Biochemical specialization of an ATG8 isoform in Nicotiana benthamiana

ATG8 is a highly-conserved ubiquitin-like protein that modulates autophagy pathways by binding autophagic membranes and numerous 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 followed by mass spectrometry, 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, particularly a single amino acid polymorphism, underpins binding specificity to the protein PexRD54. This isoleucine to valine substitution perturbed the hydrophobic pocket that accommodates the conserved ATG8 interacting motif of PexRD54. Additional proteomics experiments indicated that the N-terminal β-strand impacts ATG8 interactor profile by defining interaction specificity with about ~100 plant proteins. Our findings are consistent with the view that ATG8 isoforms comprise one layer of specificity in the regulation of selective autophagy pathways in plants.

ATG8是一类高度保守的泛素样蛋白（ubiquitin-like protein），通过结合自噬膜及货物受体、核心自噬组分等多种蛋白调控自噬通路。在植物演化历程中，ATG8已从藻类中的单一蛋白，扩展为高等植物中的多种同工型。但目前尚不清楚ATG8同工型在功能特化以结合不同蛋白的具体程度。本研究通过植物体内（in planta）免疫沉淀结合质谱分析获取全面的蛋白-蛋白互作资源，以此解析马铃薯ATG8互作组（interactome）。研究发现，不同ATG8同工型结合的植物蛋白子集存在差异，且互作重叠程度各不相同。基于该发现，我们通过体内蛋白互作实验与体外定量结合亲和力分析，对比两种马铃薯ATG8同工型，以阐明ATG8功能特化的生化基础。实验结果显示，N端β折叠链（β-strand），尤其是单个氨基酸多态性，决定了其与蛋白PexRD54的结合特异性。该异亮氨酸向缬氨酸的氨基酸置换，破坏了容纳PexRD54保守ATG8互作基序（ATG8 interacting motif）的疏水口袋。额外的蛋白质组学实验表明，N端β折叠链通过定义约100种植物蛋白的互作特异性，影响ATG8的互作谱。本研究结果支持“ATG8同工型构成植物选择性自噬通路调控特异性的一个层面”这一观点。