The Significance Of Interaction Between Snf7 And Atg17 On Phagophore Closure In Saccharomyces Cerevisiae

Autophagy is a degradation pathway that exists widely in eukaryotic cells.The process of autophagy generally includes the induction of autophagy,nucleation of phagophore,membrane extension of phagophore,sealing of phagophore,maturation of autophagosomes,the fusion of mature autophagosomes with vacuole/lysosome.Autophagy is regulated by a series of autophagy-related proteins(Atg).They function in various stages of autophagy and cooperate to degrade the cargoes smoothly.In addition,some proteins involved in vesicle transport also have roles in autophagy.For example,our laboratory reported that Vps21,a Rab5 homologous protein in S.cerevisiae,and the endosomal sorting complex required for transport(ESCRT)all participates in the sealing process of autophagosomes.Further screening revealed that the ESCRT subunit Snf7 was recruited to the phagophore through interaction with the autophagy related protein Atg17,and Snf7-Atg17 interaction was dependent on Vps21.If the unclosed phagophore in the vps21? mutant was partly due to the weak interaction between Snf7 and Atg17,then whether the forced interaction between Snf7 and Atg 17 in the vps21? mutant can fully or partially recover the autophagy defect in this mutant?In addition,which domains of Atg 17 mainly interact with Snf7,and whether such interactions can also compensate the autophagy deficient phenotype in vps21? mutant if such interactions exist?This study explored the significance of the Snf7-Atg17 interaction mediated by Vps21,and obtained the following main results:1.The interaction of Snf7-Atg17 mediated by GBP-GFP can partially bypass the autophagy defects caused by Vps21 depletionThe absence of Vps21 leads to a decrease in the interaction between Snf7 and Atg 17,and also a decrease in their colocalization in the cell.At the same time,the autophagy defect in vps21? mutant is serious,resulting in unsealed phagophores.This study tried two methods to force Snf7 to interact with Atg17 artificially in vps21? strain.First,a traditional method to construct a fusion protein in one plasmid was applied.In this case,the base sequence of Snf7 and Atg17 was connected one after another to form a fusion protein.The results showed that the function of Snf7 was lost.The goal was not achieved through this way.Next,this study tried a new protein-protein interaction method,which is using the specific affinity of GFP binding protein(GBP)for GFP.The GBP was installed between Snf7 and mCherry to form Snf7-GBP-mCherry and integrated into the chromosome,Then,the Atg17-containing plasmid labeled with GFP is also integrated on the chromosome for co-expression.As GBP will get close to GFP and bind,Snf7 and Atg17 can interact with each other even in vps21?mutant.Fluorescence observation showed that the co-localization of Snf7 and Atg17 in vps21? mutant was enhanced under nitrogen deficiency,and immunoblotting assay showed that the mature defect of Apel in it was greatly restored.Meanwhile,the relative degradation amount of Ams1,another cargo protein,in vps21? mutant with co-localized Snf7 and Atg17 in comparison with wild-type,decreased significantly compared with the control groups without co-localized Snf7 and Atg17,most likely through the restored autophagy to degrade.2.The ?3-?4 domains of Atg17 are very important for the interaction of Snf7-Atg17Previous results in this laboratory found that the membrane binding domain of Snf7 is important for its interaction with Atg17.This study will further explore the interaction between different domains of Atg17 and Snf7.According to the literature,Atg17 is divided into four domains,?l-?4.By constructing bimolecular fluorescent complementary assay(BiFC)plasmids to conduct BiFC experiments,this study found that the ?3-?4 domains of Atg17 were very critical for the interaction of Snf 7-Atg17,and this type of interaction still occurs on phagophores.However,based on the functional verification experiments,such domains are not enough to restore autophagy defects in atg17? mutant,indicating the other domains of Atg17 are also important to perform the function in autophagy after Snf7 interacts with Atg17.The above experimental results indicate that the Vps21-dependent interaction of Snf 7Atg17 is significant for phagophore closure,because the artificially forced Snf7-Atg17 interaction can partially bypass the autophagy defect caused by the depletion of Vps21.In addition,although the ?3-?4 domains of Atg17 can also interact with Snf7,they are not enough to restore the autophagy defect in atg17?.mutant.This study provides solid experimental evidence for the roles of Snf7-Atg17 interaction in the molecular mechanism of phagophore closure,and also provides methods and a reference for similar future studies.