A Study On The Mechanism Of How Whi2 Regulates Autophagy In Yeast

Aim:In this study,we investigated the mechanism by which yeast Whi2 regulates autophagy.Methods:Yeast strains with deleted genes were constructed by homologous recombination.Yeast strains overexpressed with certain proteins were constructed through transformation with specific plasmids.The prATG8-GFP plasmid was used to detect the transcription level of the autophagy gene ATG8 under specific nutritional conditions,and the expression level of GFP indicated by western blot indirectly reflected the level of intracellular autophagy induction.The prATG8-GFP-ATG8 plasmid was used to detect the level of autophagy flux in cells under the condition of low leucine,and the ratio of free GFP to total GFP(the sum of free GFP and GFP-Atg8 fusion protein)can reflect the level of autophagy flux in cells.The results were analyzed according to epistasis analysis method to construct the signaling pathway.Results:The prATG8-GFP-ATG8 plasmid was used to detect the level of autophagy flux in the cells.It was found that the expression level of GFP-Atg8 in ?whi2 was significantly lower than that in WT under both low leucine and nitrogen deprivation conditions.PEP4 was deleted in WT and ?whi2 to inhibit the degradation of autophagosomes.Compared with ?pep4y the expression of GFP-Atg8 is significantly reduced in?whi2?pep4.At the same time,the prATG8-GFP plasmid detected that ATG8 transcription level in ?whi2 is lower than that in WT,and ?whi2?pep4 had a lower ATG8 transcription level than ?pep4.PKA has an inhibitory effect on autophagy induction and autophagy flux,and inhibition of PKA activity in ?whi2 has no effect on autophagy induction and autophagy flux levels,while overexpression of Whi2 in ?tpk3 cells can still promote autophagy induction and improve the level of autophagy flux.Under conditions of low leucine and nitrogen deprivation,Whi2 cannot improve the autophagy-induction level in ?ume6.However,Ume6 can inhibit ATG8 transcription in ?whi2.At the same time,the ATG8 transcription level cannot be improved by overexpressing Whi2 in ?rim15.ATG8 transcription level in ?msn2?msn4 is similar to ATG8 transcription level in ?whi2.Overexpression of Msn2 or Msn4 in ?whi2 cells can increase the transcription level of ATG8,both under low leucine condition and nitrogen deprivation condition.The TORC1 activity of?msn2?msn4 is much lower compared with ?whi2,and the level of autophagy flux is higher in ?msn2?msn4 than in ?whi2.Conclusion:1.Whi2 promotes the transcription of autophagy gene ATG8.2.Whi2 is downstream of PKA,and PKA inhibits autophagy induction and autophagy flux through Whi2.3.Whi2 is upstream of Rim15-Ume6 and inhibits Ume6 to promote autophagy induction by activating Rim15.4.Msn2 has functional similarity with Msn4 in promoting autophagy induction.5.Msn2/Msn4 is downstream of Whi2,and does not depend on Whi2 to regulate autophagy induction.