Spontaneous clustering of polyubiquitin cargos on the lipid membranes

Lipid membranes define the boundaries of cellular organelles, which work as platforms to perform biochemical reactions. Vesicular trafficking is the transport of materials between membrane-bound organelles and is one of the essential processes to maintain life. Ubiquitination, a small protein modification of the membrane proteins, is commonly used in various membrane trafficking and signaling processes in living cells. In this report, we studied the spontaneous clustering of polyubiquitin cargos on the lipid membranes and their interaction with various lipid compositions. We used membrane reconstitution and fluorescence imaging to study how ubiquitin and sumo cargos organize on the synthetic giant unilamellar vesicles mimicking the composition of plasma membranes and endosomes. Lipid membranes at certain compositions may separate into composition domains, often referred to as lipid raft domains, to segregate cargo molecules. Protein cargos anchored on the lipid membrane can be sorted by fluidic domain phase separation. A group of proteins may promote protein condensate domain formation, such as multivalent binding proteins and intrinsically disordered proteins. Fluorescence image analysis shows that polyubiquitin and polysumo cargos on the membranes spontaneously cluster when interacting with various binding partner proteins. It implies that cargo molecules themselves may sort without the aid of hard coat trafficking proteins to affect the downstream trafficking processes, which is a canonically neglected role of cargo molecules in vesicular trafficking. The observed clusters both included fluidic and solid phases affected by lipid compositions, which suggests that lipid compositions and protein structures can collaboratively promote ubiquitin cargo cluster formation or reverse it.