TECPR1 provides E3-ligase like activity to the ATG5-ATG12 complex to conjugate LC3/ATG8 to damaged lysosomes

Abstract Autophagosomes deliver cytosolic material to lysosomes to provide amino acids during starvation and to degrade damaged proteins and organelles to maintain tissue homeostasis. Delivery to lysosomes requires LC3/ATG8 (LC3), the major membrane protein of the autophagosome, that uses adaptor proteins to capture cargo and recruits tethering and SNARE proteins to promote fusion with lysosomes. LC3 is also recruited to endo-lysosome compartments in response to increases in vacuolar pH to facilitate degradation of material entering cells by endocytosis. A series of ubiquitin-like reactions conjugate LC3 to amino groups exposed by phosphatidylethanolamine (PE) or phosphatidylserine (PS) in target membranes. The E1 and E2-ubiquitin-like activities of ATG7 and ATG3 use thioester bonds to transfer LC3 to the ATG5-ATG12 conjugate. At the same time binding of ATG16L1 to ATG5-ATG12 provides the E3 ubiquitin-ligase like activity necessary to conjugate LC3 to PE or PS. The ATG5-ATG12 conjugate can also bind TECPR1 (tectonin beta propeller repeat-containing protein) which shares an ATG5 interaction (AIR) region with ATG16L1 and can binds LC3 directly through a LC3 interaction region (LIR). In this study we have used cells lacking ATG16L1 to determine if TECPR1 can substitute for ATG16L1 during LC3 conjugation. The results show that ATG16L1-/-MEFS can conjugate LC3 to lysosomes damaged by chloroquine or LLOMes and conjugation is dependent on the ubiquitin-like enzymes ATG3, ATG5 and ATG7 upstream of ATG16L1. TECPR1, ATG5 and galectin 3 are recruited to LAMP positive damaged lysosomes in the absence of ATG16L1 suggesting that TECPR-1 recruits ATG5-ATG12 to conjugate LC3 to damaged lysosomes. This was confirmed when truncation of TECPR1 at the central PH domain required for lysosome binding prevented LC3 conjugation, and LC3 conjugation could be restored by full length TECPR1. Recruitment of TECPR1 to damaged lysosomes required the N-terminal LIR motif and was partially dependent on the central PH domain that binds PI4P exposed during lysosome repair. TECPR1 can therefore conjugate LC3 to damaged lysosomes independently of ATG16L1 by providing E3 ligase-like activity to ATG5-ATG12. Direct conjugation of LC3 by TECPR1 may contribute to the autophagosome tethering functions reported for TECPR1 by increasing recruitment of cargo receptors, tethering proteins and SNARE proteins required for fusion with lysosomes and protein degradation. TECPR1-dependent conjugation of LC3 may also facilitate lysosome repair pathways involving autophagic lysosomal reformation and transcriptional activation of autophagy by TFEB.