The Tolerance Of Yeast Cells Generating Unclosed Autophagosomes To Nitrogen Starvation And The Identification Of Fusion Of Unclosed Autophagosomes With Vacuoles

Autophagy is an important pathway for degradation and reuse of substance in eukaryotic cells.It plays an important role in maintaining homeostasis and improving survival rate of cells under stresses.The process of autophagy in budding yeast(Saccharomyces cerevisiae)can be divided into five stages:the formation of phagophore assembly site,the extension of phagophore,the closure of phagophore,the fusion of autophagosomes with the vacuole and the degradation of autophagic bodies.However,the molecular mechanisms of many of these stages are still unclear.Recent studies in our lab focused on the regulators and mechanisms for phagophore closure.We demonstrated that Rab GTPase Vps21,a molecular switch which regulates intracellular vesicle transport,collaborates with the endosomal sorting complex required for transport(ESCRT),which regulates the formation of multivesicle bodies,to regulate phagophore/autophagosome closure.Snf7 and Vps4 are main subunits of ESCRT.The absence of Vps21,Snf7 or Vps4 results in defect in autophagosome closure to accumulate unclosed autophagosomes as clusters outside the vacuole.However,the percent of cells with accumulated unclosed autophagosomes varied according to the culture conditions,this can not exclude the possibility that partial unclosed autophagosomes enter vacuoles.This study determined the tolerance of yeast cells generating unclosed autophagosomes to nitrogen starvation and intensively explored whether unclosed autophagosomes can fuse with vacuoles to enter them.The main results are shown below:1.The tolerance of yeast cells generating unclosed autophagosomes to nitrogen starvation decreases.The autophagy defective vps21?,snf7? and vps4? strains,which generate unclosed autophagosomes under nitrogen starvation,were determined for their tolerance to nitrogen starvation by Trypan Blue assay and cell growth assay to measure the survival rate.We found that these strains had decreased tolerance to nitrogen starvation.However,their tolerance to nitrogen starvation increased if the cognate proteins Vps21,Snf7 and Vps4 were overexpressed in vps21?,snf7? and vps4?,respectively.These results are consistent to the previous findings of overexpressing cognate proteins to complement autophagic defects.2.The degradation of unclosed autophagosomes is related to the cell growth concentration,the nitrogen starvation time and the expression levels of vacuolar proteolytic enzymes.In the previous experiments,our lab found that the percentage of cells with accumulation of unclosed autophagosomes outside vacuoles after nitrogen starvation decreased significantly if the cells were grown to high cell growth concentration(OD600>3),compared with cells grown in the exponential growth phase before nitrogen starvation.Taking the cells lacking Vps21 as an example,the gradient cell growth concentration(OD600=0.5,1,2,3,4)of cells before nitrogen starvation was set up.It was found that,with the increase of cell growth concentration,the degradation of unclosed autophagosomes after nitrogen starvation increased significantly.Autophagy was completely blocked in the atg1? strain,which did not produce autophagosome and GFP-Atg8 degradation in it was almost not affected by cell concentration and nitrogen starvation time.As the content of vacuolar proteolytic enzyme increased as the cell growth concentration increased in S.cerevisiae,if the increased content of vacuolar proteolytic enzyme could facilate the entery of unclosed autophagosomes to vacuoles,then the increased entery of unclosed autophagosomes to vacuoles for degradation could be observed by prolonging the nitrogen starvation time to increase the content of vacuolar proteolytic enzyme in yeast cells generating unclosed autophagosomes.Taking the cells lacking Vps4 as an example,the time of nitrogen starvation was set up as a gradient(5 min,30 min,2 h,4 h,6 h 12 h,24 h).It was found that,with the extension of time of nitrogen starvation,the degradation of unclosed autophagosomes indeed increased significantly.If the increased content of vacuolar proteolytic enzyme indeed promoted the degradation of unclsed autophagosomes in budding yeast,then deleted the genes encoding the vacuolar proteolytic enzyme or overexpressed the vacuolar proteolytic enzyme in vps21??snf7? and vps4?,which generate unclosed autophagosomes,will decrease or increased the degradation of unclosed autophagosomes under nitrogen starvation.It was found that the lack of vacuolar proteolytic enzyme promotes the degree of accumulation of unclosed autophagosomes and overexpression of vacuolar proteolytic enzyme promotes the degradation of unclosed autophagosomes.In brief,increasing the cell growth concentration,prolonging the time of nitrogen starvation,as well as increasing the content of vacuolar proteolytic enzyme,all can promote the entery of unclosed autophagosomes into vacuole for degradation,while the absence of vacuolar proteolytic enzyme increases the accumulation of unclosed autophagosomes and decreases their degradation.Take together,the content of vacuolar proteolytic enzyme can regulate the accumulation of unclosed autophagosomes outside the vacuoles or the entry of them into vacuoles.3.Prolonged nitrogen starvation promoted the fusion of unclosed autophagosomes with vacuoles to be degraded in vacuoles.In the yeast cells generating unclosed autophagosomes,do the unclosed autophagosomes fuse with the vacuole to enter them to be degraded or the unclosed autophagosomes close first to form mature autophagosomes before they fuse with the vacuole?To answer this question,a condition to facilate a mass of entry of unclosed autophagosomes to vacuoles,and to inhibit the degradation of these unclosed autophagosomes in vacuoles should be met.Based on the above experimental results,we realized this by prolonging the time of nitrogen starvation and depleting two of vacuolar proteolytic enzymes.We deleted PEP4 and PRB1,both encoding vacuolar proteolytic enzymes,from the yeast cells generating unclosed autophagosomes,then grew the cells till OD600=1 and starved cells in nitrogen starvation medium for 2-8 h to meet the condition.The closure characteristic of the autophagic membranes accumulated outside vacuoles(SD-N 2 h)or inside vacuoles(SD-N 8 h)was detected by protease protection assay for isolated autophagosomes.We found that the unclosed autophagosomes outside the vacuoles remained unclosed when they entered the vacuoles,i.e.,the unclosed autophagosomes fuse with the vacuoles to enter them with an unknown way.In addition,the process of phagophore closure to mature into autophagosomes is companied with releasing some autophagy-related proteins(Atg),including Atg11,to the cytoplasm,while they are still on the unclosed autophagosomes.By observing with a laser confocal fluorescence microscopy,we found that Atg11 also entered vacuole and co-localized with the unclosed autophagosomes in the vacuoles in vps21?,which supported the conclusion in another way that unclosed autophagosomes can fuse with vacuole.Different from the traditional view that phagophores need to be closed to mature into autophagosomes before they can fuse with the vacuole to enter it for degradation,this study proposed a new model that unclosed autophagosomes can also fuse with the vacuole to enter it for degradation.Obviously,the molecular mechanism and physiolgy relevance of this new way still need further investigations.