An ATG12‐ATG5‐TECPR1 E3‐like complex regulates unconventional LC3 lipidation at damaged lysosomes

Abstract

Lysosomal membrane damage represents a threat to cell viability. As such, cells have evolved sophisticated mechanisms to maintain lysosomal integrity. Small membrane lesions are detected and repaired by the endosomal sorting complex required for transport (ESCRT) machinery while more extensively damaged lysosomes are cleared by a galectin‐dependent selective macroautophagic pathway (lysophagy). In this study, we identify a novel role for the autophagosome‐lysosome tethering factor, TECPR1, in lysosomal membrane repair. Lysosomal damage promotes TECPR1 recruitment to damaged membranes via its N‐terminal dysferlin domain. This recruitment occurs upstream of galectin and precedes the induction of lysophagy. At the damaged membrane, TECPR1 forms an alternative E3‐like conjugation complex with the ATG12‐ATG5 conjugate to regulate ATG16L1‐independent unconventional LC3 lipidation. Abolishment of LC3 lipidation via ATG16L1/TECPR1 double knockout impairs lysosomal recovery following damage.

ESCRT recruitment to damaged lysosomes is dependent on the ATG8 E3-like ligases

“…Ca 2+ leakage will induce the scrambling of sphingomyelin (SM) from the luminal to cytoplasmic membrane surface of the lysosomal membrane (24). SM will subsequently recruit the TECPR1-ATG5-ATG12 E3-like ligase to the damaged membrane (21)(22)(23). Here we show that ATG8 E3-like ligase translocation is an essential prerequisite to ESCRT recruitment, thereby placing the ATG8 E3-like ligases in the role of damage sensors for the ESCRT-mediated membrane repair pathway.…”

“…We and others recently identified a TECPR1-dependent autophagy E3-like ligase complex which functions independently of ATG16L1 to regulate unconventional ATG8 lipidation at damaged lysosomal membranes (21)(22)(23). Double knockout of both ATG16L1 and TECPR1 was shown to compromise the repair of damaged membranes, suggesting a functionally redundant role for both E3-like ligase complexes in the repair process (21).…”

“…We and others recently identified a TECPR1-dependent autophagy E3-like ligase complex which functions independently of ATG16L1 to regulate unconventional ATG8 lipidation at damaged lysosomal membranes (21)(22)(23). Double knockout of both ATG16L1 and TECPR1 was shown to compromise the repair of damaged membranes, suggesting a functionally redundant role for both E3-like ligase complexes in the repair process (21). To determine the mechanism through which the E3-like ligase complexes contribute to membrane repair, we To further characterize the mechanism behind E3-like ligase-dependent ESCRT recruitment, E3-DKO addback cell lines were generated which stably express wild type (WT) TECPR1, or one of two TECPR1 mutants.…”

“…To determine the mechanism through which the E3-like ligase complexes contribute to membrane repair, we To further characterize the mechanism behind E3-like ligase-dependent ESCRT recruitment, E3-DKO addback cell lines were generated which stably express wild type (WT) TECPR1, or one of two TECPR1 mutants. TECPR1 Δ1-377 is lacking the N-terminal dysferlin domain required for lysosomal translocation in response to membrane damage (21). TECPR1 ΔAIR is lacking the ATG5 interaction region (AIR) (26), preventing co-recruitment of ATG5 to the damaged membrane (Fig S3).…”

“…In the context of endolysosomal damage, ATG16L1 is recruited to damaged membranes via interaction with the Vacuolar type ATPase (V-ATPase), activated in response to damageinduced loss of the proton gradient (18,19) (a process referred to as V-ATPase-ATG16L1induced ATG8 lipidation (VAIL) (20)). Recently, we and others identified a second E3-like ligase complex which utilizes the autophagosome-lysosome tethering factor, Tectonin betapropeller repeat containing 1 (TECPR1), in place of ATG16L1 (21)(22)(23). TECPR1 recognizes damaged membranes via interaction with sphingomyelin, a membrane lipid that translocates from the luminal to cytoplasmic membrane surface in response to damage (24) (a process that we call sphingomyelin-TECPR1-induced ATG8 lipidation (STIL)).…”