Here, we describe a purification protocol for isolating clathrin-coated vesicles (CCVs) from adult rat brain by using differential centrifugation coupled with Ficoll-sucrose and DO-sucrose density gradient centrifugation and an additional linear sucrose step gradient at the end to separate CCVs from contaminating membranes present in the crude microsomal fraction.
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Unlabelled: Endocytic recycling of transmembrane proteins is essential to cell signaling, ligand uptake, protein traffic and degradation. The intracellular domains of many transmembrane proteins are ubiquitylated, which promotes their internalization by clathrin-mediated endocytosis. How might this enhanced internalization impact endocytic uptake of transmembrane proteins that lack ubiquitylation? Recent work demonstrates that diverse transmembrane proteins compete for space within highly crowded endocytic structures, suggesting that enhanced internalization of one group of transmembrane proteins may come at the expense of other groups.
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