Regulation of NRG1 and NRG2 RNA levels in response to carbon source. Cultures of wild-type (WT) and snf1Δ strains (MCY4702 and MCY4758) were grown in YEP containing high (2%) glucose (Hi) and were shifted to low (0.05%) glucose (Lo) for 1 h. Wild-type cells were also grown in 2% glycerol-2% ethanol (GE); snf1Δ cells do not grow in a nonfermentable carbon source. RNA was prepared and subjected to Northern blot analysis. Blots were hybridized with probes specific for NRG1, NRG2, and the loading control, U3 RNA. Autoradiograms are shown. The panel at the right shows a shorter exposure for two lanes.

Regulation of Nrg1 and Nrg2 protein levels in response to carbon source. Wild-type (WT) and mutant strains of the indicated genotype (Table ) expressed Nrg1-HA or Nrg2-HA from the chromosomal locus (+) or expressed the wild-type untagged proteins (−). Cultures were grown in YEP containing high (2%) glucose (Hi) and were shifted to low (0.05%) glucose (Lo) for 1 h or were grown in 2% glycerol-2% ethanol (GE). For the experiments shown in panels C and D, the shift to low glucose was for 3 h. Proteins were resolved by SDS-12% polyacrylamide gel electrophoresis, and proteins were blotted and detected with anti-HA antibody 12CA5 (A and B) or Babco HA.11 (C and D). Panel D shows two blots from different experiments. Arrows indicate the Nrg-HA proteins.

Chromatin immunoprecipitation to detect Nrg1-HA and Nrg2-HA binding. Cells expressed Nrg1-HA or Nrg2-HA from the chromosomal locus (MCY4744 and MCY4751, respectively) or expressed native, untagged Nrg1 (MCY4702). (A to C) Cells were grown in YEP containing 2% glucose (Hi). Cells expressing HA-tagged Nrg proteins were also shifted to 0.05% glucose (Lo) for 3 h or were grown in 2% glycerol-2% ethanol (GE). (D) Cells were grown in YEP containing 2% glycerol-2%ethanol and were shifted to 2% glucose for 5 min. DNA was purified from total chromatin (input) and chromatin that was immunoprecipitated with anti-HA (IP). DNA from the input was diluted 1:1,000, and DNA from the immunoprecipitation was used undiluted or diluted 1:10, where indicated. DNA was also used at a 1:3 dilution to confirm linearity (data not shown). Five microliters was used as a template in PCRs with primers specific for the control POL1 coding sequence and two regions of the RPI1 promoter, spanning nucleotides −1610 to −1352 and −1137 to −697 (CCCCT sequences are at positions −1571, −1086, and −731) or the CYC7 promoter (−480 to −188).