The endoplasmic reticulum (ER) undergoes continuous remodelling via a selective autophagy pathway, known as ER-phagy¹. ER-phagy receptors have a central role in this process², but the regulatory mechanism remains largely unknown. Here we report that ubiquitination of the ER-phagy receptor FAM134B within its reticulon homology domain (RHD) promotes receptor clustering and binding to lipidated LC3B, thereby stimulating ER-phagy. Molecular dynamics (MD) simulations showed how ubiquitination perturbs the RHD structure in model bilayers and enhances membrane curvature induction. Ubiquitin molecules on RHDs mediate interactions between neighbouring RHDs to form dense receptor clusters that facilitate the large-scale remodelling of lipid bilayers. Membrane remodelling was reconstituted in vitro with liposomes and ubiquitinated FAM134B. Using super-resolution microscopy, we discovered FAM134B nanoclusters and microclusters in cells. Quantitative image analysis revealed a ubiquitin-mediated increase in FAM134B oligomerization and cluster size. We found that the E3 ligase AMFR, within multimeric ER-phagy receptor clusters, catalyses FAM134B ubiquitination and regulates the dynamic flux of ER-phagy. Our results show that ubiquitination enhances RHD functions via receptor clustering, facilitates ER-phagy and controls ER remodelling in response to cellular demands.

内质网 (ER) 通过称为 ER-phagy ¹的选择性自噬途径进行连续重塑。ER 吞噬受体在此过程中起着核心作用², 但监管机制在很大程度上仍然未知。在这里，我们报告了 ER 吞噬受体 FAM134B 在其网状同源域 (RHD) 内的泛素化促进受体聚集和与脂化 LC3B 的结合，从而刺激 ER 吞噬。分子动力学 (MD) 模拟显示泛素化如何扰乱模型双层中的 RHD 结构并增强膜曲率诱导。RHD 上的泛素分子介导相邻 RHD 之间的相互作用，形成致密的受体簇，促进脂质双层的大规模重塑。用脂质体和泛素化的 FAM134B 在体外重建膜重塑。使用超分辨率显微镜，我们在细胞中发现了 FAM134B 纳米团簇和微团簇。定量图像分析显示泛素介导的 FAM134B 寡聚化和簇大小增加。我们发现 E3 连接酶 AMFR 在多聚体 ER 吞噬受体簇中催化 FAM134B 泛素化并调节 ER 吞噬的动态通量。我们的结果表明，泛素化通过受体聚集增强 RHD 功能，促进 ER 吞噬并控制 ER 重塑以响应细胞需求。