Phase Separation Clustering of Poly Ubiquitin Cargos on the Ternary Mixture Lipid Membranes by Synthetically Cross-Linked Ubiquitin Binder Peptides.
Ubiquitylation is involved in various physiological processes in our bodies such as signaling and vesicle trafficking, thus Ubiquitin (UB) is an important medical target system of interest. Polymeric addition of UB allows cargo molecules to be recognized specifically by multivalent binding interaction with UB binding proteins leading to various downstream processes. Recently, implication of protein condensate formation by Ubiquitylated proteins have 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 binder motifs and polymeric UB cargos by synthetic bioconjugation and protein purification. Giant unilamellar vesicles with lipid raft composition were created to reconstitute polymeric UB cargo organization on the membranes. Fluorescence imaging was used to observe the outcome. We found that polymeric UB cargos clustered on the membranes by forming phase separation codomains in interaction with multivalent UB binder conjugate. This phase separation was valency dependent and was strongly correlated to their potency to form protein condensate droplets in solution. Multivalent UB binding interactions showed a general trend toward the formation of phase separated condensates, and the resulting condensates were either in liquid-like or solid-like state depending on the conditions and interactions used. It implies that polymeric UB cargos on the plasma and endosomal membranes may use the codomain phase separation to assist clustering of UB cargos on the membranes for cargo sorting. Our work also shows that model systems of such phase behavior can be created by modular synthetic approach that can potentially be used to further engineer biomimetic interactions in vitro. Graphical abstracts (TOC) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/608403v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@eca49dorg.highwire.dtl.DTLVardef@2b64d2org.highwire.dtl.DTLVardef@e82b98org.highwire.dtl.DTLVardef@862558_HPS_FORMAT_FIGEXP M_FIG C_FIG