Centre for Protein Science and Crystallography, State Key Laboratory of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China.
Centre for Cell and Developmental Biology, State Key Laboratory of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China.
Autophagy. 2022 Jun;18(6):1350-1366. doi: 10.1080/15548627.2021.1976965. Epub 2021 Oct 17.
In selective macroautophagy/autophagy, cargo receptors are recruited to the forming autophagosome by interacting with Atg8 (autophagy-related 8)-family proteins and facilitate the selective sequestration of specific cargoes for autophagic degradation. In addition, Atg8 interacts with a number of adaptors essential for autophagosome biogenesis, including ATG and non-ATG proteins. The majority of these adaptors and receptors are characterized by an Atg8-family interacting motif (AIM) for binding to Atg8. However, the molecular basis for the interaction mode between ATG8 and regulators or cargo receptors in plants remains largely unclear. In this study, we unveiled an atypical interaction mode for Arabidopsis ATG8f with a plant unique adaptor protein, SH3P2 (SH3 domain-containing protein 2), but not with the other two SH3 proteins. By structure analysis of the unbound form of ATG8f, we identified the unique conformational changes in ATG8f upon binding to the AIM sequence of a plant known autophagic receptor, NBR1. To compare the binding affinity of SH3P2-ATG8f with that of ATG8f-NBR1, we performed a gel filtration assay to show that ubiquitin-associated domain of NBR1 outcompetes the SH3 domain of SH3P2 for ATG8f interaction. Biochemical and cellular analysis revealed that distinct interfaces were employed by ATG8f to interact with NBR1 and SH3P2. Further subcellular analysis showed that the AIM-like motif of SH3P2 is essential for its recruitment to the phagophore membrane but is dispensable for its trafficking in endocytosis. Taken together, our study provides an insightful structural basis for the ATG8 binding specificity toward a plant-specific autophagic adaptor and a conserved autophagic receptor. ATG, autophagy-related; AIM, Atg8-family interacting motif; BAR, Bin-Amphiphysin-Rvs; BFA, brefeldin A; BTH, benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester; CCV, clathrin-coated-vesicle; CLC2, clathrin light chain 2; Conc A, concanamycin A; ER, endoplasmic reticulum; LDS, LIR docking site; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; LIR, LC3-interacting region; PE, phosphatidylethanolamine; SH3P2, SH3 domain containing protein 2; SH3, Src-Homology-3; UBA, ubiquitin-associated; UIM, ubiquitin-interacting motif.
在选择性的巨自噬/自噬中，货物受体通过与 Atg8（自噬相关 8 家族）蛋白相互作用被募集到正在形成的自噬体中，并促进特定货物的选择性隔离进行自噬降解。此外，Atg8 与许多对自噬体生物发生至关重要的衔接蛋白相互作用，包括 ATG 和非 ATG 蛋白。这些衔接蛋白和受体的大多数都具有用于与 Atg8 结合的 Atg8 家族相互作用基序（AIM）。然而，植物中 ATG8 和调节剂或货物受体之间相互作用模式的分子基础在很大程度上仍不清楚。在这项研究中，我们揭示了拟南芥 Atg8f 与植物特有的衔接蛋白 SH3P2（SH3 结构域包含蛋白 2）之间的一种非典型相互作用模式，但与其他两种 SH3 蛋白没有这种相互作用模式。通过对未结合形式的 Atg8f 进行结构分析，我们在 Atg8f 结合到已知的植物自噬受体 NBR1 的自噬反应的 AIM 序列时，鉴定了 Atg8f 的独特构象变化。为了比较 SH3P2-Atg8f 与 Atg8f-NBR1 的结合亲和力，我们进行了凝胶过滤实验，结果表明 NBR1 的泛素相关结构域与 SH3P2 的 SH3 结构域竞争 Atg8f 的相互作用。生化和细胞分析表明，Atg8f 分别使用不同的界面与 NBR1 和 SH3P2 相互作用。进一步的亚细胞分析表明，SH3P2 的 AIM 样基序对于其募集到吞噬泡膜是必需的，但对于其在胞吞作用中的运输是可有可无的。总的来说，我们的研究为 Atg8 与植物特异性自噬衔接蛋白和保守的自噬受体的结合特异性提供了一个有见地的结构基础。ATG，自噬相关；AIM，Atg8 家族相互作用基序；BAR，Bin-Amphiphysin-Rvs；BFA，布雷菲德菌素 A；BTH，苯并-[1,2,3]-噻二唑-7-羧酸 S-甲酯；CCV，网格蛋白包被小泡；CLC2，网格蛋白轻链 2；Conc A，康那霉素 A；ER，内质网；LDS，LIR 对接位点；MAP1LC3/LC3，微管相关蛋白 1 轻链 3；LIR，LC3 相互作用区域；PE，磷脂酰乙醇胺；SH3P2，SH3 结构域包含蛋白 2；SH3，Src-Homology-3；UBA，泛素相关；UIM，泛素相互作用基序。
