SCFPop1p-Pop2p mediated Rum1p ubiquitylation in vitro (2A) Various Cdc2p complexes were affinity purified from cells co-expressing 6 × His-Myc-tagged Cdc2p and HA-tagged cyclins on nickel NTA resin. Kinase complexes were incubated in the presence of ATP with Rum1p produced by combined in vitro transcription/translation in rabbit reticulocyte lysate (lanes denoted with "+"). Reactions only containing buffer were used as negative controls (lanes denoted "-"). Reaction products were separated on SDS gels and detected by fluorography (left panel). Both, Cdc2p/Cdc13p and Cdc2p/Cig1p produced a shift in Rum1p migration indicative of phosphorylation, although it is unclear whether both kinases target the same residues . In a confirmatory experiment, bacterially expressed Rum1p was purified to apparent homogeneity by column chromatography as described  (middle panel), and phosphorylated by incubation with Cdc2p/Cig1p complexes in the presence of radiolabeled ATP (right panel). Reaction products were separated by SDS gel electrophoresis and detected by autoradiography. (2B) Phosphorylated Rum1p interacts with Pop1p-Pop2p complexes. 6 × His-Myc-tagged Pop2p and HA-Pop1p were co-overexpressed in wild-type fission yeast, and Pop1p-Pop2p complexes were affinity-purified on Ni-NTA resin. Bound complexes were incubated with bacterially expressed phosphorylated Rum1p. After the incubation, beads were washed extensively, followed by boiling in SDS sample buffer and electrophoresis. Rum1p bound to Pop1p-Pop2p was visualized by autoradiography (lane 1, top panel). As controls, 6 × His-tagged Pop1p or Pop2p individually overexpressed in pop1 pop2 double mutants were employed in the Rum1p binding assay (lanes 2, and 3; top panel). As a further negative control, lysate of the untransformed pop1 pop2 double mutant was absorbed to Ni-NTA beads (lane 4, top panel). The bottom panel shows the affinity-purified Pop1p and Pop2p complexes used in the binding assay. (2C) 6 × His-tagged human E1 and UBC3 were expressed in bacteria and purified by affinity binding to nickel NTA resin, followed by subsequent chromatographic steps (MonoQ and gel filtration). The preparations used in the ubiquitylation assays are shown. (2D) Rum1p ubiquitylation assay. Bacterially expressed, phosphorylated Rum1p, derived from the preparation shown in Fig. 2A was incubated with Pip1p complexes immunopurified from the indicated strains, human E1, UBC3 (hUBC3), ATP, and ubiquitin for 90 min at 30°C. Reactions were stopped by the addition of SDS sample buffer, and reaction products were separated by SDS PAGE and visualized by autoradiography. Lanes 2, 4, and 6 included mutant UBC3 in which the catalytic cysteine residue was replaced (UBC3-ΔCys). Mutant ubiquitin, in which all lysine residues were replaced by arginine was present in lane 7. The assays presented in the right panel (lanes 8 – 11) were performed with Pip1p complexes purified from csn5 mutants and contained the indicated human or S. pombe UBCs.