Peroxisomes are ubiquitous organelles that house various metabolic reactions and are essential for human health^1,2,3,4. Luminal peroxisomal proteins are imported from the cytosol by mobile receptors, which then recycle back to the cytosol by a poorly understood process^1,2,3,4. Recycling requires receptor modification by a membrane-embedded ubiquitin ligase complex comprising three RING finger domain-containing proteins (Pex2, Pex10 and Pex12)^5,6. Here we report a cryo-electron microscopy structure of the ligase complex, which together with biochemical and in vivo experiments reveals its function as a retrotranslocation channel for peroxisomal import receptors. Each subunit of the complex contributes five transmembrane segments that co-assemble into an open channel. The three ring finger domains form a cytosolic tower, with ring finger 2 (RF2) positioned above the channel pore. We propose that the N terminus of a recycling receptor is inserted from the peroxisomal lumen into the pore and monoubiquitylated by RF2 to enable extraction into the cytosol. If recycling is compromised, receptors are polyubiquitylated by the concerted action of RF10 and RF12 and degraded. This polyubiquitylation pathway also maintains the homeostasis of other peroxisomal import factors. Our results clarify a crucial step during peroxisomal protein import and reveal why mutations in the ligase complex cause human disease.

过氧化物酶体是普遍存在的细胞器，可容纳各种代谢反应，对人类健康至关重要^1,2,3,4 。管腔过氧化物酶体蛋白通过移动受体从细胞质中输入，然后通过一个鲜为人知的过程再循环回细胞质中^1,2,3,4 。回收需要通过膜嵌入的泛素连接酶复合物对受体进行修饰，该复合物包含三个含环指结构域的蛋白质（Pex2、Pex10 和 Pex12） ^5,6 。在这里，我们报告了连接酶复合物的冷冻电子显微镜结构，该结构与生化和体内实验一起揭示了其作为过氧化物酶体输入受体的逆转位通道的功能。该复合物的每个亚基都贡献五个跨膜片段，这些跨膜片段共同组装成一个开放通道。三个无名指结构域形成一个胞质塔，其中无名指 2 (RF2) 位于通道孔上方。我们建议将再循环受体的 N 末端从过氧化物酶体腔插入孔中，并通过 RF2 进行单泛素化，以便能够提取到细胞质中。如果回收受到损害，受体会因 RF10 和 RF12 的协同作用而被多泛素化并降解。这种多泛素化途径还维持其他过氧化物酶体输入因子的稳态。我们的结果阐明了过氧化物酶体蛋白输入过程中的关键步骤，并揭示了连接酶复合物中的突变为何会导致人类疾病。