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 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.

The selective autophagy cargo receptor NBR1 enhances the disassembly of cell-matrix focal adhesions during cell migration.

Summary Autophagy traditionally sustains metabolism in stressed cells via promoting intracellular catabolism and nutrient recycling. Here, we demonstrate that in response to stresses requiring increased glycolytic demand, the core autophagy machinery also facilitates glucose uptake and glycolytic flux by promoting cell surface expression of the glucose transporter Glut1/Slc2a1. During metabolic stress, LC3+ autophagic compartments bind and sequester the RabGAP protein TBC1D5 away from its inhibitory interactions with the retromer complex, thereby enabling retromer recruitment to endosome membranes and Glut1 plasma membrane translocation. In contrast, TBC1D5 inhibitory interactions with the retromer are maintained in autophagy-deficient cells, leading to Glut1 mis-sorting into endolysosomal compartments. Furthermore, TBC1D5 depletion in autophagy deficient cells rescues retromer recruitment to endosomal membranes and Glut1 surface recycling. Hence, TBC1D5 shuttling to autophagosomes during metabolic stress facilitates retromer-dependent Glut1 trafficking. Overall, our results illuminate key interconnections between the autophagy and endosomal pathways dictating Glut1 trafficking and extracellular nutrient uptake.