Traditionally viewed as an autodigestive pathway, autophagy also facilitates cellular secretion; however, the mechanisms underlying these processes remain unclear. Here, we demonstrate that components of the autophagy machinery specify secretion within extracellular vesicles (EVs). Using a proximity-dependent biotinylation proteomics strategy, we identify 200 putative targets of LC3-dependent secretion. This secretome consists of a highly interconnected network enriched in RNA-binding proteins (RBPs) and EV cargoes. Proteomic and RNA profiling of EVs identifies diverse RBPs and small non-coding RNAs requiring the LC3-conjugation machinery for packaging and secretion. Focusing on two RBPs, heterogeneous nuclear ribonucleoprotein K (HNRNPK) and scaffold-attachment factor B (SAFB), we demonstrate that these proteins interact with LC3 and are secreted within EVs enriched with lipidated LC3. Furthermore, their secretion requires the LC3-conjugation machinery, neutral sphingomyelinase 2 (nSMase2) and LC3-dependent recruitment of factor associated with nSMase2 activity (FAN). Hence, the LC3-conjugation pathway controls EV cargo loading and secretion.

自噬传统上被视为一种自体消化途径，但它也促进细胞分泌。然而，这些过程背后的机制仍不清楚。在这里，我们证明自噬机制的组成部分指定细胞外囊泡（EV）内的分泌。使用邻近依赖性生物素化蛋白质组学策略，我们确定了 200 个 LC3 依赖性分泌的推定靶标。该分泌蛋白组由高度互连的网络组成，富含 RNA 结合蛋白 (RBP) 和 EV 货物。 EV 的蛋白质组学和 RNA 分析可识别需要 LC3 缀合机制进行包装和分泌的不同 RBP 和小非编码 RNA。重点关注两种 RBP，即异质核核糖核蛋白 K (HNRNPK) 和支架附着因子 B (SAFB)，我们证明这些蛋白质与 LC3 相互作用，并在富含脂化 LC3 的 EV 中分泌。此外，它们的分泌需要 LC3 结合机制、中性鞘磷脂酶 2 (nSMase2) 和 LC3 依赖性招募与 nSMase2 活性相关的因子 (FAN)。因此，LC3 缀合途径控制 EV 货物的装载和分泌。