Furthermore, immunoprecipitation experiments also showed glucose starvation did not increase the association of Atg1 with Atg13 in = 300 cells pooled from three independent experiments. starvation-induced autophagy. Open in MK-2206 2HCl a separate window FIGURE 1 MK-2206 2HCl Mitochondrial fusion machinery is specifically involved in glucose starvation-induced autophagy. GFP-Atg8 plasmids were expressed in yeast strains accordingly listed from (ACF). Yeast cells were grown to the log-growth phase, then subjected to glucose starvation (SD-G) or nitrogen starvation (SD-N) for 4 h. MK-2206 2HCl Autophagic activity was detected by western blot using anti-GFP antibody. Mitochondrial Fusion Machinery Regulates the Recruitment of Atg1 and Other Autophagic Proteins to PAS and the Association of Atg1 With Atg13 Under Glucose Starvation Next, to study which steps during autophagy is mitochondria fusion machinery involved upon glucose starvation, we knocked out and genes, respectively, in yeast strain co-expressing PAS marker Atg17-2XCherry and other autophagy-related proteins labeled with 2XGFP. In yeast cells, Atg17/FIP200, Atg31, and Atg29 proteins form a stable complex independent of nutritional status. Atg1, Atg11, and Atg13 form polymer complex with Atg17-Atg31-Atg29 as a platform for the recruitment of other ATG proteins (Araki et al., 2017). As shown in Supplementary Figures S1A,B, under nitrogen starvation and glucose starvation, Atg17 protein appears as puncta in the and knockout in response to nitrogen starvation (Figures 2ACC). Furthermore, immunoprecipitation experiments also showed glucose starvation did not increase the association of Atg1 with Atg13 in = 300 cells pooled from three independent experiments. Data are presented as means SD. *** 0.001; NS, not significant; two-tailed Students = 3 independent experiments were quantified. Data are presented as means SD. *** 0.001; * 0.05; NS, not significant; two-tailed Students = 3 independent experiments were quantified. Data are presented as means SD. MK-2206 2HCl *** 0.001; two-tailed Students in yeast cells co-expressing mitochondrial marker Om45-Cherry and GFP-Mec1, respectively. Image data showed that upon 4 h of glucose starvation, Mec1 dissociates from mitochondria in wild type and and greatly inhibited the dissociation of Mec1 from mitochondria (Figure 4A). Statistical analysis indicated that Mec1 puncta was still mostly associated with mitochondria under prolonged glucose starvation (Figures 4B,C). Open in a separate window FIGURE 4 Mitochondrial fusion machinery is required for the dissociation of Mec1 from mitochondria during prolonged starvation. (A,D) Co-expression of GFP-Mec1 and Om45-Cherry were expressed in the indicated yeast strains. Cells were cultured in SD-G medium for 0, 1, and 4 h, and then viewed by laser confocal microscopy. Scale bar, 2 m. (B,E) Strains from (A,D) were analyzed for the number of cells with Mec1 puncta. = 300 cells pooled from three independent experiments. Data are presented as means SD. *** 0.001; NS, not significant; two-tailed Students in the wild-type co-expressing Om45-Cherry and GFP-Mec1 yeast cells, and then re-introduced empty vector, Snf1 wild-type, Snf1 kinase dead plasmid into and knockout completely inhibited the increase of ALP activity, suggesting that Atg5 and Atg17 are essential for glucose starvation-induced autophagy. To clarify the existence of autophagosome in vacuole, electron microscopy experiments were carried out on Rabbit Polyclonal to Claudin 2 = 3 independent experiments. Error bars indicate standard deviation (SD). (B) EM analysis of autophagy in = 3 independent experiments. Error bars indicate standard deviation (SD). *** 0.001; two-tailed Students to detect the degradation of endogenous mitochondrial protein Porin. Western blot results showed that in the wild type yeast strain, glucose starvation induces the cleavage of GFP-Atg8, but Porin is.