FIGURE 4.

Met32-1 is inactive as a transcription factor but does not affect Met4 deubiquitination. A, met31 met32 double mutants expressing the proteins indicated under control of the GAL1 promoter were grown at 30 °C in galactose-containing media with repressive amounts of methionine (1 mm). Cells were collected by filtration, washed with prewarmed galactose medium lacking methionine (SC-Gal/-Met), and resuspended in SC-Gal/-Met medium to induce expression of MET genes. Cells were collected 15, 30, and 60 min after induction and expression levels of the three MET genes indicated were analyzed by RT-qPCR. Expression levels are represented normalized to ACT1 expression. The highest expression level was arbitrarily set to 100%. B, cells as described for A were grown in SC-Gal/+Met medium, collected by filtration, washed, and resuspended in SD-Gal/-Met medium to activate Met4. Cell lysates were prepared after incubation in SD-Gal/-Met for the time indicated. Total cell lysates were separated by SDS-PAGE and analyzed by immunoblotting using polyclonal antibodies directed against Met4. The proteasome subunit Rpt1 was detected as a loading control. Met4 activation is evident by loss of ubiquitinated forms and the appearance of phosphorylated Met4.