The Sainsbury Laboratory, University of East Anglia, Norwich, United Kingdom.
Department of Biological Chemistry, John Innes Centre, Norwich, United Kingdom.
PLoS Biol. 2019 Jul 22;17(7):e3000373. doi: 10.1371/journal.pbio.3000373. eCollection 2019 Jul.
Autophagy-related protein 8 (ATG8) is a highly conserved ubiquitin-like protein that modulates autophagy pathways by binding autophagic membranes and a number of proteins, including cargo receptors and core autophagy components. Throughout plant evolution, ATG8 has expanded from a single protein in algae to multiple isoforms in higher plants. However, the degree to which ATG8 isoforms have functionally specialized to bind distinct proteins remains unclear. Here, we describe a comprehensive protein-protein interaction resource, obtained using in planta immunoprecipitation (IP) followed by mass spectrometry (MS), to define the potato ATG8 interactome. We discovered that ATG8 isoforms bind distinct sets of plant proteins with varying degrees of overlap. This prompted us to define the biochemical basis of ATG8 specialization by comparing two potato ATG8 isoforms using both in vivo protein interaction assays and in vitro quantitative binding affinity analyses. These experiments revealed that the N-terminal β-strand-and, in particular, a single amino acid polymorphism-underpins binding specificity to the substrate PexRD54 by shaping the hydrophobic pocket that accommodates this protein's ATG8-interacting motif (AIM). Additional proteomics experiments indicated that the N-terminal β-strand shapes the broader ATG8 interactor profiles, defining interaction specificity with about 80 plant proteins. Our findings are consistent with the view that ATG8 isoforms comprise a layer of specificity in the regulation of selective autophagy pathways in plants.
自噬相关蛋白 8（ATG8）是一种高度保守的泛素样蛋白，通过与自噬膜和许多蛋白质（包括货物受体和核心自噬成分）结合来调节自噬途径。在植物进化过程中，ATG8 从藻类中的单一蛋白扩展到高等植物中的多种同工型。然而，ATG8 同工型在结合不同蛋白质方面的功能专业化程度尚不清楚。在这里，我们描述了一个全面的蛋白质-蛋白质相互作用资源，该资源是使用植物体内免疫沉淀（IP） followed by mass spectrometry（MS）获得的，用于定义马铃薯 ATG8 相互作用组。我们发现 ATG8 同工型结合不同的植物蛋白组，具有不同程度的重叠。这促使我们通过使用体内蛋白质相互作用测定和体外定量结合亲和力分析比较两种马铃薯 ATG8 同工型，来定义 ATG8 专业化的生化基础。这些实验表明，N 端 β-链，特别是一个单一的氨基酸多态性，通过塑造容纳该蛋白质的 ATG8 相互作用基序（AIM）的疏水性口袋，为与底物 PexRD54 的结合特异性提供了基础。额外的蛋白质组学实验表明，N 端 β-链塑造了更广泛的 ATG8 相互作用蛋白谱，与大约 80 种植物蛋白的相互作用特异性有关。我们的发现与以下观点一致，即 ATG8 同工型在植物中选择性自噬途径的调节中构成了特异性的一层。
