Specific autophagy and ESCRT components participate in the unconventional secretion of CFTR

Abstract

The most common mutation in cystic fibrosis patients is a phenylalanine deletion at position 508 (ΔF508) in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. This mutation impairs cell-surface trafficking of CFTR. During cellular stress, core-glycosylated CFTRΔF508 is transported to the cell surface from the endoplasmic reticulum (ER) via an unconventional route that bypasses the Golgi. However, the mechanisms for this unconventional secretory pathway of CFTR are not well delineated. Here, we report that components of the macroautophagy/autophagy and ESCRT (endosomal sorting complex required for transport) pathways are involved in unconventional secretion of CFTR. In mammalian cells, we found that autophagic pathways were modulated by conditions that also stimulate unconventional secretion, namely ER stress and an ER-to-Golgi transport blockade. Additionally, we found that knockdown of early autophagy components, ATG5 and ATG7, and treatment with pharmacological autophagy inhibitors, wortmannin and 3-methyladenine, abolished the unconventional secretion of CFTR that had been stimulated by ER stress and an ER-to-Golgi blockade. Interestingly, immunoelectron microscopy revealed that GORASP2/GRASP55, which mediates unconventional CFTR trafficking, is present in multivesicular bodies (MVB) and autophagosomal structures under ER stress conditions. A custom small-interfering RNA screen of mammalian ESCRT proteins that mediate MVB biogenesis showed that silencing of some ESCRTs, including MVB12B, inhibited unconventional CFTRΔF508 secretion. Furthermore, MVB12B overexpression partially rescued cell-surface expression and Cl channel function of CFTRΔF508. Taken together, these results suggest that components involved in early autophagosome formation and the ESCRT/MVB pathway play a key role in the stress-induced unconventional secretion of CFTR.

Lysosomal Exocytosis, Exosome Release and Secretory Autophagy: The Autophagic- and Endo-Lysosomal Systems Go Extracellular

“…Interestingly, membrane proteins have also been shown to be recruited to the plasma membrane by secretory autophagy. Indeed, specific autophagy and ESCRT components participate in cystic fibrosis transmembrane conductance regulator (CFTR) unconventional secretion, thus suggesting that the autophagic machinery may play a specific role in cystic fibrosis pathogenesis [101].…”

“…Conversely, other components of the macroautophagic machinery appear to be dispensable, such as Ulk1 [104], whereas some, such as the activation of the protein Rubicon, commit the cell to LAP, inhibiting autophagy [107]. LAP is involved in the response to pathogens [101], but also in the elimination of dead cells [108], including the finalization of the entotic process, by which cells engulf and cannibalize other cells [109].…”

GORASPs link Golgi cisternae laterally to stabilize the rims and prevent vesiculation

“…Clearly, genetics tells us that GORASPs have a role in unconventional secretion. This conclusion is now supported by a large number of reports showing the involvement of GORASPs in other systems including mammalian cells (Feinstein & Linstedt, 2008;Noh et al, 2018;van Ziel, Largo-Barrientos, Wolzak, Verhage, & Scheper, 2019;Wu, Li, & Zhao, 2020). Although transport by COPII and COPI is not required for unconventional secretion, the essential role of GORASPs in this process suggests a link to the Golgi membranes.…”

IRE1α kinase–mediated unconventional protein secretion rescues misfolded CFTR and pendrin

“…The IRE1 kinase-ASK1 cascade was suggested to play a role in the initiation of autophagosome formation by modulating Beclin-1 (38). Components of the early autophagosome formation, such as autophagy related 5 (ATG5) and autophagy related 7 (ATG7), participate in the vesicular structures involved in ER stress-associated UPS of CFTR (14). In addition, IRE1 is an upstream signal that regulates the generation and anchoring of Sec16A at the ER exit sites, which increases the ER export of core-glycosylated CFTRs during UPS in response to ER stress (15).…”

“…Although recent studies have identified several key molecules associated with the UPS of membrane proteins (i.e., Sec16A in ER exit and early autophagy/multivesicular body components in the vesicular pathway for UPS of CFTR) (14,15), the entire complex of UPS regulatory mechanisms remains unknown. In general, most UPS is not constitutive but instead is stress induced (16).…”