How Whi2 Regulates Autophagy

Aim:In this study,we would investigate how yeast Whi2 regulates autophagy.Methods:Determine yeast TORC1 activity by detecting the immunoblot of the phosphorylation of ribosomal protein S6 under different nutrient conditions.Use double knockout strain ?whi2?gtrl made by homologous recombination to determine the position of Whi2 in TORC1 signaling pathway.Determine the effect of Whi2 on different stages of nonselective autophagy under different nutrient conditions by transforming prATG8-GFP,prPGK-PGK-GFP,PrPGK-GFP-ATG8,prATG8-GFP-ATG8 plasmids into Awhi2 strain.Determine how Whi2 affects mitophagy by using prATG32-GFP plasmid and tagging GFP after Om45 ORF through homologous recombination.Determine whether Whi2 regulates autophagy through PKA signaling pathway by constructing ?whi2?tpk3 double knockout strain through homologous recombination and overexpression of Pde2 protein.Results:The growth phenotype and TORC1 activity of the double knockout strain?whi2Agtrl is between ?whi2 and ?gtrl.Under low leucine and nitrogen deprivation conditions,Induction of ATG8 gene expression requires Whi2 but not Npr2,Npr3,and the induction of ATG8 gene expression by Whi2 is independent of TORC1.Under low leucine condition,Whi2,Npr2 and Npr3 are required to activate autophagic flux through inhibiting TORC1.Under nitrogen deprivation condition,autophagic flux relies on inhibition of TORC1 by Npr2 and Npr3,but doesn't require Whi2.Whi2,Npr2,and Npr3 are all required in mitophagy under low leucine and nitrogen deprivation conditions.Progression of mitophagy requires a certain concentration of Atg8 and a core machinery for maintaining autophagic flux.Whi2 does not induce ATG8 expression by inhibiting PKA activity.Conclusion:Yeast Whi2 inhibits TORC1 activity lindependently of Gtr complex in response to low leucine.Under low leucine and nitrogen deprivation conditions,Whi2 induces the expression of the ATG8 gene through a mechanism independent of TORC1 and PKA.Under low leucine condition,Whi2 activates autophagic flow by inhibiting TORC1.Under nitrogen deprivation condition,Whi2 has no effect on autophagic flux.Under low leucine and nitrogen deprivation conditions,Whi2 is required for mitophgy.