In vitro reconstitution of SCF substrate ubiquitination with purified proteins.
The development of in vitro systems to monitor ubiquitin ligase activity with highly purified proteins has allowed for new insights into the mechanisms of protein ubiquitination to be uncovered. This chapter describes the methodologies employed to reconstitute ubiquitination of the budding yeast cyclin-dependent kinase inhibitor Sic1 by the evolutionarily conserved ubiquitin ligase SCF(Cdc4) and its ubiquitin-conjugating enzyme Cdc34. Based on our experience in reconstituting Sic1 ubiquitination, we suggest some parameters to consider that should be generally applicable to the study of different SCF complexes and other ubiquitin ligases.
Petroski
Matthew D
MD
Howard Hughes Medical Institute, Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
Deshaies
Raymond J
RJ
D064173:Anaphase-Promoting Complex-Cyclosome; D018797:Cell Cycle Proteins / Q000302:isolation & purification / Q000378:metabolism*; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D004926:Escherichia coli / Q000235:genetics; D044783:F-Box Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000302:isolation & purification / Q000378:metabolism*
C080443:CDC4 protein, S cerevisiae; D018797:Cell Cycle Proteins; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D044783:F-Box Proteins; C086926:SIC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; C577449:CDC34 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases