Figure 3.

The loss of nuclear export results in constitutively nuclear Gln3 that is hyperphosphorylated. (A–D) Wild-type strain JK9-3da was transformed with Cen II-based plasmids containing either wild-type Gln3-Myc¹³ (pRR536) or the gln3 NES mutant (pRR752). Cells for (A and B) were cultured and treated as described in Figure 2, A and B. Cells for (C and D) were cultured in YNB-glutamine medium (Gln) to A_{600 nm} ∼0.5. Cultures were then treated for 15 min with 200 nM rapamycin (+Rap). Sample preparation and data collection were as described in Materials and Methods and Figure 2, A and B. Data in (A–D) are averages of two and five biological replicates, respectively. SD of the measurements are indicated as error bars. (E and F) Wild-type (strain JK9-3da) transformants containing plasmid pRR536 or pRR752 were cultured as in (A–D). Samples were then collected and processed for western blot analyses as described in Materials and Methods.