The Molecular Mechanism Of Phagophore Closure By The Vps21 Module Collaborating With The ESCRT Complex In Saccharomyces Cerevisiae

As an important degradation and recycling pathway in eukaryote cells,autophagy plays an essential role in maintaining cell survival under stress,eliminating damaged intracellular organelles and toxic proteins,and maintaining homeostasis.The autophagy process includes sequential steps of autophagy induction;the phagophore nucleation,phagophore extension and closure;autophagosome maturation;fusion of mature autophagosomes with vacuoles;degradation of autophagosomes in the vacuole and release of small molecular product to cytosol and is regulated by many autophagy-related proteins(Atg).In addition to Atg proteins,an emerging evidence indicates that quite a lot of proteins involving in vesicle trafficking are also required for autophagy process.In Saccharomyces cerevisiae,a series of small Rab GTPase,Ypt(Yeast protein transport),acting as molecular switches,regulate intracellular transport of vesicles.Ypts are activated by their GEFs(guanine nucleotide exchange factors),changing from inactive GDP-binding state to active GTP-binding state,to recruit downstream effectors to regulate vesicular trafficking.Recently,many Ypts are also reported to be involved in autophagy.The results of previous study in our laboratory showed that the Vps21 module proteins,including Vps21(also known as Ypt51,yeast Rab5 homologoe)and its GEF and effectors,were also involved in autophagy.Depletion of these proteins resulted in accumulation of autophagosome-like membrane structures besides vacuolar membrane.Protease protection assay further indicated that these autophagosome-like membrane structures were not closed,termed the phagophore.Phagophore closure is a critical step,but its regulatory factors and the underlying molecular mechanism are still not fully understood.In this study,based on the knowledge that the depletion of Vps21 module proteins affects phagophore closure,we attempted to explore the molecular mechanism of phagophore closure.In Vps21 mediated endocytic pathway in yeast,the endosomal sorting complex required for transport(ESCRT),composed of multiple subunits,functions downstream of Vps21 in the endocytic pathway in yeast.The ESCRT is an important molecular machinery for membrane scission and closure,and functions on the scission and sealing of membranous organelles in multiple life processes.Therefore,it has been speculated that the ESCRT complex may participate in the closure of phagophore during autophagy,but there is no experimental evidence for it so far.This study will focus on the effect of the absence of ESCRT complex subunits on autophagy,especially on phagophore closure.After confirming the defect of phagophore closure by the depletion of ESCRT complex subunits,the molecular mechanism of Vps21 module proteins,whose absence also leads to phagophore closure defect,on phagophore closure,will be explored through studying the coordination between Vps21 module proteins and the ESCRT complex in regulating phagophore closure.The following main results are obtained:1.The Vps21 module functions in autophagy downstream of Ypt1 and upstream of Ypt7 and is involved in phagophore closure and Atg dissociation from autophagosomes.Firstly,we used double-mutant epistasis analysis to show the relationship between Ypt1 and Vps21.Ypt1 functions in early stage of autophagy,regulating the formation of PAS and the expansion of phagophore.The mutation of Ypt1(ypt1-1)resulted in dispersed dots of Atg8 in cytosol,which did not colocalize with Atgll,indicating a defect of PAS formation.ypt1-1vps21? cells showed a similar defect as ypt1-1 cells but not as vps21?cells,suggesting that Ypt1 functions upstream of Vps21.Secondly,based on the autophagy defects observed in the Vps21 module protein-depleted strains,this study confirmed that the accumulated autophagosome-like membrane structures were unsealed phagophore through protease protection assay.Depletion of Vps21 also led to defects of Atg proteins dissociasion from autophagosome.Finally,Ypt7 functions in the late stage of autophagy and regulates the fusion of mature autophagosomes with vacuoles.Similar double-mutant epistasis analysis suggested that Vps21 functions upstream of Ypt7 in autophagic process.2.The Vps21 module functions upstream of the phosphatase Ymrl and affect the removal of PI3P from autophagosomes.After autophagosome closure,phosphatase Ymrl mediates PI3P removal from closed autophagosomes.Epistasis analysis showed that unsealed and PI3P-decorated autophagosomes accumulated beside vacuole membrane as clusters in both vps21? and vps21?ymr1? cells,while sealed autophagosomes decorated with PI3P dispersed as multiple dots in ymr1? cells.It suggested that Vps21 functions upstream of Ymr1-mediated PI3P removal from closed autophagosomes.In vps21?ymr1?,and ymr1?,PI3P on phagophore or autophagosomes could not be removed.In addition,we found that Ymrl mislocalized from phagophore in vps21? cells,which indicated that Vps21 could regulate the removal of PI3P from autophagosomes by mediating the localization of Ymr1.3.Depletion of ESCRT subunit results in defect of phagophore closure.Vps21 module proteins function upstream of the ESCRT complex in vesicle trafficking.Based on the role of Vps21 module proteins in phagophore closure and the ability of the ESCRT complex to seal membranous organelles,we speculated that the Vps21 module proteins most likely use the ESCRT complex for phagophore closure.In order to investigate whether the Vps21 modular proteins participate in the closure of phagophore through the ESCRT complex,we first examined the effect of depletion of the ESCRT complex subunit on autophagy.Through the detection of localization and degradation of GFP-Atg8 in the single deletion strains of ESCRT complex subunit,we found that all mutations with autophagy defects to different degrees,indicating that the ESCRT complex does not function only through a unique subunit in autophagy.After that,we mainly studied the autophagic defects in the depletion of key subunits of the ESCRT complex,Snf7 and Vps4,and found that autophagosome-like membrane structures clustered on the edge of the vacuoles.Protease protection assay revealed that the autophagosome-like membrane structures in these mutants were unclosed phagophores.The dissociation of Atg proteins(Atg2,Atg5,Atgll,Atg17,etc.)was blocked.The autophagy defect phenotypes in the strains with depletion of these ESCRT key subunits are very similar to those in the strains with depletion of Vps21 module proteins,indicating that the ESCRT complex also functions on phagophore closure.4.The ESCRT complex subunit Snf7 interacts with Atg17 and localizes to the phagophore.Previous experiments showed that there are no direct interactions between the Vps21 module proteins and Atg proteins.However,it was reported that some ESCRT-? subunits might interact with subunits of the Atgl complex.Thus,we screened the interaction between the ESCRT-? key subunit Snf7 and the subunits of the Atg1 complex by BiFC assay and found that Snf7 interacts with Atg17 at mCherry-Atg8 marked phagophore.This interaction was confirmed by GST pulldown.Absence of Atg17 significantly decreased the localization of Snf7 on mCherry-Atg8 marked phagophore,but not vice versa.In addition,the co-localization of Snf7-mCherry and Atg17-GFP was not affected by the absence of Atg8.These results suggest that Atg17 recruits Snf7 to mCherry-Atg8 marked phagophore through interactions.5.Vps21 mediates the interaction between Snf7 and Atg17 and the localization of Snf7 on phagophore.Based on the fact that the absence of either Vps21 module proteins or ESCRT complex resulted in the inability to seal the phagophore in the autophagy pathway,and ESCRT key subunit can indeed be recruited to phagophore through the interaction with Atg proteins,we hypothesized that ESCRT may seal the phagophore through the membrane scission and closure functions.In order to understand the relationship between the Vps21 modular proteins and the ESCRT complex in autophagy,we examined the effect of Vps21 depletion on the interaction between Snf7 and Atg 17,and the localization of Snf7 on phagophore.We found that the interaction between Snf7 and Atg1 7 was greatly reduced,and the ratio of Snf7 on the phagophore was significantly decreased after Vps21 depletion.Dynamic observations also showed that Snf7 rarely contact with the phagophore in the Vps21 depletion mutant.It shows that Vps21 acts in the upstream of ESCRT in autophagy pathway,mediates the interaction between Snf7 and Atg1 7,and thus affects the localization of ESCRT on phagophore,and maybe regulate the closure of phagophore.The above contents clearly indicate that both the Vps21 module proteins and the ESCRT complex have roles in phagophore closure and show the effect of Vps21 module proteins on the interaction between Snf7 and Atg17,and on the localization of Snf7 on phagophore.We propose that the Vps21 module affects the localization of ESCRT complex on phagophore by regulating the interaction between Snf7 and Atg17 for its function on phagophore closure.This study lays the foundation for a deep understanding of the regulators and molecular mechanism of phagophore closure,and also provides important data for exploring the relationship between vesicle transport and autophagy.