Isolation of functional endogenous clathrin-coated vesicles and in vitro reconstitution of pure clathrin cages

An integrated purification workflow isolates biologically active endogenous clathrin-coated vesicles from porcine brain and reconstitutes homogeneous, smooth-vesicle–free clathrin cages.

• Why it matters: Existing centrifugation methods enrich CCVs but leave intraluminal smooth vesicles and co-isolated exosomes, lowering sample homogeneity and limiting high-resolution structural and proteomic studies.
• What they did: Crude CCVs from porcine brain are enriched by differential and sucrose density gradient centrifugation, then clathrin cages are depolymerized under alkaline conditions and separated by ultracentrifugation before in vitro reassembly into uniform cages.
• The result: Purified CCVs retain Auxilin-Hsc70-dependent uncoating activity, and their enclosed smooth vesicles contain active vATPase proton pumps, enabling cryo-electron microscopy and mass spectrometry of clathrin assembly and disassembly.

AI summary of the abstract · may contain errors · not medical advice · read the paper.