Functional interaction of 13 yeast SCF complexes with a set of yeast E2 enzymes in vitro.
SCF complexes are multi-subunit ubiquitin ligases that, in concert with the E1 and E2 ubiquitination enzymes, catalyze the ubiquination of specific target proteins. Only three yeast SCFs have been reconstituted and characterized to date; each of these ubiquitinates its target protein with the E2 Cdc34. We have reconstituted and purified 1 known and 12 novel yeast SCF complexes, and explored the ability of these complexes to function with 5 different purified E2 enzymes; Ubc1, Cdc34, Ubc4, Ubc8 and Ubc11. We have found that the ubiquitination of Sic1 by the reconstituted SCF(Cdc4) complex was specifically catalyzed by two of the five E2 enzymes tested in vitro; Cdc34 and Ubc4. We also show that at least eight of the purified SCF complexes clearly ubiquitinated their F-box proteins in vitro, lending support for a regulatory mechanism in which F-box proteins catalyze their own destruction. The autoubiquitination of each F-box was in some cases catalyzed only by Cdc34, and in other cases preferentially catalyzed by Ubc4. Ubc4 thus interacts with multiple SCFs in vitro, and the interactions among SCF and E2 components of the ubiquitination machinery may allow further diversification of the roles of SCFs in vivo.
Kus
Bart M
BM
Banting and Best Department of Medical Research, University of Toronto, Ontario.
Caldon
Catherine E
CE
Andorn-Broza
Ronit
R
Edwards
Aled M
AM
D064173:Anaphase-Promoting Complex-Cyclosome; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D019281:Dimerization; D008025:Ligases / Q000378:metabolism; D046911:Macromolecular Substances; D010766:Phosphorylation; D020836:Protein Structure, Quaternary; D044843:SKP Cullin F-Box Protein Ligases / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000302:isolation & purification / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism
D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D046911:Macromolecular Substances; C086926:SIC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; C577449:CDC34 protein, S cerevisiae; C066752:UBC1 protein, S cerevisiae; C114862:UBC11 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; C063109:ubiquitin-conjugating enzyme UBC4; C409686:ubiquitin-conjugating enzyme UBC8; D064173:Anaphase-Promoting Complex-Cyclosome; D044843:SKP Cullin F-Box Protein Ligases; D008025:Ligases