Phase Separation Clustering of Poly Ubiquitin Cargos on Ternary Mixture Lipid Membranes by Synthetically Cross-Linked Ubiquitin Binder Peptides.

Ubiquitylation is involved in various physiological processes, such as signaling and vesicle trafficking, whereas ubiquitin (UB) is considered an important clinical target. The polymeric addition of UB enables cargo molecules to be recognized specifically by multivalent binding interactions with UB-binding proteins, which results in various downstream processes. Recently, protein condensate formation by ubiquitylated proteins has been reported in many independent UB processes, suggesting its potential role in governing the spatial organization of ubiquitylated cargo proteins. We created modular polymeric UB binding motifs and polymeric UB cargos by synthetic bioconjugation and protein purification. Giant unilamellar vesicles with lipid raft composition were prepared to reconstitute the polymeric UB cargo organization on the membranes. Fluorescence imaging was used to observe the outcome. The polymeric UB cargos clustered on the membranes by forming a phase separation codomain during the interaction with the multivalent UB-binding conjugate. This phase separation was valence-dependent and strongly correlated with its potent ability to form protein condensate droplets in solution. Multivalent UB binding interactions exhibited a general trend toward the formation of phase-separated condensates and the resulting condensates were either in a liquid-like or solid-like state depending on the conditions and interactions. This suggests that the polymeric UB cargos on the plasma and endosomal membranes may use codomain phase separation to assist in the clustering of UB cargos on the membranes for cargo sorting. Our findings also indicate that such phase behavior model systems can be created by a modular synthetic approach that can potentially be used to further engineer biomimetic interactions in vitro.