自噬体生物发生是一个复杂的过程,由Atg(自噬相关)蛋白之间的动态相互作用协调,以特定货物的周转为特征。随着时间的推移,这可能会有所不同,这取决于自噬是如何被刺激的。蛋白质组学分析是揭示蛋白质-蛋白质相互作用网络的核心,当与邻近依赖的生物素化或邻近标记(PL)方法结合时,它们还允许检测瞬时和弱相互作用。然而,目前酿酒酵母的PL程序,自噬研究的主要模型之一,不允许保持时间特异性,因此在自噬诱导后的精确时间点识别相互作用和货物。这里,我们提出了一种新的基于抗坏血酸过氧化物酶2(APEX2)的PL方案,该方案适用于酵母,该方案保留了时间特异性,并允许通过蛋白质印迹或蛋白质组学发现相邻蛋白质。作为概念的证明,我们应用这种新方法来鉴定Atg8和Atg9相互作用物,并在富氮和氮饥饿条件下检测到已知的结合伴侣以及潜在的未表征伴侣。此外,作为概念的证明,我们证实了Atg8和Faa1之间的空间邻近相互作用。我们相信,该协议将是所有研究酵母自噬机制和作用的研究人员的一个新的重要实验工具,还有这个模型生物中的其他细胞途径。缩写:APEX2,抗坏血酸过氧化物酶2,Atg,自噬相关;BP,生物素苯酚;Cvt,细胞质到液泡靶向;ER,内质网;LN2,液氮;MS,质谱;PAS,噬菌体组装位点;PL,邻近标签;PE,磷脂酰乙醇胺;PPINs,蛋白质-蛋白质相互作用网络;PPI,蛋白质-蛋白质相互作用;RT,室温;SAR,选择性自噬受体;WT,野生型。
Autophagosome biogenesis is a complex process orchestrated by dynamic interactions between Atg (autophagy-related) proteins and characterized by the turnover of specific cargoes, which can differ over time and depending on how autophagy is stimulated. Proteomic analyses are central to uncover protein-protein interaction networks and when combined with proximity-dependent biotinylation or proximity labeling (PL) approaches, they also permit to detect transient and weak interactions. However, current PL procedures for yeast Saccharomyces cerevisiae, one of the leading models for the study of autophagy, do not allow to keep temporal specificity and thus identify interactions and cargoes at a precise time point upon autophagy induction. Here, we present a new ascorbate peroxidase 2 (APEX2)-based PL protocol adapted to yeast that preserves temporal specificity and allows uncovering neighbor proteins by either western blot or proteomics. As a proof of concept, we applied this new method to identify
Atg8 and Atg9 interactors and detected known binding partners as well as potential uncharacterized ones in rich and nitrogen starvation conditions. Also, as a proof of concept, we confirmed the spatial proximity interaction between
Atg8 and Faa1. We believe that this protocol will be a new important experimental tool for all those researchers studying the mechanism and roles of autophagy in yeast, but also other cellular pathways in this model organism.Abbreviations: APEX2, ascorbate peroxidase 2, Atg, autophagy-related; BP, biotin phenol; Cvt, cytoplasm-to-vacuole targeting; ER, endoplasmic reticulum; LN2, liquid nitrogen; MS, mass spectrometry; PAS, phagophore assembly site; PL, proximity labeling; PE, phosphatidylethanolamine; PPINs, protein-protein interaction networks; PPIs, protein-protein interactions; RT, room temperature; SARs, selective autophagy receptors; WT, wild-type.