Figure 6
The low affinity phosphate transporter Pho90 is responsible for increased selenite toxicity in aft2 cells.
(A) Exponential cultures of the following strains in YPD medium were serially diluted and spotted on YPD plates containing sodium selenite: wild type (W303-1A), aft2 (MML1086), pho87 (MML2066), pho90 (MML2068), aft2 pho87 (MML2085), aft2 pho90 (MML2088), and aft2 pho87 pho90 (MML2095). Growth was recorded after 2 days of incubation at 30 °C. (B) The indicated strains growing exponentially in YPD medium were treated with 1 mM sodium selenite for 3 hours and samples were taken for determination of intracellular selenium by ICP-MS. Data are expressed as mean ± SEM from 3 to 12 independent determinations. (C) Western blot analysis of wild type (W303-1A), spl2 (ASC67) and aft2 (MML1086) cells transformed with plasmids pWS93-PHO87 (left panel) or pWS93-PHO90 (right panel), from cultures in standard SC medium without (control) or with 1 mM selenite (3 hours), or in low phosphate (0.1 mM) SC medium. Twenty-five μg of total protein were loaded per lane. Membranes were probed with anti-HA antibodies. Ponceau staining of sections of the blotted membranes is shown as loading control. Quantitative data of Pho87 or Pho90 levels (mean of three experiments) are indicated relative to the control (unit value), after being normalized by the respective Ponceau signals.
