Interaction of Tail-anchored Proteins with Liposomes in Different Cholesterol Content: Initial Steps for the Fabrication of Artificial Neuroendocrine Vesicles

A group of membrane proteins endowed with a single C-terminal hydrophobic domain capable of insertion into lipid bilayer is known as tail-anchored (TA) proteins. We analyzed the integration of the TA domain of holo-form of human cytochrome b5 (HCYTb5) into protein-free liposomes different in a lipid composition. The integration of holo-b5 occurred efficiently into membranes with low cholesterol content and, once incorporated, the proteoliposomes were very stable. For membranes with high cholesterol content, incorporation efficiency was not so high. However, carbonate-extraction followed by discontinuous sucrose gradient showed that HCYTb5 was tightly incorporated into both types of vesicle membranes. Tryptic digestion of proteoliposomes containing HCYTb5 and free HCYTb5 showed a clear difference in the cleavage sites. The former cleavage site was identical to that occurred for native holo-b5 upon treatment of ER microsomes with trypsin