ABSTRACT

Reticulophagy, the autophagic degradation of the endoplasmic reticulum, is crucial to maintain ER homeostasis during stress. Although several reticulophagy receptors have been discovered recently, most of them have been studied using nutrient starvation. How macroautophagy/autophagy cross-talks with other ER-quality control mechanisms is largely unknown. Using ATG8-based affinity proteomics in the model plant Arabidopsis thaliana, we identified AT5G06830/C53, a soluble protein that directly interacts with ATG8. Biochemical and biophysical characterization of C53-ATG8 interaction using both human (CDK5RAP3) and Arabidopsis proteins revealed that C53 binds ATG8 via shuffled Atg8-family interacting motifs (sAIMs) located at its intrinsically disordered region (IDR). C53 is recruited to phagophores, precursors to autophagosomes, during ER stress in an autophagy-dependent manner. Consistently, c53 mutants are highly sensitive to ER stress treatments. C53 senses ER stress by forming a tripartite receptor complex that involves UFL1, the E3 ligase that mediates ufmylation, and its ER-resident adaptor protein DDRGK1. C53 activity is regulated by another ubiquitin-like protein, UFM1, which is transferred from C53 to the ribosomes upon ribosome collision/stalling at the ER, thereby activating the C53 pathway to recycle stalled nascent chains. Altogether our findings suggest C53 forms an ancient quality control pathway that links ribosome-associated quality control with selective autophagy at the ER.

摘要

网状吞噬是内质网的自噬降解，对于在应激期间维持内质网稳态至关重要。尽管最近发现了几种网状噬菌体受体，但其中大部分都是通过营养饥饿来研究的。巨自噬/自噬如何与其他 ER 质量控制机制相互作用在很大程度上是未知的。在模型植物拟南芥中使用基于 ATG8 的亲和蛋白质组学，我们确定了 AT5G06830/C53，一种可直接与 ATG8 相互作用的可溶性蛋白质。使用人类 (CDK5RAP3) 和拟南芥蛋白对 C53-ATG8 相互作用的生化和生物物理学表征表明，C53 通过位于其固有无序区域 (IDR) 的改组 Atg8 家族相互作用基序 (sAIM) 结合 ATG8。C53 在 ER 应激期间以自噬依赖的方式被募集到吞噬细胞，即自噬体的前体。一贯地，c53 突变体对 ER 应激处理高度敏感。C53 通过形成涉及 UFL1 的三方受体复合物来感知 ER 压力，UFL1 是介导 ufmylation 的 E3 连接酶，及其 ER 驻留衔接蛋白 DDRGK1。C53 活性受另一种泛素样蛋白 UFM1 调节，该蛋白在 ER 核糖体碰撞/停滞时从 C53 转移到核糖体，从而激活 C53 通路以回收停滞的新生链。总之，我们的研究结果表明 C53 形成了一个古老的质量控制途径，将核糖体相关的质量控制与 ER 的选择性自噬联系起来。