Figure 7

Dephosphorylation of Mig1 and Snf1 upon overexpression of Ppz1. (a) Left panel. Changes in phosphorylation of Mig1 at residues Ser311, 314 derived from the phosphoproteomic experiments. Data represent the mean ± SEM from 4 experiments. Right panel. Cells expressing a HA-tagged version of Mig1 were exposed to Ppz1 inducing conditions and the mobility of Mig1 monitored by SDS-PAGE (10% polyacrylamide) of protein extracts (40 µg of proteins) followed by immunoblot with anti-HA antibodies. Ponceau staining of the membrane is shown for comparison of loading and transfer efficiency. (b) Wild type (BY4741) and ZCZ01 cells were transformed with an episomal plasmid bearing a GFP-tagged version of Mig1. Cultures were treated for Ppz1 induction as described in Material and Methods. Cells were collected at different times, stained with DAPI to reveal the position of the nucleus, and observed in a fluorescence microscope. The graph on the right shows the abundance of cells (as %) in which Mig1 was retained in the nucleus. Data are the mean ± SEM (n = 3) from an average of 167 to 426 cells counted per experiment. (c) Changes in Snf1 phosphorylation induced by Ppz1 overexpression. Left panel. BY4741 (WT) and ZCZ01 cells were treated as in panel A and protein extracts electrophoresed, transferred to membranes and probed with anti-Ppz1, anti-AMPK-P^T172 (Snf1-P), or anti-polyHis (Snf1) antibodies. Right panel. Quantification of the P-Snf1/Snf1 ratio by integration of the signals from three independent experiments. The result for the wild type strain at time = 0 is defined as the unit and the mean ± SEM is represented.