Fig. 4: Calcium accumulation in ER caused blockage of autophagy in Csg2 deleted yeast cells.

a Schematic diagram of ER calcium accumulation in csg2Δ cells. When the transport directions of calcium (into the ER by Pmr1 and out of the ER by Csg2) was disrupted by Csg2 deletion, calcium levels were increased in the ER lumen. b, c ER calcium probe Stt3-jGCaMP7c were investigated by fluorescence assays in indicated cells after starvation in SD-N medium for 3 h. Fluorescence parameters were set the same to compare the signal intensities. Representative images (b) and fluorescence intensity quantification (c) were shown. Scale bars: 5 μm. Two-tailed t-test, n = 3 independent experiments. p values: 2.902E-05, 0.1626. ***p < 0.001. ns: p > 0.05, not significant. Error bars, mean ± SD. d WT cells and csg2Δ cells with or without deletion of ER calcium channel Pmr1 were analyzed for the autophagic degradation of GFP-Atg8 after starvation in SD-N medium. 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. e Autophagic marker Cherry-Atg8 was expressed and investigated by fluorescence examination in WT cells, csg2Δ cells, pmr1Δ cells, and csg2Δpmr1Δ cells before and after starvation in SD-N medium. The experiment was repeated independently for three times and representative images were shown. Scale bars: 5 μm. f WT or D53A mutant Pmr1 was expressed in csg2Δpmr1Δ cells and the degradation of GFP-Atg8 after starvation in SD-N medium was analyzed. The blots were probed with anti-GFP antibody and HA antibody, Pgk1 was used as a loading control. The samples derive from the same experiment and gels were processed in parallel. g Starvation resistance conferred by autophagy in csg2Δpmr1Δ cells was prohibited by exogenous expression of Pmr1 but not by D53A mutant of Pmr1 that lose calcium channel ability. The experiment was repeated independently for three times with similar results.