The Study Of Vps21 In Autophagy Through PI3P And Its Binding Protein Atg21 On Interactions Among Atgs In Saccharomyces Cerevisiae

Macroautophagy is a process that transports surplus products,such as proteins and other harmful substances,to lysosomes/vacuoles for degradation to reuse in eukaryotes.The general autophagy process includes induction,nucleation,expansion,closure,fusion with lysosomes,autophagosome degradation and release.Our previous studies have found that the endocytic and autophagy pathways converge at the site regulated by Rab5/Vps21 GTPase module in yeast.Most importantly,the Rab5/Vps21 GTPase module is important for autophagsome closure and is required for the recruitment of endosomal sorting complex required for transport(ESCRT)to phagophore to catalyze autophagosome closure.Furthermore,the absence of Vps21 module proteins significantly affects the localization of Vps34,a component of phosphoinositide 3-kinase(PI3K)complex,which functions in both endocytic and autophagy pathways.However,it is unknown how Vps21 affects autophagy through PI3K.In this study,we found that Vps21 regulates a subsequent serious of events,including the localizations on Snf7 marked endosomes and(or)Apel marked preautophagosome assembly site(PAS)of PI3K complex,PI3P,Atg21 and Atg12-Atg5-Atg16 complex and the interactions between Vps34 and Vps8,Atg2l and Atg16 or Atg8.These changes may partly contribute to the roles of Vps21 module proteins in autophagosome closure except that Vps21 module proteins regulate ESCRT to phagophores to close them.However,the detailed regulation mechanism still needs further investigation.The main results are as follows:1.The absence of Vps21 results in decreased localization of PI3K complex,PI3P,Atg21 and Atg12-Atg5-Atg16 complex on endosomes or PASPhosphoinositide 3-phosphate(PI3P)produced by phosphorylation of phosphatidylinositol(PI)by PI3K complex subunit Vps34 recruits PI3P binding protein Atg21 and others to endosomes and PAS,meanwhile Atg21 further recruits the Atg12-Atg5-Atg16 complexe to PAS.To understand how Vps21 regulates autophagy,especially phagophore closure through PI3K and a series of following issues,this study detected the localizations of these related proteins on Snf7 marked endosomes or Apel marked PAS.We found that the localizations of Vps38,Vps34 and Atg14,the subunits of PI3K complex,and PI3P binding protein Atg21 on endosomes or(and)PAS are all induced by nitrogen starvation and regulated by Vps21.What's more,Vps38,Vps34 or Atg21 could locate on the dual localizations marked by Snf7-mTu and RFP-Apel,but significantly decreased when Vps21 was absent,although this decrease is more obvious on endosomes.In addition,the localization of Atg12-Atg5-Atg16 complex on PAS is also induced by nitrogen starvation and regulated by Vps21.2.The decreased localization of PI3K complex,Atg21 and Atg12-Atg5-Atg16 complex in vps21? is not due to the decreased protein levels.In order to explore whether the localization changes of the above proteins are caused by the decreased protein expression levels,this study examined the expression levels of the subunits of PI3K complex,Atg21 as well as Atg5 and Atg16 in Vps21-absent mutant under nitrogen starvation condition.The results showed that the expression levels of these proteins were not decreased significantly when Vps21 was absent,indicating that the decreased localizations of these proteins were not due to the decreased protein levels,but to some unknown mechanisms.3.The absence of Vps21 weakens the interactions between Vps8 and Vps34,Atg21 and Atg16,Atg21 and Atg8,and the latter two could occur on the dual localizations marked by Snf7-mTu and mCherry-Atg8.The Vps21-dependent interactions between Vps8 and Vps34 were detected by BiFC and Y2H assays,and the interactions occur on Snf7 marked endosomes but not Atg8 marked autophagosomes in this lab.These results were further verified in yeast by GST pulldown assay in this study.Previously,the interactions between Atg21 and Atg16 or Atg8 were also detected by BiFC,and the interactions occurred on Atg8 marked autophagosomes and were Vps21-dependent.As the endosomes and autophagosomes can get close and even fuse during autophagy,it is important to explore whether the interactions also occurred on endosomes.The results showed that these interactions occurred on endosomes,and some even on dual localizations marked by Snf7-mTu and mCherry-Atg8,although the proportion is less than 16%,depending on Vps21.