Roles Of Partial Escrt Subunits In Macroautophagy And Lipophagy In Saccharomyces Cerevisiae

Autophagy is a conserved intracellular degradation pathway in eukaryotes.In this process,proteins and damaged organelles are isolated in a bilayer membrane vesicles and transported into lysosomes in mammalian cells(into vacuole in yeast or plants).Autophagy is divided into non-selective or selective pathway and the latter include Cytoplasm to vacuole targeting pathway,Mitophagy and Lipophagy.Under normal physiological conditions,autophagy occurs at a basic level and along with a variety of selective degradation pathway to maintain the normal cell life activities.When the cell is exposed to stress stimuli,autophagy is further induced to help cell survive by degradating and recycling of intracellular materials.With the ever-increasing research,it is found that many diseases in higher eukaryotes are associated with autophagy.Endosomal sorting complex required for transport complex(ESCRT)is a machinery of late endosome budding to form multivesicular bodies.The ESCRT machinery consists of five complexes,ESCRT-0,?,?,?,? and two accessory subunits,Brol and Doa4.Different subunit has upsteam and downsteam relation-ship.Their main function are to transport ubiquitin proteins into vacuole for degradation,and also involved in autophagy and Virus budding.At present,the study of the relationship between ESCRT and autophagy is widely focused.In recent years,studies have shown that autophagy defects will increase ESCRT proteins expression.But others said that autophagy defects in ESCRT mutants may be caused by MVB formation indelectly.In this study,we investigate the roles of ESCRT subunits in autophagy and lipophagy.Three main results are showed below:1.The absence of subunits of ESCRT-1 affects autophagy.Y2H show that Vps23 interact with Vps38,Vps15,Vps34 and Atg17,Atg20,Atg24The degradation of GFP-Atg8 and maturation of prApel can be used to reflect the situation of autophagy in yeast.In the mutant with the absence of ESCRT-I single subunit,some GFP-Atg8 cannot be transported into the vacuole and accumulated at the PAS,especially in vps23? mutants.Autophagy is strongly blocked in ESCRT-I all deletion mutants.2.Vps23 interact with Vps38,Vps15,Vps34 and Atg17,Atg20,Atg24Y2H assay showed that Vps23 interact with Vps38,Vps 15 and Vps34.What's more,Vps23 also interact with Atg17,Atg20 and Atg24.Then,we comfirm that Vps23 could interact with Atg17,Atg20 and Atg24 by BiFC assay,and the interaction posizition localizes on endosome and PAS site.To further investigate these interaction in autophagy,we constructed double deletion mutants vps23?atg17?,vps23?atg20?,vps23?atg24?.The degradation of GFP-Atg8 and maturation of prApe1 in these mutants significantly decreased compared with single mutant or wild type.3.The influence of localization between interaction proteinsGFP-Atg8 accumulated at PAS in vps23?atg17?mutant,which is similar to the phenotype in atg17?.Vps23 deficiency has no effects on Atg17 localization,while Atg17 deficiency causes Vps23 diffusion cytoplasm.At early stage of autophagy,Atg17 accumulates at the PAS as a scaffold protein firstly and recruit other Atg proteins.So we speculate that Atg17 is likely to be directly recruit Vps23 and then recruit other ESCRT proteins in autophagy.GFP-Atg8 is observed to accumulate on vacuole membrane as crescent-like structures in vps23?atg20? vps23?atg24?,which is similar to the phenotype in vps23?.These results show that autophagy is blocked at very late stage.In Vps23 deficiency cells,Atg20 and Atg24 accumulate on Class E compartment,while Atg20 and Atg24 have no effects on Vps23 localization.It has been reported that Atg20 and Atg24 participate in Cvt pathway.Atg20 and Atg24 deficiency could enhance autophagy defects in vps23?,showing that Atg20 and Atg24 might also involved in non-selective autophagy.4.The deficiency of ESCRT core subunits Snf7,Vps4 promotes lipophagyTriglycerides(TAG)in lipid droplets can be transported into lysosome through autophagy pathway and be dissolved by acid lipase,which is called lipophagy.Atg15 is a phospholipase in Saccharomyces cerevisiae,localizing in the vacuole.Recent study reported Atg 15 is involved in lipophagy.Atg15 is responsible for the degradation of triglycerides(TAG),which play an important role in maintaining the cycle of intracellular lipid droplets.Previous data in our lab showed that Atg15 accumulate at Class E in ESCRT mutants.So we are eager to explore the relationship between ESCRT proteins and lipophagy.Main results are showed below:The degradation rate of Erg6-GFP in snf7?,vps4? is 39%and 46%,while it is only 16%in wild type.In atg15?snf7?,atg15?snf7? or in atg15?,there is little degradation Erg6-GFP.Fluorescence results show that cells with Erg6 transported into vacuole make up 22%and 27%in snf7? and vps4?,much higher than that of wild type.These results suggest that Snf7,Vps4 deficiency promotes lipophagy.Tg13 and Tg14 is lipase in Saccharomyces cerevisiae.And they are responsible for degradation of triglycerides(TAG)into diacylglycerol(DAG),localizing on the lipid droplets.The colocalization rate between Tg13,Tg14 and LDs decrease in snf7?,vps4?.Western blot shows that the protein level of Tg13 and Tg14 decline compared with that of wild type.During transport,lipid droplets could contact with some organelles,such as mitochondria and endosome.Some think that this contact may provide convenience for the exchange of lipid between organelles.Under the condition of lipophagy,the colocalization between LDs and late endosome in vps4?significantly decline compared to wile type.This result shows that lipid droplets could't contact with late endosome normally.Above all,we showed that the absence of partial ESCRT subunits affect the normal autophagy as well as lipophagy in Saccharomyces cerevisiae.These studys will provide important evidence for the mechanism of ESCRT in autophagy and lipophagy.