Excess Met1-linked ubiquitination leads to solid aggregate formation.

The ubiquitin ligase HOIL-1 regulates the formation of Met1-linked (linear) ubiquitin chains through its coordination with the E3 ligase HOIP within the Linear Ubiquitin Chain Assembly Complex (LUBAC). While HOIP-dependent Met1-linked ubiquitination is well established in inflammation and immunity, the physiological importance of its quantitative control remains unclear. Here, we show that cells expressing catalytically inactive HOIL-1 accumulate increased α-synuclein, tau, and amyloid-β aggregates. This is associated with defective late-stage autophagic flux, characterized by impaired delivery of p62-positive aggregates to lysosomes. In parallel, p62 bodies undergo a biophysical transition from dynamic, liquid-like condensates to rigid, solid-like structures. Elevation of Met1-linked ubiquitin chains, either through HOIL-1 inactivation or depletion of the Met1-specific deubiquitinase OTULIN, phenocopies these defects. Together, our findings identify HOIL-1 as a key regulator of aggregate clearance and proteostasis through quantitative control of Met1-linked ubiquitination.