Systematically defining selective autophagy receptor-specific cargo using autophagosome content profiling

Autophagy deficiency in fed conditions leads to the formation of protein inclusions highlighting the contribution of this lysosomal delivery route to cellular proteostasis. Selective autophagy pathways exist that clear accumulated and aggregated ubiquitinated proteins. Receptors for this type of selective autophagy (aggrephagy) include p62, NBR1, TOLLIP and OPTN which all possess LC3-interacting regions and ubiquitin-binding domains (UBDs), thus working as a bridge between LC3/GABARAP proteins and ubiquitinated substrates. However, the identity of aggrephagy substrates and the redundancy of aggrephagy and related UBD-containing receptors remains elusive. Here, we combined proximity labeling and organelle enrichment with quantitative proteomics to systematically map the autophagic degradome targeted by UBD-containing receptors under basal and proteostasis challenging conditions. We therewith identified various new autophagy substrates, some of which were differentially engulfed by autophagosomal and endosomal membranes via p62 and TOLLIP, respectively. Overall, this resource will allow to dissect autophagy’s proteostasis contribution to numerous individual proteins.

在饱食状态下自噬功能的缺失导致蛋白质包涵体的形成，凸显了此溶酶体递送途径对细胞蛋白质稳态的贡献。存在选择性自噬途径，能够清除累积和泛素化的蛋白质。此类选择性自噬（聚集自噬）的受体包括p62、NBR1、TOLLIP和OPTN，它们均具备LC3相互作用区域和泛素结合域（UBDs），因而充当LC3/GABARAP蛋白与泛素化底物之间的桥梁。然而，聚集自噬底物的身份以及聚集自噬和相关UBD含有受体的冗余性仍属未知。在本研究中，我们结合邻近标记和细胞器富集技术，以及定量蛋白质组学，系统性地绘制了在基础状态和蛋白质稳态挑战条件下，由UBD含有受体靶向的自噬降解图谱。因此，我们识别了多种新的自噬底物，其中一些底物分别通过p62和TOLLIP被自噬体和内质膜选择性吞噬。总体而言，这一资源将有助于解析自噬对众多个体蛋白质蛋白质稳态贡献的作用。