Abstract Lysosomes are the major cellular organelles responsible for nutrient recycling and degradation of cellular material. Maintenance of lysosomal integrity is essential for cellular homeostasis and lysosomal membrane permeabilization (LMP), induced by lysosomotrophic agents, sensitizes towards cell death. Damaged lysosomes are repaired or degraded via lysophagy, during which glycans, exposed on ruptured lysosomal membranes, are recognized by galectins leading to K48- and K63-linked poly-ubiquitination (poly-Ub) of lysosomal proteins followed by recruitment of the autophagic machinery and degradation. Linear (M1) poly-Ub, catalyzed by the E3 ligase linear ubiquitin chain assembly complex (LUBAC) and removed by the OTU domain-containing deubiquitinase with linear linkage specificity (OTULIN) exerts important functions in immune signaling and cell survival, but the role of M1 poly-Ub in lysosomal homeostasis remains largely unexplored. Here, we demonstrate that damaged lysosomes are decorated with M1 poly-Ub in a LUBAC-, OTULIN- and K63-dependent manner. LMP-induced M1 poly-Ub at damaged lysosomes contributes to lysosome degradation, recruits nuclear factor κ-B (NF-κB) essential modulator (NEMO) and locally activates inhibitor of NF-ĸB kinase (IKK) to trigger NF-κB activation in a K63 poly-Ub-dependent manner. Inhibition of lysosomal degradation enhances LMP- and OTULIN-dependent cell death, indicating pro-survival functions of LMP and potentially lysophagy. Finally, we demonstrate that M1 poly-Ub occurs at L-leucyl-leucine methyl ester (LLOMe)-damaged lysosomes in primary mouse neurons and induced pluripotent stem cell (iPSC)-derived primary human dopaminergic neurons. Together, our results reveal novel functions of M1 poly-Ub during lysosomal homeostasis, LMP and degradation of damaged lysosomes, with important implications for NF-κB signaling, inflammation and cell death.

Linear ubiquitination at damaged lysosomes induces local NF-κB activation and controls cell survival