proteins in the process of ubiquitylation, resulting in a variety of functional outcomes. In particular, the most commonly-associated and well-studied fate for proteins modified with ubiquitin is their ultimate destruction: degradation by the 26S proteasome via the ubiquitin-proteasome system, or digestion in lysosomes by proteolytic enzymes. From the earliest days of ubiquitylation research, a reliable and versatile “cell-in-a-test-tube” system has been employed in the form of cytoplasmic extracts from the eggs and embryos of the frog Xenopus laevis. Biochemical studies of ubiquitin and protein degradation using this system have led to significant advances particularly in the study of ubiquitin-mediated proteolysis, while the versatility of Xenopus as a developmental model has allowed investigation of the in vivo consequences of ubiquitylation. Here we describe the use and history of Xenopus extract in the study of ubiquitin-mediated protein degradation, and highlight the versatility of this system that has been exploited to uncover mechanisms and consequences of ubiquitylation and proteolysis.
extract system, ubiquitin, degron, protein degradation, 26S proteasome, Xenopus