The Study Of Interactions Between Vps21 Module Proteins And PI3K Copmplex Subunits In Saccharomyces Cerevisiae

Autophagy is an evolutionarily conserved catabolic process widely existing in eukaryotes.In yeast,some cytoplasmic compartments,such as proteins,sugars,lipids,damaged organelles and so on,which were encapsuled to form autophagosomes to be transported to lysosome(vacuole)to be degraded.At last,the degraded materials will be released to cytoplasm for recycling.Studies have shown that the process of autophagy is very important for maintaining cellular homeostasis and other physiological activities.During development process,the defect of autophagy can seriously affect the normal differentiation of embryonic cells and development of embryos.The autophagy is also associated with the occurrence of many diseases,such as tumor,neurodegenerative disease,infectious diseases and so on.The formation and regulation of autophagy is a multi-step and extremely complex process under the regulation of a lot of autophagy related gene.In addition to the regulation of autophagy related gene,recent literatures have reported that Rab/Ypt proteins,which regulate vesicle transport,were also involved in autophagy.For example,in mammalian cells,Rab5 regulates the formation of autophagosome through Vps34.Recently our lab reported that the mutants of yeast Rab5 homologue Vps21 and its module proteins can affect autophagy,but the underlying mechanism is unclear.Our latest data showed that the mutants of Vps21 module proteins might affect autophagosome closure,and further study showed that the mutants of Vps21 module proteins affected the localization of Vps34,a subunit of the PI3K complexes.It was known that Vps34 belongs to the subunit of yeast PI3K complex ? and ?,which regulate autophagy and vesicle transport,respectively.To further interpret which interact with the Vps21 module proteins and its function in cells,we investigated the interactions between Vps21 module proteins and PI3K complex subunits by yeast two hybrid and other methods.The following main results are obtained:1.The absence of Vps21 module proteins affects the localization of Vps34,but the absence of Vps34 does not affect the localization of Vps21 at PASWe found that the absence of Vps21 module proteins resulted in the diffusion of most Vps34 on vacuolar membranes,but some still at PAS with accumulation.On the contrary,on the basis of Vps21 localizing to PAS,we first observed the localization of Vps21 under starvation when Vps34 was absent.The result shows that the absence of Vps34 did not affect the localization of Vps21 to PAS.These data indicate that Vps21 is not recruited to PAS by Vps34,but by other proteins.We propose that Vps21 module proteins may interact with PI3K complex subunits.2.Y2H to detect the interactions between some Vps21 module proteins and PI3K complex subunitsTo test whether the Vps21 module proteins interact with PI3K complex subunits,we performed Y2H assay.The results showed that Vps8,Vacl,Pep12,Vps39,Vps41 and Vam3 interacted with Vps15.Among them,Vps8 strongly interact with Vps15.The Y2H results also showed that Vacl,Vps8 and Vam3 interacted with Vps30,but the strength of ineraction is much weaker.Furthermore,only Vps8 interacted with Vps34.These interactions implicate that Vps21 module proteins may regulate autophagy through their interactions with the PI3K complex subunits.3.Vps8 interacts with Vps34 on endosome depending on Vps21.Furthermore,Vps8 also colocalizes with the subunits of PI3K complex under starvationThe interaction between Vps8 and Vps34 is further confirmed by bimolecular fluorescence complementation assay(BiFC)and GST-pull down.We found that the interaction between Vps8 and Vps34 was induced by starvation and the interaction site localized with Atg21 mainly on endosome.We further found that Vps21 was required for the interaction between Vps8 and Vps34 and the N terminal of Vps8 is important for the interaction.We also found that Vps8 colocalized with the subunits of PI3K complex under starvation condition.Taken together,we peopose that the Vps21 module proteins may mainly function through the interactions between Vps8 and Vps34 to recuit other proteins to seal autophagosomes at the late stage of autophagy.However,more data are required to prove this hypothesis.