You are studying the binding of proteins to the cytoplasmic face of a cultured n

Question

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.

Explanation / Answer

(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.

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