Fig. 9.

Nuclear localization of Gal83 isoform is required for alkaline stress response. (A) Coomassie stained gel of kinase substrates purified from bacteria. (B) In vitro kinase assay of the different Snf1 isoforms incubated with [γ-³²P]ATP and GST, GST-Mig1 or GST-Mig2 as the substrates. Sak1 kinase was present in all reactions as indicated. (C) Western blot of HA-tagged Mig2 before and 10 minutes after alkaline stress in wild type cells (WT) and cells lacking one of the three Snf1-activating kinases. The mobility of Mig2 and phosphorylated Mig2 (Mig2-P) is indicated. Ratio of phospho-Mig2 over total Mig2 is plotted on the right. (D) Localization of Gal83-GFP before and after alkaline stress in wild type cells and cells lacking Sak1. Position of the vacuoles was determined by staining with CMAC. White arrows indicate the fluorescent signal is excluded from the nucleus and red arrows indicate the fluorescent is enriched in the nucleus.