Functional interaction of 13 yeast SCF complexes with a set of yeast E2 enzymes in vitro

Abstract

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.

The F-Box Protein Dia2 Regulates DNA Replication

“…It has previously been shown that recombinant Dia2 can assemble with SCF components (Kus et al, 2004) and that overexpressed Dia2 assembles with Skp1 and Cdc53 in yeast cells (Ho et al, 2002). We also observed Dia2 assembly with SCF components using an HA 3 -tagged Dia2 baculovirus for expression in insect cells.…”

Nonredundant Roles of Mitochondria-associated F-Box Proteins Mfb1 and Mdm30 in Maintenance of Mitochondrial Morphology in Yeast

“…What might be the role of Mfb1 and Mdm30 in mediating turnover of Fzo1 and other mitochondrial target proteins? Mdm30 has been shown to interact with Skp1 and Cdc53 in the yeast two-hybrid system (Uetz et al, 2000), recombinant Mdm30 assembles with Skp1, Cdc53, and Rbx1 into an SCF complex in vitro (Kus et al, 2004), and Mfb1 has been found in a complex with Skp1 in large-scale identification of yeast protein complexes by mass spectrometry (Ho et al, 2002) and in coimmunoprecipitation experiments (Kondo-Okamoto et al, 2006). However, Mfb1-Skp1 complexes apparently do not contain Cdc53 (Kondo-Okamoto et al, 2006).…”

Yeast Chfr homologs retard cell cycle at G1and G2/M via Ubc4 and Ubc13/Mms2-dependent ubiquitination

“…Plasmids containing the constructs for Histagged Ubc4 and Ubc11 were kindly provided by Aled Edwards. 22 Plasmid pC105 was constructed by amplifying wild-type CHF2 with added PstI and XhoI sites for insertion into pRS425GAL1. Plasmid pC110 was generated by amplifying the yeast MMS2 gene from a yeast cDNA library with NdeI and XhoI sites for insertion into plasmid pSGA04, 50 which provides an N-terminal His tag.…”

“…The enzymes were purified as described 22 with minor modifications. Cell pellets were resuspended in 20 mM HEPES, pH 8.0, 500 mM NaCl, 10% glycerol, 10 μM ZnCl 2 , 5 mM imidazole, 0.5 mM Tris (2-carboxyethyl) phosphine hydrochloride, and protease inhibitor tablets (Roche), and then lysed by sonication.…”

“…4,5,20 Biochemical analysis indicates that individual E2 enzymes can functionally pair with multiple E3 ligases 21 while several purified E3 ligases function in vitro with multiple E2 conjugating enzymes. 22 However, the apparent promiscuity of in vitro reactions casts doubt on reactions that have not been validated by genetic analysis.…”