Key Laboratory of Molecular Biophysics of Ministry of Education, Hubei Bioinformatics and Molecular Imaging Key Laboratory, Center for Artificial Intelligence Biology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei China.
Nanjing University Institute of Artificial Intelligence Biomedicine, Nanjing, Jiangsu China.
Autophagy. 2021 Dec;17(12):4453-4476. doi: 10.1080/15548627.2021.1898749. Epub 2021 Mar 15.
In , Atg9 is an important autophagy-related (Atg) protein, and interacts with hundreds of other proteins. How many Atg9-interacting proteins are involved in macroautophagy/autophagy is unclear. Here, we conducted a multi-omic profiling of Atg9-dependent molecular landscapes during nitrogen starvation-induced autophagy, and identified 290 and 256 genes to be markedly regulated by in transcriptional and translational levels, respectively. Unexpectedly, we found most of known Atg proteins and autophagy regulators that interact with Atg9 were not significantly changed in the mRNA or protein level during autophagy. Based on a hypothesis that proteins with similar molecular characteristics might have similar functions, we developed a new method named inference of functional interacting partners (iFIP) to integrate the transcriptomic, proteomic and interactomic data, and predicted 42 Atg9-interacting proteins to be potentially involved in autophagy, including 15 known Atg proteins or autophagy regulators. We validated 2 Atg9-interacting partners, Glo3 and Scs7, to be functional in both bulk and selective autophagy. The mRNA and protein expressions but not subcellular localizations of Glo3 and Scs7 were affected with or without during autophagy, whereas the colocalizations of the 2 proteins and Atg9 were markedly enhanced at early stages of the autophagic process. Further analyses demonstrated that Glo3 but not Scs7 regulates the retrograde transport of Atg9 during autophagy. A working model was illustrated to highlight the importance of the Atg9 interactome. Taken together, our study not only provided a powerful method for analyzing the multi-omics data, but also revealed 2 new players that regulate autophagy. ALP: alkaline phosphatase; Arf1: ADP-ribosylation factor 1; Atg: autophagy-related; Co-IP: co-immunoprecipitation; Cvt: cytoplasm-to-vacuole targeting; DEM: differentially expressed mRNA; DEP: differentially expressed protein; DIC: differential interference contrast; E-ratio: enrichment ratio; ER: endoplasmic reticulum; ES: enrichment score; FC: fold change; FPKM: fragments per kilobase of exon per million fragments mapped; GAP: GTPase-activating protein; GFP: green fluorescent protein; GO: gene ontology; GSEA: gene set enrichment analysis; GST: glutathione S-transferase; HA: hemagglutinin; iFIP: inference of functional interacting partners; KO: knockout; LR: logistic regression; OE: over-expression; PAS: phagophore assembly site; PPI: protein-protein interaction; RFP: red fluorescence protein; RNA-seq: RNA sequencing; RT-PCR: real-time polymerase chain reaction; SCC: Spearman's correlation coefficient; SD-N: synthetic minimal medium lacking nitrogen; THANATOS: The Autophagy, Necrosis, ApopTosis OrchestratorS; Vsn: variance stabilization normalization; WT: wild-type.
在酵母中，Atg9 是一种重要的自噬相关（Atg）蛋白，与数百种其他蛋白质相互作用。在氮饥饿诱导的自噬过程中，有多少与 Atg9 相互作用的蛋白质参与巨自噬/自噬尚不清楚。在这里，我们对氮饥饿诱导自噬过程中依赖 Atg9 的分子景观进行了多组学分析，分别鉴定了 290 个和 256 个基因在转录和翻译水平上受到显著调控。出乎意料的是，我们发现大多数已知的与 Atg9 相互作用的 Atg 蛋白和自噬调节剂在自噬过程中其 mRNA 或蛋白水平没有明显变化。基于一种假设，即具有相似分子特征的蛋白质可能具有相似的功能，我们开发了一种名为功能相互作用伙伴推断（iFIP）的新方法，以整合转录组、蛋白质组和相互作用组数据，并预测了 42 种可能参与自噬的 Atg9 相互作用蛋白，包括 15 种已知的 Atg 蛋白或自噬调节剂。我们验证了 2 种 Atg9 相互作用伙伴 Glo3 和 Scs7 在整体和选择性自噬中均具有功能。Glo3 和 Scs7 的 mRNA 和蛋白表达在自噬过程中受到影响，但亚细胞定位不受影响，而这 2 种蛋白与 Atg9 的共定位在自噬过程的早期阶段显著增强。进一步的分析表明，Glo3 而不是 Scs7 调节自噬过程中 Atg9 的逆行运输。一个工作模型被阐明，以突出 Atg9 相互作用组的重要性。总之，我们的研究不仅提供了一种分析多组学数据的强大方法，还揭示了 2 种新的调节自噬的因子。ALP：碱性磷酸酶；Arf1：ADP-核糖基化因子 1；Atg：自噬相关；Co-IP：共免疫沉淀；Cvt：细胞质到液泡靶向；DEM：差异表达 mRNA；DEP：差异表达蛋白；DIC：微分干涉对比；E-ratio：富集比；ER：内质网；ES：富集分数；FC：倍数变化；FPKM：每百万映射片段的外显子片段的每千碱基片段；GAP：GTPase 激活蛋白；GFP：绿色荧光蛋白；GO：基因本体论；GSEA：基因集富集分析；GST：谷胱甘肽 S-转移酶；HA：血凝素；iFIP：功能相互作用伙伴推断；KO：敲除；LR：逻辑回归；OE：过表达；PAS：噬菌斑组装位点；PPI：蛋白质-蛋白质相互作用；RFP：红色荧光蛋白；RNA-seq：RNA 测序；RT-PCR：实时聚合酶链反应；SCC：Spearman 相关系数；SD-N：缺乏氮的合成最小培养基；THANATOS：自噬、坏死、凋亡协调器；Vsn：方差稳定归一化；WT：野生型。
