FIG. 5.

Snf1 is activated by phosphorylation on threonine-210 after transfer from glucose to galactose medium but not after HU treatment. snf1Δ cells transformed with a centromeric plasmid expressing Snf1-HA (41) were grown to log phase in synthetic complete-glucose medium lacking uracil. HU was added to a concentration of 200 mM to one aliquot of cells, whereas a second aliquot of cells was harvested, washed, and resuspended in synthetic complete-galactose (Gal) medium lacking uracil. Cells were harvested at the indicated times after medium changes, Snf1-HA was immunoprecipitated from protein extracts, and Snf1 activation was monitored by immunoblotting using antibodies directed against phospho-T210 (41). The efficiency of Snf1-HA immunoprecipitation was monitored by immunoblotting using anti-HA antibodies (lower panel).