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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.Leidal et al. show that the LC3-conjugation pathway, which is part of the autophagy machinery, controls extracellular vesicle cargo loading and secretion of RNA-binding proteins.

CRISPR–Cas systems are adaptive immune systems that protect bacteria from bacteriophage (phage) infection 1 . To provide immunity, RNA-guided protein surveillance complexes recognize foreign nucleic acids, triggering their destruction by Cas nucleases 2 . While the essential requirements for immune activity are well understood, the physiological cues that regulate CRISPR–Cas expression are not. Here, a forward genetic screen identifies a two-component system (KinB–AlgB), previously characterized in the regulation of Pseudomonas aeruginosa alginate biosynthesis 3 , 4 , as a regulator of the expression and activity of the P. aeruginosa Type I-F CRISPR–Cas system. Downstream of KinB–AlgB, activators of alginate production AlgU (a σ E orthologue) and AlgR repress CRISPR–Cas activity during planktonic and surface-associated growth 5 . AmrZ, another alginate regulator 6 , is triggered to repress CRISPR–Cas immunity upon surface association. Pseudomonas phages and plasmids have taken advantage of this regulatory scheme and carry hijacked homologs of AmrZ that repress CRISPR–Cas expression and activity. This suggests that while CRISPR–Cas regulation may be important to limit self-toxicity, endogenous repressive pathways represent a vulnerability for parasite manipulation. The identification of the KinB–AlgB two-component system, known to modulate alginate biosynthesis, together with downstream proteins that repress the Type I-F CRISPR–Cas system in Pseudomonas aeruginosa , elucidates how bacteria control the expression of nucleolytic host defence systems to minimize the potential risks of self-targeting.

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.

The endosome-lysosome (endolysosome) system plays central roles in both autophagic degradation and secretory pathways, including the exocytic release of extracellular vesicles and particles (EVPs). Although previous work has revealed important interconnections between autophagy and EVP-mediated secretion, our molecular understanding of these secretory events during endolysosome inhibition remains incomplete. Here, we delineate a secretory autophagy pathway upregulated in response to endolysosomal inhibition that mediates the EVP-associated extracellular release of autophagic cargo receptors, including p62/SQSTM1. This extracellular secretion is highly regulated and critically dependent on multiple ATGs required for the progressive steps of early autophagosome formation as well as Rab27a-dependent exocytosis. Furthermore, the disruption of autophagosome maturation, either due to genetic inhibition of the autophagosome-to-autolyosome fusion machinery or blockade via the SARS-CoV2 viral protein ORF3a, is sufficient to induce robust EVP-associated secretion of autophagy cargo receptors. Finally, we demonstrate that this ATG-dependent, EVP-mediated secretion pathway buffers against the intracellular accumulation of autophagy cargo receptors when classical autophagic degradation is impaired. Based on these results, we propose that secretory autophagy via EVPs functions as an alternate route to clear sequestered material and maintain proteostasis in response to endolysosomal dysfunction or impaired autophagosome maturation.

CRISPR-Cas systems are adaptive immune systems that protect bacteria from bacteriophage (phage) infection. To provide immunity, RNA-guided protein surveillance complexes recognize foreign nucleic acids, triggering their destruction by Cas nucleases. While the essential requirements for immune activity are well understood, the physiological cues that regulate CRISPR-Cas expression are not. Here, a forward genetic screen identifies a two-component system (KinB/AlgB), previously characterized in regulating Pseudomonas aeruginosa virulence and biofilm establishment, as a regulator of the biogenesis of the Type I-F CRISPR-Cas surveillance complex. Downstream of the KinB/AlgB system, activators of biofilm production AlgU (a σE orthologue) and AlgR, act as repressors of CRISPR-Cas surveillance complex expression during planktonic and surface-associated growth. AmrZ, another biofilm activator, functions as a surface-specific repressor of CRISPR-Cas activity. Pseudomonas phages and plasmids have taken advantage of this regulatory scheme, and carry hijacked homologs of AmrZ, which are functional CRISPR-Cas repressors. This suggests that while CRISPR-Cas regulation may be important to limit self-toxicity, endogenous repressive pathways represent a vulnerability for parasite manipulation. Footnotes * Figure 2 is revised to include both measurements of Cas3 and Csy complex, Figure 4 is revised to include functional characterization of 4 more mobile AmrZ homologs