Figure 4. Enhanced Ypk1 phosphorylation of Lac1 and Lag1 under sphingolipid and heat stress.
(A) 3xHA-Lac1 (yAM165–A) cells were grown to early exponential phase in YPD. Cultures were then treated with sublethal doses of myriocin (0.625 μM) or methanol (vehicle) or aureobasidin A (1.8 μM) or ethanol (vehicle) for 2 hr. (B) 3xHA-Lac1 (yAM165–A) cells were grown to exponential phase in YPD at 30°C. A sample of this culture was then harvested. The remaining culture was then moved to 42°C to initiate heat shock and samples were harvested at the indicated time points. Whole cell extracts were prepared from each sample, resolved by Phos-tag SDS-PAGE, and immunoblotted as in Figure 3. P-Lac1 indicates the band corresponding to S23 S24 phosphorylation. * indicates a non-specific band that appears in HA blots of yeast whole cell extracts. (C) LAC1 LAG1 (yAM205–A), Lac1SSAA Lag1SSAA (yAM207B) and Lac1SSEE Lag1SSEE (yAM210) were grown to exponential phase in YPD. Serial dilutions of each culture were made and spotted on YPD plates containing vehicle or the indicated concentration of myriocin or aureobasidin A. Cells were allowed to grow for 3 days at 30°C prior to imaging. (D) 3xHA-Lac1::HIS3 3xFLAG-Lag1::LEU2 (yAM168) 3xHA-Lac1(S23A S24A)::HIS3 3xFLAG-Lag1(S23A S24A)::LEU2 (Lac1SSAA Lag1SSAA) (yAM184) and 3xHA-Lac1(S23E S24E)::HIS3 3xFLAG-Lag(S23E S24E)1::LEU2 (Lac1SSEE Lag1SSEE) (yAM192–A) cells were grown to mid-exponential phase and then treated with 1.0 μM myriocin or 18.2 nM aureobasidin A for 2 hr. Whole cell extracts were prepared from each sample, resolved by SDS-PAGE, and immunoblotted as indicated.