Multi-omic Analysis Of The Atg9 Molecular Network In Regulating Yeast Autophagy

Autophagy is an important catabolic process in cells and regulates cellular homeostasis through formation of autophagic vesicles in a bilayer structure,and delivery of cytoplasmic contents such as damaged or superfluous organelles,misfolded proteins,aggregates or intercellular pathogens,into lysosome(in animals)or vacuole(in yeasts or plants)for the degradation and recycling.Saccharomyces cerevisiae is a classic model organism for autophagy research,and currently 37 autophagy-related(ATG)genes have been discovered,including 24 that are conserved in mammalians with orthologous sequences.Thus,the core machinery of autophagy is highly conserved in eukaryotes.Atg9 is an important ATG in S.cerevisiae,and its protein product contains 6 transmembrane ?helices.In the stage of autophagic initiation,Atg9 physically interacts with other Atg proteins and autophagy regulators to form complex molecular networks and synergestically orchestrate early autophagic events such as membrane transport,phagophore nucleation and autophagosome formation.Currently,it's unclear that how many proteins in the Atg9 interacting network are involved in regulating autophagy.Also,how these proteins in coordination with Atg9 also remain to be studied.In this project,we selected wild-type(WT)and ATG9 knockout(atg9?)haploid yeast strains in BY4741 background.After nitrogen starvation t for 0,1 and 2 hours,we conducted the transcriptomic and proteomic profiling,and quantified 6,473 m RNAs and3,678 proteins,respectively.Further analyses demonstrated that the expression levels of290 m RNAs and 256 proteins were significantly changed in atg9? yeasts,including 11 genes significantly changed in both m RNA and protein levels.Combined with the results of enrichment analyses,we found that membrane transport-related processes were mainly enriched in the protein level,whereas translation-related processes were enriched in the m RNA level,indicating a different role of Atg9 in shaping transcription and translation.Together with protein-protein interactions in public databases,we obtained 341 known Atg9-interacting proteins,and found the m RNA or protein expression levels of known Atg proteins and autophagy regulators were not markedly altered.In order to find new autophagy regulator,we raised a new hypothesis that proteins with similar molecular characteristics might have similar functions,integrated the transcriptomic,proteomic and interactomic data,and designed a new algorithm named inference of functional interacting partners(i FIP).This algorithm integratively evaluated the similarity between candidate proteins and known Atg proteins or autophagy regulators in the transcriptional,translational and/or interacting levels,and in total predicted 42 Atg9-interacting proteins as potential autophagy regulators,including 15 reported Atg proteins or autophagy regulators.Using the library of Yeast Deletion Clones,we screened 17 Atg9-interacting proteins with corresponding knockout strains,and identified Glo3,a GTPase-activating protein(GAP),and a ceramide hydroxylase Scs7 to be functional in both selective and non-selective autophagy.During the nitrogen starvation-induced autophagic process,Atg9 markedly influences the m RNA and protein expression levels of Glo3 and Scs7,but does not change their sub-cellular localizations.The co-immunoprecipitation assay demonstrated that both Glo3 and Scs7 directly interact with Atg9,and their co-localizations with Atg9 are markedly enhanced in early stages of the autophagy process.Further experiments revealed that Glo3 but not Scs7 participates in regulating the retrograde transport of Atg9 during autophagy.Combined with 44 known and newly identified Atg9-interacting proteins that were involved in regulating yeast autophagy,we re-constructed the Atg9 molecular network with 6 functional modules,including protein/membrane transport,autophagosome assembly,autophagosome formation/maturation,vacuole fusion,Tor signaling and RNA regulation.Taken together,this research not only provided a new computational method for analyzing the mult-omic data,but also identified two new autophagy regulators,Glo3 and Scs7,through further experiments,as well as the elucidation of potential mechanisms of Atg9 molecular network in regulating yeast autophagy.We believe the techniques and methods used in this study can be extended to analyze other important molecular networks.