Generation of Mad3 phosphorylation mutants

Phosphorylation mutants were created from pJK-Mad3, which is based on the vector pJK148 [73]. It contains 500 bp of 5’UTR, followed by the mad3 ORF and 500 bp of 3’UTR. All mad3 strains were generated by integration of pJK-Mad3 vectors into a mad3Δ strain at the endogenous mad3 locus. Mad3 gene synthesis (GeneArt) was used to generate the 18A allele (see Fig 1). The C9A and N9A alleles were created from the 18A vector by sub-cloning, using an Xho1 site present in the Mad3 gene. All other point mutations were introduced using the Quikchange Kit for Site Directed Mutagenesis (Stratagene). For the APC/C binding assays C-terminal GFP tags were introduced using the Bahler cassette system (Bahler et al. 1998)

Recombinant wild-type (wt) and phospho-mimic Mad3 mutants as well as the closed Mad2 mutant (cMad2 [39]) used for the APC/C activity assays were constructed via Gateway cloning using the donor vector pDONR201 (Life technologies) and the destination vector pHMGWA described previously [74] containing 6xHis and Maltose Binding Protein (MBP) tags. Proteins were expressed in BL21 RIL cells with 0.25 mM IPTG at 18°C for 16hrs. Cells were harvested by centrifugation and pellets were frozen at -20°C until further use. Cells were lysed by sonication in lysis buffer A containing 50 mM potassium phosphate pH 7.0, 300 mM NaCl, 5% glycerol, 1mM DTT and 1% glucose supplemented with 1 mM Pefabloc (SIGMA Aldrich) and a cocktail protease inhibitor tablet (Roche). cMad2 and Mad3 proteins were affinity purified using amylose beads (NEB) and eluted with buffer A containing 15 mM maltose. Eluted fractions were pooled together and further purified by size exclusion chromatography using a Superdex S200 column (GE Healthcare) either in buffer B (for cMad2) containing 25 mM HEPES pH 7.5, 150 mM NaCl, or in buffer C (for Mad3 proteins) containing 25 mM potassium phosphate pH 7.0, 200 mM NaCl, 5% glycerol, 1mM DTT, 1% glucose and 1 mg/ml NVOY polymer (Expedeon). Protein purity was visualised by SDS-PAGE.