Question

Two different membrane preparations have been created from the plasma membrane of red blood cells (1) right-side-out membrane vesicles (ROVs) and (2) insideout membrane vesicles IOVs) in which the exoplasmic leaflet faces the interior of the vesicles. a. Annexin V is a protein that binds to phosphatidylserine, one of the phospholipids present in cell membranes. Fluorescently labeled annexin V is mixed with each vesicle preparation. The vesicles are washed to remove unbound annexin V and then examined by fluorescence microscopy. Presence of annexin V is detected by green fluorescence, as shown above. What can be deduced from these data? b. A monoclonal antibody has been prepared against band 3, an integral membrane protein of the red blood cell plasma membrane. The antibody recognizes an epitope on the carboxy terminus of band 3. Incubation of the two membrane vesicle preparations with this antibody followed by staining with a fluorescently labeled secondary antibody generates data similar to that shown in the micrographs above. What can you deduce about the structure of band 3 from these data?

Answer 1

a) Since annexin-V bound more intensely to the inside-out vesicles (to the surface supposed to be inside in RBCs), it can be concluded that phosphatidylserine is more prevalent in the inner leaflet of the plasma membrane than the outer one.

Annexin-V is therefore used to study apoptosis. If a cell is undergoing apoptosis, its membrane integrity is compromised and hence, annexin-V can bind to the the membrane.

b) Since the antibody bound to the inside-out vesicles (to the surface supposed to be inside RBCs), the carboxy-terminal domain of band 3 faces towards the cytosol in RBCs.