The ATG5 interactome links clathrin-mediated vesicular trafficking with the autophagosome assembly machinery.
The ATG5 interactome links clathrin-mediated vesicular trafficking with the autophagosome assembly machinery.
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DOI：
10.1080/27694127.2022.2042054
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发表时间：
2022
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自噬体的形成涉及保守的ATG蛋白的顺序动作，以协调在新生的吞噬细胞膜的泛素样修饰剂Atg 8家族蛋白的脂化。尽管驱动这一过程的分子步骤已经很好地理解，但用于扩大吞噬细胞的膜的来源及其传递模式现在才开始被揭示。在这里，我们已经使用定量SILAC为基础的蛋白质组学，以确定与ATG 12-ATG 5共轭，在Atg 8家族蛋白质脂质化的关键球员相关联的蛋白质。我们的数据集揭示了网格蛋白介导的囊泡运输，包括网格蛋白重链和轻链，和几个网格蛋白衔接子的监管机构的强烈富集。还确定了PIK 3C 2A（一种参与网格蛋白介导的内吞作用的磷酸肌醇3-激酶）和HIP 1 R（网格蛋白囊泡的一种组分），这些蛋白质中任何一种的缺乏都会改变基于细胞的饥饿试验中的自噬通量。为了确定ATG 12-ATG 5缀合物是否影响内吞区室内的运输，我们在进食和饥饿条件下捕获了自噬能力和自噬能力低下的小鼠胚胎成纤维细胞的细胞表面蛋白质组。我们报告了单个细胞表面蛋白相对比例的变化，并表明SLC 7A 5-SLC 3A 2氨基酸转运蛋白的细胞表面水平受到自噬能力的影响。我们的数据提供了证据，直接监管耦合之间的ATG 12-ATG 5缀合物和网格蛋白膜运输系统，并建议候选的膜蛋白，其细胞内的运输可能是由自噬机制调制。缩略语：ATG，自噬相关; BafA 1，巴弗洛霉素A1; GFP，绿色荧光蛋白; HIP 1 R，亨廷顿蛋白相互作用蛋白1相关; MEF，小鼠胚胎成纤维细胞; PIK 3C 2A，磷脂酰肌醇-4-磷酸3-激酶催化亚基2 α型; SILAC，培养物中氨基酸的稳定同位素标记; SQSTM 1，多价螯合体1; STRING，用于检索相互作用基因/蛋白质的搜索工具
Autophagosome formation involves the sequential actions of conserved ATG proteins to coordinate the lipidation of the ubiquitin-like modifier Atg8-family proteins at the nascent phagophore membrane. Although the molecular steps driving this process are well understood, the source of membranes for the expanding phagophore and their mode of delivery are only now beginning to be revealed. Here, we have used quantitative SILAC-based proteomics to identify proteins that associate with the ATG12–ATG5 conjugate, a crucial player during Atg8-family protein lipidation. Our datasets reveal a strong enrichment of regulators of clathrin-mediated vesicular trafficking, including clathrin heavy and light chains, and several clathrin adaptors. Also identified were PIK3C2A (a phosphoinositide 3-kinase involved in clathrin-mediated endocytosis) and HIP1R (a component of clathrin vesicles), and the absence of either of these proteins alters autophagic flux in cell-based starvation assays. To determine whether the ATG12–ATG5 conjugate reciprocally influences trafficking within the endocytic compartment, we captured the cell surface proteomes of autophagy-competent and autophagy-incompetent mouse embryonic fibroblasts under fed and starved conditions. We report changes in the relative proportions of individual cell surface proteins and show that cell surface levels of the SLC7A5-SLC3A2 amino acid transporter are influenced by autophagy capability. Our data provide evidence for direct regulatory coupling between the ATG12–ATG5 conjugate and the clathrin membrane trafficking system and suggest candidate membrane proteins whose trafficking within the cell may be modulated by the autophagy machinery. Abbreviations: ATG, autophagy related; BafA1, bafilomycin A1; GFP, green fluorescent protein; HIP1R, huntingtin interacting protein 1 related; MEF, mouse embryo fibroblast; PIK3C2A, phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 alpha; SILAC, stable isotope labelling with amino acids in culture; SQSTM1, sequestosome 1; STRING, search tool for the retrieval of interacting genes/proteins