Fig. 6: Accumulated phytosphingosine caused blockage of autophagy in csg2Δ cells.

a Hypothesis of mechanism of increased calcium in ER caused autophagy blockage through disturbing sphingolipid synthesis pathway. b WT and temperature sensitive tsc10ts, lip1ts, and aur1ts yeast cells were checked by GFP-processing assays using GFP-Atg8 after starvation in SD-N medium for 16 h at permissive 24 °C and nonpermissive 30 °C or 37 °C conditions. The blots were probed with anti-GFP antibody and Pgk1 was used as a loading control. The samples derive from the same experiment and gels were processed in parallel. c Schematic diagram showed effects of mutations of sphingolipid synthesis enzymes Tsc10, Lip1, and Aur1 on autophagy. d, e GFP-processing assays using GFP-Atg8 were checked in tsc10ts, lip1ts, and aur1ts yeast cells combining with Csg2 deletion after starvation in SD-N medium for 16 h at nonpermissive 37 °C. The blots were carried out as in (b). f Autophagic degradation of GFP-Atg8 in indicated yeast cells was analyzed after starvation in SD-N medium for 16 h. The blots were carried out as in (b). g Autophagic degradation of GFP-Atg8 in indicated yeast cells was analyzed after starvation in SD-N medium for 16 h. The blots were carried out as in (b). h Cell viability of indicated yeast cells was analyzed before and after starvation in SD-N medium for 72 h. The experiment was repeated independently for three times with similar results. i Hypothesis of mechanism of increased calcium in ER caused blockage of autophagy. j, k Autophagic degradation of GFP-Atg8 was analyzed in indicated yeast cells after starvation in SD-N medium with addition of PHS (5 μM, solved in EtOH) or control EtOH for 16 h. The blots were carried out as in (b). l Autophagic degradation of GFP-Atg8 was analyzed in indicated yeast cells after starvation in SD-N medium with addition of PHS (5 μM, solved in EtOH) or control EtOH at the presence of EDTA (5 mM) or EGTA (20 mM) for 16 h. The blots were carried out as in (b).