Endolysosomal Inhibition Induces the Autophagy-Dependent Secretion of Autophagosomal Cargo

Autophagy is traditionally viewed as a degradative pathway in which cytoplasmic material is sequestered in double-membrane organelles called autophagosomes and delivered to the lysosome for breakdown. However, autophagy-related proteins (ATGs) have also been implicated in promoting unconventional secretion of proteins. We uncovered a role for the ATG conjugation system in the loading and secretion of proteins in small extracellular vesicles (EVs) separate from the canonical degradative autophagy pathway. To determine the interrelationships between degradative and secretory autophagy, I utilized a proteomic strategy to evaluate the secretome in response to pharmacologically inhibiting endolysosomal acidification, thereby blocking autophagic degradation. In contrast to secretion from cells with baseline autophagic activity, the secretome in response to endolysosomal inhibition is significantly enriched with autophagy-related proteins and autophagic cargoes. This suggests that impaired autophagic degradation in cells results in the redirection of degradative cargo to be secreted outside of the cell. To further investigate this pathway, I characterized the secretion of the autophagy regulator LC3 and its interacting cargo receptors (p62, NBR1, OPTN, NDP52), which selectively deliver cargo to the growing autophagosome but have not previously been implicated in secretory autophagy. These autophagy cargo receptors are secreted as unprotected EV-associated protein in an autophagosome formation-dependent manner, indicating a distinct mechanism requiring classical autophagosome formation for the secretion of these proteins. Furthermore, genetic knockdown of two separate SNARE proteins mediating autophagosome-lysosome fusion increases secretion of LC3 and autophagy cargo receptors. Overall, these findings establish a new relationship between degradative autophagy and secretory autophagy, in which the impaired degradation of autophagic cargo due to the lack of autophagosome-lysosome fusion results in a concomitant increase in EV-associated secretion of cargo receptors