Reduced TPI activity protects against diamide by increasing NADPH. (a) S. cerevisiae strains MR101 and MR105 were grown in duplicate to mid-log phase, pyridine nucleotides were extracted, and LC-MS/MS measurements were performed in triplicate. MR105 cells expressing TPIIle170Val had a higher overall NADPH/NADP+ ratio compared with MR101 cells expressing wild-type TPI. (b) S. cerevisiae strain BY4741 was transformed with an empty 2μ plasmid or with a 2μ plasmid encoding K. lactis GDP1 (p1696). Afterwards, single transformants were selected, grown overnight and the same number of cells were spotted as fivefold serial dilutions on agar plates supplemented with different concentrations of diamide. Growth was monitored after plates were incubated at 30°C for 3 days. (c) The isogenic yeast strains MR101 expressing wild-type human TPI or MR105 expressing human TPIIle170Val were transformed with plasmids for expression of K. lactis GDP1 and processed as described in (b).
Ralser et al. Journal of Biology 2007 6:10 doi:10.1186/jbiol61