A Study On The Mechanism For Recycling Of Autophagosomal Membrane Components

objective Autophagy is a response of cells to changes from internal and external environment,and is a degraded/recycled system widely present in eukaryotic cells.The fusion of autophagosomes encapsulating the substrate to be degraded with lysosomes is to form autolysosomes.The substrate in autolysosomal lumens is degraded into small molecules such as amino acids under action of various hydrolases,then enters cells for recycling.The autolysosomal membrane component mainly includes two parts,one part comes from autophagosomes,the other part comes from lysosomes.The membrane component from lysosomes undergoes budding and stretching to reform pro-lysosomes,but membrane components of autophagosomes by taking up membranes from multiple organelles including the endoplasmic reticulum,mitochondria,plasma membrane,Golgi and so on are more complicated and the fate of autophagosomal membrane components is still unknown.However,no matter which step of autophagy is disordered or blocked,it will lead to neurodegenerative diseases,tumors,immune damage,aging and other major diseases.Therefore,this study focuses on discussing the fate of autophagosomal components and explore the mechanism,which will build a solid foundation for further improving the autophagic process and serving the clinic.Method 1.Stable cell lines were constructed by the technology of lentiviral packaging.2.Dynamic phenomena in cells were observed by confocal microscopy.3.The localization of target protein in cells was detected by immunofluorescence(IF).4.The function of the target protein was detected by si RNA.5.The function of the target protein was verified by CRISPR Cas9.6.The interaction between intracellular proteins and proteins was examined by coimmunoprecipitation(IP).7.The direct interaction between proteins and proteins in vitro was verified by pull-down experiments.8.The distribution of the target protein in cells was detected by cell fractionation.9.The membrane protein was identified by proteinase K assay.10.The autophagic flux was detected by immunoblotting and flow cytometry.Results 1.Autophagosomal transmembrane protein Syntaxin 17(STX17)is not degraded during autophagy,but recycling from autolysosomal membranes.2.Sorting nexin 4(SNX4)and Sorting nexin 5(SNX5)are required for the recycling of STX17 from autolysosomal membranes.3.SNX4 and SNX5 form a heterodimer through their BAR domain to assist STX17 to be sorted and recycled from autolysosomal membranes.4.Sorting nexin 17(SNX17)acts as an adaptor to link STX17-SNX4-SNX5 and dyneindynactin(DCTN1)together to pull STX17 off from autolysosomal membranes.5.SNX4,SNX5 and SNX17 forms a new complex,named recycler,to help STX17 recycling from autolysosomal membranes.6.ATG9 A is retrieved from autolysosomes by the recycler complex.7.Recycler deficiency inhibits the degradation of autophagic substrates and blocks the fusion of autophagosomes and lysosomes.Conclusion During autophagy,autophagosomal components(STX17 and ATG9A)are not degraded,but are sorted and recycled by a new complex composed of SNX4,SNX5 and SNX17.In this study,the recycling of autophagosomal components from autolysosomal membranes is named "ACR"(Autophagosomal Components Recycling),and the new complex with SNX4,SNX5 and SNX17 constituted is called "recycler".Recycler deficiency will result in accumulating of cargoes(STX17 and ATG9A)on autolysosomal membranes and blocking autophagy.Therefore,this study discovers a new phenomenon on autolysosomal membranes-the recycling of autophagosomal components and clearly reveals its recycling mechanism and significance,files the gap in the autophagy process and provide a new direction for exploring the relationship between autophagy and disease.