You are studying the binding of proteins to the cytoplasmic

You are studying the binding of proteins to the cytoplasmic face of a cultured neuroblastoma cells and have found a method to create small liposomes containing lipids and proteins from the plasma membrane. Your preparations always contain both inside-out and right-side-out vesicles. To separate these, a friend suggests that you pass your vesicles over an affinity column made of lectin coupled to solid beads. The diagrams below show the orientation of a membrane protein in the original plasma membrane, and how the membrane protein would be oriented in an inside-out vs. a right-side-out vesicle. You can assume the membrane lipids are similarly oriented. What is the function of lectins? Do you predict a lectin-based column would be able to bind to inside-out and/or right-side-out vesicles? How does this achieve the goal of separating the inside-out and right-side-out vesicles? Explain your reasoning. Another friend suggests you stain the vesicle mixture with fluorescently-labeled Annexin V, which is a protein that binds to phosphatidylserine lipids. Which set of vesicles, inside-out or right-side-out, would you expect to show greater fluorescence intensity after this staining? Explain your reasoning.

Solution

(a) Lectins serve to recognize and bind the sugars present on the glycosylated lipids and membrane proteins.
(b) Since these modifications are located on the extracellular (non-cytosolic) face of the membrane, glycosylation sites would face inward on the inside-out vesicles and outward on the right-side-out vesicles. So, the column of beads having lectin will selectively bind to the right-side-out vesicles while ensuring a pure set of inside-out vesicles to pass through.