Mammalian Atg9 Contributes To The Post-Golgi Transport And Maturation Of Lysosomal Hydrolases By Interacting With Adaptor Protein-1

Autophagy is a highly conserved catabolic pathway dependent on lysosomes in eukaryotic cells.The biomacromolecules and damaged organelles can be sequestered into autophagosomes for lysosomal degradation.Autophagy plays a critical role in maintaining cellular homeostasis and responsing to environmental stress.More than thirty autophagy related proteins(Atgs)have been discovered.Atgs are involved in different stages of autophagy,such as the initiation,elongation,closure of autophagosomes and the fusion of autophagosomes with lysosomes.Atg9 is the only known transmembrane protein essential for autophagy,which contains six conserved transmembrane domains with both N-and C-terminus exposed to cytoplasm.Mammalian Atg9(mAtg9)is mainly distributed in the trans-Golgi network(TGN),and cycles between TGN,endosomes and plasma membrane.In response to starvation,mAtg9 vesicles are targeted to the initiation sites to provide membrane source for autophagosomes.However,under physiological conditions,the significance and molecular mechanism of mAtg9 cycling remain elusive.The structural characters,location and transport process of mAtg9 in the formation of autophagosomes indicate a potential role in intracellular protein and membrane trafficking.In this study,we found that in autophagy deficient ATG5 knockout(KO)and ATG7 knockout cells,mAtg9 is still localized in endosomes.The degradation of EGFR(epidermal growth factor receptor)and DQ Red BSA were impaired in mAtg9-depleted cells.Depletion of mAtg9 inhibited the maturation of lysosomal hydrolases cathepsin D/cathepsin L and resulted in the excretion of cathepsins.We found that mAtg9 can not only bind to adaptor protein-1(API)of clathrin vesicles,but also interact specifically with cation-independent mannose-6-phosphate receptor(CIMPR)by immunoprecipitation.We identified new API-binding motifs of mAtg9.Using mutants that unable to interact with AP1 and ATG9A knockout cells,we found that the interaction between mAtg9 and API can significantly improve the affinity between CIMPR and API,thereby promoting API oligomerization and cathepsin D maturation.These results indicate that under normal physiological conditions,mAtg9 located in TGN plays an important role in the transport of lysosomal hydrolases cathepsin D from TGN to late endosomes/lysosomes through interaction with AP1 and CIMPR.Our study found the new function of mAtg9 other than autophagy,suggesting that mAtg9 may serve as a co-receptor of lysosomal hydrolases for their TGN export by cycling between the TGN and endosomes.