Fig 1. Amino acid substitutions in Hxk2 confer resistance to 2DG.

A. 2DG-resistance assay in hxk2Δ cells transformed with low-copy plasmids encoding wild type (WT) HXK2, HXK2 with the indicated amino acid substitutions, the wca and wrf alleles [18] or empty vector (hxk2Δ). Three independent cultures were grown in 2% glucose in media with and without 0.1% 2DG with percent growth plotted relative to growth in the absence of 2DG. Individual data points are shown with the mean ± SD indicated with a solid bar. Values statistically different from wild type are indicated. B. Western blot of Hxk2 proteins tagged with three copies of the V5 epitope at the C-terminus. Cells were grown in synthetic complete medium with 2% galactose. Quantitation of the western signal in arbitrary units (AU) is shown. As a control for equal loading, Coomassie stained gel of the same extracts is shown below. C. Catalytic activity of hexokinase was measured in protein extracts of cells lacking all three hexokinase genes (hxk1Δ hxk2Δ glk1Δ). Cells were transformed with the same low-copy plasmids in A and grown as in B. Activity is expressed in nmoles per minute normalized to the amount of protein in the extract as determined by western blotting. Values statistically different from wild type are indicated. D. Invertase activity measured in three independent transformants of hxk2Δ cells grown in media with 2% glucose and with the same plasmids used in A. Values statistically different from wild type are indicated. E. Snf1 kinase activation was measured in duplicate cultures before and after treatment with 2DG. Ratio of phosphorylated Snf1 over total Snf1 is plotted with representative western blots shown below.