Fig. 2. Met4p is destabilized specifically in repressive growth conditions. (A) Cells carrying a plasmid coding for the indicated HA–tagged proteins under the regulation of the GAL1 promoter were grown in raffinose and expression was induced by resuspending the cells in fresh galactose-containing medium (2% galactose) for 90 min. Cultures were then divided in two, filtered, transferred to glucose-containing medium in the presence or absence of 1 mM l–methionine, and samples taken at the times indicated and immunoblotted with anti-HA antibodies. The non-specific band revealed by anti-HA antibodies (indicated by an arrow in the case of Met4p stability determination) was used as a control of the amount of loaded extracts in each experiment (T = extract of cells expressing no HA-tagged protein). (B and C) Total RNA was extracted from cells expressing either the HA-Met4 or the HA-Met28 fusion proteins grown as in (A) and analyzed with MET4, MET16, MET25 and MET28 probes. (D) Induction of Met4p modification was followed by first growing the cells in raffinose-containing medium. Cells were then transferred to galactose-containing medium (2% galactose) to induce GAL1–HA-MET4 expression, and samples were taken at the times indicated.