FIG 2.

Psy2 promotes HXT gene repression in the absence of glucose. (A) The phosphorylation state of Rgt1 was analyzed by immunoblotting using wild-type (WT) and psy2Δ mutant cells grown in Gal-containing medium and shifted to Glc-containing medium. The position of unphosphorylated Rgt1 is indicated by a line, whereas the lines with asterisks denote the most highly phosphorylated species of Rgt1. The membrane was stained for protein prior to development for the loading control. (B) Similar analysis was performed with WT and psy2Δ mutant cells shifted from Glc- to Gal-containing medium for the time points indicated. An immunoblot with anti-PSTAIRE antibody was used for the loading control. (C) Quantitative RT-PCR analysis of HXT3 RNA with samples collected as described in panel B at the indicated time points. (D) Chromatin immunoprecipitation assay was performed using wild-type (WT) and psy2Δ mutant cells treated as in panel B, after cross-linking and immunoprecipitation of the Rgt1-HA-DNA complex using anti-HA monoclonal antibodies. The qPCR data represent the average of HXT3 RNA samples analyzed in triplicate and normalized to ACT1 RNA. HXT3 RNA is plotted relative to the wild-type time zero sample.