RESUMEN
The endolysosome system plays central roles in both autophagic degradation and secretory pathways, including the release of extracellular vesicles and particles (EVPs). Although previous work reveals important interconnections between autophagy and EVP-mediated secretion, our understanding of these secretory events during endolysosome inhibition remains incomplete. Here, we delineate a secretory autophagy pathway upregulated in response to endolysosomal inhibition, which mediates EVP-associated release of autophagic cargo receptors, including p62/SQSTM1. This secretion is highly regulated and dependent on multiple ATGs required for autophagosome formation, as well as the small GTPase Rab27a. Furthermore, disrupting autophagosome maturation, either via genetic inhibition of autophagosome-to-autolysosome fusion or expression of SARS-CoV-2 ORF3a, is sufficient to induce EVP secretion of autophagy cargo receptors. Finally, ATG-dependent EVP secretion buffers against the intracellular accumulation of autophagy cargo receptors when classical autophagic degradation is impaired. Thus, we propose secretory autophagy via EVPs functions as an alternate route to clear sequestered material and maintain proteostasis during endolysosomal dysfunction or impaired autophagosome maturation.
Asunto(s)
Autofagia , Vesículas Extracelulares , Lisosomas , Proteostasis , Autofagosomas/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Lisosomas/metabolismo , SARS-CoV-2 , Proteína Sequestosoma-1 , Proteínas Viroporinas , Proteínas rab27 de Unión a GTPRESUMEN
CRISPR-Cas systems are adaptive immune systems that protect bacteria from bacteriophage (phage) infection1. To provide immunity, RNA-guided protein surveillance complexes recognize foreign nucleic acids, triggering their destruction by Cas nucleases2. 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 biosynthesis3,4, 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 σE orthologue) and AlgR repress CRISPR-Cas activity during planktonic and surface-associated growth5. AmrZ, another alginate regulator6, 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.
Asunto(s)
Alginatos/metabolismo , Bacterias/metabolismo , Bacterias/virología , Bacteriófagos/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Bacterias/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sistemas CRISPR-Cas , Proteínas de Unión al ADN/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Genes Reguladores/genética , Inmunidad , Fagos Pseudomonas/genética , Pseudomonas aeruginosa/metabolismo , Factores de Transcripción , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Autofagosomas/metabolismo , Autofagia/genética , Vesículas Extracelulares/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Unión al ARN/genética , Proteínas Adaptadoras del Transporte Vesicular/deficiencia , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Autofagosomas/química , Proteína 7 Relacionada con la Autofagia/deficiencia , Proteína 7 Relacionada con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/deficiencia , Proteínas Relacionadas con la Autofagia/genética , Transporte Biológico , Biotinilación , Vesículas Extracelulares/química , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células HEK293 , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lisosomas/química , Lisosomas/metabolismo , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteómica/métodos , Células RAW 264.7 , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , Proteínas de Unión al ARN/clasificación , Proteínas de Unión al ARN/metabolismo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/metabolismoRESUMEN
Autophagy-related (ATG) proteins have increasingly demonstrated functions other than cellular self-eating. In this issue, Mauthe et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201602046) conduct an unbiased RNA interference screen of the ATG proteome to reveal numerous noncanonical roles for ATG proteins during viral infection.