Abstract 14-3-3 proteins are among the most abundant proteins in the brain and bind a large number of proteins in a phosphorylation dependent manner, including proteins prone to aggregate in neurodegenerative diseases. Binding of 14-3-3 is reported to facilitate the function, promote solubility, and coordinate the assembly of client proteins. For the microtubule-associated protein Tau, a neuronal client of 14-3-3, we show that phosphorylation-dependent stoichiometric binding of 14-3-3ζ dimers inhibits Tau assembling into biomolecular condensates, prevents its aggregation, and realizes efficient dissociation of Tau from microtubules. In contrast, at sub-stoichiometric 14-3-3 concentrations, multivalent electrostatic interactions promote the co-condensation of 14-3-3ζ with Tau in a phosphorylation-independent manner, offering an additional level in regulating the interactions of both proteins. These findings offer long-sought mechanistic insights into how 14-3-3 proteins regulate substrate solubility and highlight their importance for maintaining Tau protein functionality in the brain.

14-3-3 binding regulates Tau assembly and microtubule association