Figure S4.

Combinatorial effects of SUMOylation on pericentromeric proteins. (A) Sgo1-4RΔdb showed reduced SUMOylation compared with Sgo1-Δdb. SUMO pull-down was performed for sgo1-Δdb-9MYC (AMy29896) and sgo1-4R-Δdb-9MYC (AMy29996). (B) sgo1-4RΔdb showed a metaphase delay similar to sgo1-4R. Strains imaged contained CDC14-GFP and YFP-TUB1 and were wild type (AMy24174), sgo1-4R (AMy29305), and sgo1-4RΔdb (AMy30024). Shown are the average of three to four independent experiments, and error bars represent standard error. Statistics: one-tailed Student’s t test (*, P < 0.05; **, P < 0.01). IB, immunoblot. (C) Components of condensin and CPC complex are SUMOylated in metaphase, but Rts1 and Mps1 are not SUMOylated in metaphase. Strains used carried pMET-CDC20 and were transformed with His-SMT3 or empty vector: BRN1-6HA (AMy8955), YCS4-6HA (AMy8953), IPL1-6HA (AMy6937), BIR1-6HA (AMy6941), RTS1-13MYC (AMy8951), and MPS1-6HA (AMy7450). Vector controls were harvested as cycling cells (Cyc). HIS-SMT3 transformants were metaphase arrested in the presence or absence of tension. SUMO pull-down was performed as described in Materials and methods. (D) bir1-3R and sgo1-4RΔdb single- and double-mutant cells grew similarly to wild type or with mildly improved growth on benomyl. Serially diluted cells of wild type (AMy1176), sgo1Δ (AMy827), sgo1-4R (AMy21705), sgo1-4RΔdb (AMy29901), bir1-3R (AM29717), bir1-3R sgo1-4R (AM29735), and bir1-3R sgo1-4RΔdb (AMy30060) were plated on medium containing 10 µg/ml benomyl or DMSO (solvent).