Fig. 1: Csg2 is identified essential for autophagy.

a Scheme of calcium pumps and channels in yeast cells. b WT and calcium pumps/channels-deleted yeast cells including csg2Δ cells, pmr1Δ cells, spf1Δ cells, pmc1Δ cells, vcx1Δ cells, yvc1Δ cells, mid1Δ cells, rch1Δ cells, ecm7Δ cells, cch1Δ cells were starved for different times in SD-N medium and then detected for cell viability by serial dilution spotting and growth on YPD plates. The experiment was repeated independently for three times with similar results. c N-terminal GFP-tagged Atg8 with the ADH promoter was transferred into WT and calcium pumps/channels-deleted yeast cells to test its autophagic degradation after 16 h of 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. d Validation of degradation of autophagic substrate GFP-Atg8 in WT cells, atg1Δ cells, and csg2Δ cells following starvation in SD-N medium was performed at the indicated time points. 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 Validation of Csg2-dependent degradation of autophagic substrate GFP-50Q (50 poly-glutamine) as in (d). f N-terminal RFP-tagged Ape1 was transferred into vacuoles in WT cells after starvation, but not in atg1Δ cells and csg2Δ cells detected by fluorescence assays. The experiment was repeated independently for three times with similar results. Representative images were shown. Scale bars: 5 μm. g Detection of Csg2-dependent transportation of the autophagic substrate Pho8Δ60 by ALP assays. Indicated yeast cells were subject to starvation in SD-N medium. After transfer into vacuoles by autophagy, Pho8Δ60 activities were measured. Two-tailed t-test, n = 3 independent experiments. p values: 0.00518, 0.00217. **p < 0.01. Error bars, mean ± SD. h Autophagic marker GFP-Atg8 under control of endogenous ATG8 gene promoter was investigated by fluorescence examination in WT cells, atg1Δ cells and csg2Δ cells before and after starvation. The experiment was repeated independently for three times with similar results. Representative images were shown. Scale bars: 5 μm.