The Gcn2 Regulator Yih1 Interacts with the Cyclin Dependent Kinase Cdc28 and Promotes Cell Cycle Progression through G2/M in Budding Yeast
Fig 6
Cdc28 co-precipitates with GST-Yih1.
(A) In vivo GST-pull-down assay. yih1Δ strains (MSY-Y2) expressing GST-Yih1 or GST alone from the galactose inducible promoter were grown to log-phase and harvested. Equal amounts of WCEs (2 mg) were subjected to glutathione-mediated GST pull-down assays. The precipitates (100% of the bound proteins – right-panel) and the input (1/100th of the input – left panel) were assessed by immunoblot to detect the indicated proteins. (B) GST-Yih1 purified from E. coli co-precipitates endogenous Cdc28 from yeast WCEs. Full-length Yih1 fused to GST or GST alone were expressed in E. coli, purified, immobilized on glutathione-Sepharose beads and incubated with equal amounts of glutathione-Sepharose pre-cleared WCEs (1.25 mg) prepared from a yih1Δ strain (MSY-Y2). After extensive washes the precipitates (100% of the bound proteins) and the input (1/50th of the input) were analyzed by immunoblot to detect the indicated proteins. The Ponceau staining of the membrane is shown (lower panel). One representative blot from two independent experiments performed in duplicate is shown.