ESCRT Negatively Regulates Lipophagy By Suppressing The Enrichment Of Atg14 On Vacuolar Membranes In Saccharomyces Cerevisiae

Lipid droplets(LDs)are ubiquitous dynamic organelles that store intracellular neutral lipid such as triacylglycerol(TAG)and sterol esters(SE).They play a vital role in the storage,transport,distribution and consumption of lipids and further affect various life activities such as energy maintenance,redox homeostasis and membrane homeostasis.The degradation of lipid droplets was mainly attributed to two pathways:lipolysis and lipophagy.Lipolysis is catalysed by neutral lipases localizing on the surface of lipid droplets,whereas lipophagy is a process that autophagic machinery delivers lipid droplets to the vacuole(in yeast)or lysosome(in mammal)for degradation.The regulation of lipophagy in response to stress conditions in Saccharomyces cerevisiae has been reported to be associated with a variety of proteins including some Atg proteins and vacuolar protein sorting(Vps)proteins.The endosomal sorting complex required for transport(ESCRT)machinery,constituting mainly of Vps proteins,was divided into four subcomplexes(ESCRT-0,ESCRT-?,ESCRT-? and ESCRT-?)and other associated proteins such as ATPase Vps4.Several evidences have suggested that in yeast,ESCRT machinery plays a pivotal role in lipophagy under lipid stress,nitrogen starvation or after diauxic shift.However,it is unknown whether the ESCRT machinery is required for microlipophagy induced by glucose starvation.In addition,the vacuole was shown to uptake lipid droplets through its specific sterol enriched sites under glucose starvation and the formation of this site is mediated by Atg14 localization.Thus,if the ESCRT machinery is required for glucose starvation induced lipophagy,will it function through regulating Atg 14 localization?In this study,we investigated the roles of ESCRT subunits and Atg14 in glucose starvation induced lipophagy.Three main results are showed below:1.LD numbers significantly decreased in ESCRT mutant cells with the absence of subunit Vps27,Snf7 or Vps4Atgl5 is required for the vacuolar degradation of neutral lipids in LDs and autophagic bodies.It was also known that the targeting of Atg 15 to vacuoles depends on the ESCRT machinery.This raises the possibility that the ESCRT machinery may regulate the degradation of LDs.To investigate whether the ESCRT machinery plays a role in LD degradation,we observed the LDs stained with Nile Red,a lipophilic dye,in wild type,atg15? and representative ESCRT mutant(vps27?,snf7? and vps4?)cells by fluorescence microscopy.The results indicated that LD numbers significantly decreased in ESCRT mutant cells upon culturing to stationary phases under glucose restriction(GR),although not decrease as obviously as that in atg15? cells.2.Lipophagy is facilitated in an Atg15-dependent manner in representative ESCRT mutant cells under GRWe proposed that the observed decrease of LD numbers in ESCRT mutant cells under GR might be due to LD degradation in vacuoles through lipophagy.So we tagged LD protein Erg6 at its C-terminal with GFP and investigated the degradation of Erg6-GFP in wild type and ESCRT mutants and conducted epistasis analysis for atg15?snf7? and atg15?vps4? double mutants.We found that the degradation of Erg6-GFP was facilitated in ESCRT mutant(vps27?,snf7? and vps4?)cells and this process was dependent on Atg 15.Furthermore,when gradual glucose restriction(GGR)did not facilitate the degradation of Erg6-GFP,the acute glucose restriction(AGR)did.On the other hand,it has been reported that the absence of Vps4 blocks nitrogen starvation induced lipophagy.This study also investigated this type of lipophagy in ESCRT mutants under nitrogen starvation and found that after nitrogen starvation for 14 hours,Erg6-GFP entered the vacuoles of wild type cells and partially degraded while these processes were blocked in ESCRT mutants and atg15? cells,suggesting that lipophagies under GR and nitrogen starvation are regulated by different pathways and the ESCRT machinery plays opposite roles under these two conditions.In addition,to exclude the possibility that Erg6 is transported into the vacuoles and degraded by macroautophagy under GR,we also examined the macroautophagy marker protein,GFP-Atg8,for its delivery to vacuoles and degradation under GR.Fluorescence and immunoblot assay results showed that the degradation of GFP-Atg8 and maturation of prApel in ESCRT mutants were not facilitated under GR at 42 hours culturing,indicating that the promoted Erg6-GFP processing in ESCRT mutants is achieved by selective lipophagy,not macroautophagy.3.Atg14 aggregates as multiple puncta in ESCRT mutant cells and its localization on vacuolar membrane was further enriched under GR culture conditionsSince Atg14 has been reported to initiate lipophagy by redistributing from ER exit sites onto liquid-ordered membrane domains on vacuolar membranes,we next determined whether ESCRT regulated lipophagy through affecting Atg14 under GR.As the expression of endogenous Atg14 is very low,to clearly observe the localization of Atg14,we constructed a plasmid pRS415-CUP1pro-yEGFP-Atg14 to induce with copper ion to overexpress yEGFP-Atg14.The function of this plasmid in macroautophagy and lipophagy were validated After it was transformed into wild type and ESCRT mutants,we found that under GR Atg 14 mainly diffused in cytoplasm in wild type cells while it aggregated as multiple puncta and enriched onto the vacuole membranes in ESCRT mutants.Further cognate complementary assay of Snf7 in Snf7 mutant confirmed that this Atg14 distribution pattern in the ESCRT mutant cells is indeed coming from the absence of ESCRT subunits.Based on the above results,we proposed that the absence of ESCRT subunit facilitated lipophagy under prolonged GR through the enrichment of Atg 14 on vacuolar membranes in them,however,the absence of ESCRT subunit inhibited lipophagy under prolonged nitrogen starvation,indicating that the ESCRT complex machinery used different mechanisms to regulate lipophagy under prolonged GR and nitrogen starvation.