ATXN3 regulates lysosome regeneration after damage by targeting K48-K63-branched ubiquitin chains

The cellular response to lysosomal damage involves fine-tuned mechanisms of membrane repair, lysosome regeneration and lysophagy, but how these different processes are coordinated is not fully understood. Here we show in human cells that the deubiquitinating enzyme ATXN3 helps restore integrity of the lysosomal system after damage by targeting K48-K63-linked branched ubiquitin chains on regenerating lysosomes. We find that ATXN3 translocates to lysosomes after different types of damage and there colocalizes with its partner, VCP/p97. ATXN3 recruitment occurs late after the initial repair of a subset of lysosomes and after phagophore formation on terminally damaged lysosomes. Of note, inactivation of ATXN3 by induced degradation, depletion or knock-out impairs clearance of damaged lysosomes and full restoration of lysosomal capacity. Mechanistically, ATXN3, along with VCP/p97, turns over K48-K63-branched ubiquitin conjugates on LAMP1-positive, phosphatidylinositol-(4,5)-bisphosphate-decorated compartments that are not yet fully re-acidified indicating involvement in lysosome regeneration. Our findings identify a key role of ATXN3 in restoring lysosomal function after lysosomal membrane damage and uncover K48-K63-branched ubiquitin chain-regulated regeneration as a critical element of the lysosomal damage stress response.