FIG. 3.

Protein levels, subcellular localization, and Snf1 catalytic activity for Gal83W184A,R214Q. (A) Protein extracts were prepared from cultures of MCY4626 expressing Gal83, Gal83W184A,R214Q, or no protein (vector) at the 9-h time point (OD₆₀₀ = 0.6) of the experiment depicted in Fig. 2D. Immunoblot analysis was carried out, and proteins were detected with anti-GFP antibody. (B) Strain MCY4622 (gal83Δ) expressing GFP-tagged Gal83W184A,R214Q (pHW39) was grown to mid-log phase in SD plus 2% glucose (2% Glu) and shifted to 0.1% glucose (0.1% Glu) or 2% glycerol plus 3% ethanol (Gly+Eth) for 20 min. GFP was visualized by fluorescence microscopy. Arrows indicate representative nuclei identified by DAPI staining. (C) Immunoprecipitation kinase assay. Extracts were prepared from strain MCY4622 (gal83Δ) expressing GFP-tagged Gal83 or Gal83W184A,R214Q (pRT13 or pHW40) and LexA-tagged Snf1 or Snf1T210A (pRJ55 and pRJ217). Proteins were immunoprecipitated with anti-LexA, incubated in kinase buffer containing [γ-³²P]ATP, and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. The protein marker is in kilodaltons. Levels of Snf1 and Gal83 were comparable by immunoblotting (data not shown).