Ubiquitination of Alzheimer's-related tau protein affects liquid-liquid phase separation in a site- and cofactor-dependent manner

The formation of biomolecular condensates has emerged as a crucial player both in neuronal physiology and neurodegeneration. Phase separation of the Alzheimer's related protein tau into liquid condensates is facilitated by polyanions and is regulated by post-translational modifications. Given the central role of ubiquitination in proteostasis regulation and signaling, we investigated the behavior of monoubiquitinated tau during formation of condensates. We ubiquitinated the lysine-rich, four-repeat domain of tau either unspecifically via enzymatic conjugation or in a position-specific manner by semisynthesis. Ubiquitin conjugation at specific sites weakened multivalent tau/RNA interactions and disfavored tau/heparin condensation. Yet, heterogeneous ubiquitination was tolerated during phase separation and stabilized droplets against aggregation-linked dissolution. Thus, we demonstrated that cofactor chemistry and site of modification affect the mesoscopic and molecular signatures of ubiquitinated tau condensates. Our findings suggest that ubiquitination could influence the physiological states and pathological transformations of tau in cellular condensates.