Experiments were repeated at least three times. DNA fragmentation following a shift to SD-N medium was quantified using flow cytometry and bd facsdiva software after terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (Madeo et al., 1997; Büttner et al., 2007). For sphingolipid labeling, yeast cultures were incubated in SD-N-inositol containing [3H]myo-inositol
[1 μCi mL−1; American Radiolabelled Chemicals (St. Louis, MO)], after which sphingolipids were extracted and analyzed (Thevissen et al., 2005). Ceramide and sphingoid base analysis was performed by Lipidomics CORE at Medical University of South Carolina as described previously using a sphingolipidomics approach (Bielawski et al., see more 2006; Aerts et al., 2008). For each condition, experiments were performed twice at least in duplicate. Statistical analysis was performed using a paired t-test. To determine whether induction of autophagy is affected in the Δipt1Δskn1
mutant as compared with the single deletion mutants and WT, we used the Pho8Δ60 assay (Klionsky, 2007). Pho8Δ60 is a truncated variant of the vacuolar alkaline phosphatase Pho8, which lacks the N-terminal transmembrane region that normally allows entry into the endoplasmic reticulum, resulting in accumulation of the mutant protein in the cytosol (Noda et al., 1995). Cytosolic Pho8Δ60 is sequestered as a nonspecific Selleck PD-L1 inhibitor cargo within autophagosomes upon induction of autophagy and delivered into the vacuole, where it is processed into an enzymatically active form due to removal of a C-terminal propeptide. Therefore, the alkaline phosphatase activity of Pho8Δ60 reflects the magnitude of autophagic cargo delivery. To this end, SKN1 and/or IPT1 were deleted in a Pho8Δ60 yeast strain background. Upon challenge with N starvation medium, Δipt1Δskn1 cells showed a significant 10–20% increase in Orotidine 5′-phosphate decarboxylase Pho8Δ60 activity as compared with the single deletion mutants or the corresponding Pho8Δ60 WT (Fig. 1), indicating increased autophagy upon deletion of both IPT1 and SKN1. This is
in contrast to the single deletion mutants in IPT1 or SKN1, which did not show significantly increased autophagy as compared with the corresponding WT under conditions of N starvation (Fig. 1). Deletion of ATG1, encoding a protein serine/threonine kinase required for autophagy (Matsuura et al., 1997), served as a negative control in this experiment and showed essentially no increase in Pho8Δ60-dependent alkaline phosphatase activity upon starvation. A functional cross-talk exists between autophagy and apoptosis (Maiuri et al., 2007). Upon challenge with N starvation medium, we observed a slight, but significant, increase in the death rate (10–15%) of all the deletion mutant strains as compared with WT (Fig. 2a), while no difference was observed when shifting to a rich medium (SD), ruling out a survival defect of the mutant strains (data not shown).