Fig. 1.

Regulation of Snf1 Thr210 phosphorylation in strains with altered glycogen metabolism. (A and B) Strains were WT and mutants, as indicated, in the W303 (Left) or BY4741 genetic background (Right); similar results were obtained for all mutants in both genetic backgrounds. (A) Glycogen content was determined. Values are averages of six determinations (bars show SD). (B) Cells were grown in SC plus 2% (H, high) glucose, and an aliquot of the culture was collected by rapid filtration. Another aliquot was collected, resuspended in SC plus 0.05% (L, low) glucose for 10 min, and collected. Extracts were prepared, and proteins (10 μg) were analyzed by immunoblotting with antiphospho-Thr-172-AMPK antibody to detect phosphorylation on Thr210 of Snf1 (pT210). Lower exposure did not reveal differences in phosphorylation in low glucose. Membranes were reprobed with antipolyhistidine antibody to detect Snf1. (C and D) W303-1A cells carrying vector pCM252 or pGSY2 expressing Gsy2-3xHA from the doxycycline-inducible tetO₇ promoter (57) were grown overnight in selective SC plus 2% glucose, diluted in fresh media to an OD₆₀₀ of 0.2 in the presence (+) or the absence (−) of 2 μg/mL doxycycline (dox), and grown for 6 h. Control cultures were treated with the drug vehicle (ethanol). Similar results were obtained with induction times up to 16 h. (C) Glycogen content was determined for induced cultures as above. (D) Aliquots were collected for analysis of phosphorylated Thr210 (pT210) and Snf1. Gsy2-3xHA was detected with 12CA5 antibody.