Figure 7. Failure of Ypk1 to upregulate ceramide synthase causes LCBP accumulation that triggers autophagy.
(A) LAC1 LAG1 (yAM205–A) or Lac1SSAA Lag1SSAA (yAM207–B) were transformed with PYPK1-Ypk1D242A (shown as Ypk1*) (pFR273) or empty vector pRS316 (EV). Transformants were grown to exponential phase in synthetic complete medium and then diluted to OD600 = 0.1 and grown in microtiter plates (lower) or on agar plates (upper). For liquid cultures, each was grown in at least quadruplicate and the error bars indicate the SEM of replicates at each time point. (B) Cells from (A) were grown to mid-exponential phase in selective synthetic complete media and then harvested. Sphingolipids were extracted and analyzed as described in ‘Materials and methods’. Values represent the mean of three independent experiments (each performed in triplicate) and error bars represent SEM. (C) LAC1 LAG1 lcb4Δ (yAM237) or Lac1SSAA Lag1SSAA lcb4Δ (yAM238–A) were transformed with Ypk1* (pFR273) and growth experiments performed as in (A). (D) Liquid growth assays were performed as in (A) for LAC1 LAG1 (yAM205–A), Lac1SSAA Lag1SSAA (yAM207–B), Lac1SSEE Lag1SSEE (yAM210), LAC1 LAG1 lcb3Δ (yGT12), Lac1SSAA Lag1SSAA lcb3Δ (yGT13) and Lac1SSEE Lag1SSEE lcb3Δ (yGT14) strains. (E) LAC1 LAG1 (yAM205–A) or Lac1SSAA Lag1SSAA (yAM207–B) or LAC1 LAG1 lcb3Δ (yGT12) strains were transformed with Ypk1* (pFR273) or pRS316 (EV) and additionally PTPI1-GFP-Atg8. Growing cultures treated with vehicle or 2 μg/ml rapamycin for 2 hr and then harvested and whole extracts prepared. Extracts were resolved by SDS-PAGE and blotted with anti-GFP to detect GFP-Atg8 and free GFP arising from GFP-Atg8 autophagic processing and anti-Pgk1 antibody. The blot is representative of triplicate samples and the quantitation of the ratio of free GFP/Pgk1 from these replicates is shown below the blot. (F) LAC1 LAG1 atg1Δ (yAM239–A) or Lac1SSAA Lag1SSAA atg1Δ (yAM240–A) sensitivity to Ypk1* was measured as in (A). (G) Cells from (F) were grown to mid-exponential phase in selective synthetic complete media and then harvested. Sphingolipids were extracted and analyzed as described in (B).