Case 13

A “Flippase” Enzyme Maintains Membrane Asymmetry

Last modified 7 Sept 2010

Focus concept

The properties of a phospholipid translocase enzyme are determined via a series of experiments with sonicated phospholipid vesicles and red blood cells.

Prerequisites

∙Phospholipid structure and nomenclature.

∙Singer-Nicholson fluid mosaic model of a membrane.

Background

Figure 13.1: Experimental protocol for shape-change experiments.

It has been well established that the cellular phospholipid membrane bilayer is asymmetric. Choline-containing lipids such as phosphatidylcholine (PC) and sphingomyelin (SM) are more likely to be found in the outer leaflet of the bilayer whereas aminophospholipids like phosphatidylserine (PS) and phosphatidylethanolamine (PE) are more likely to be found in the inner leaflet of the bilayer. Several investigators have used a variety of methods in order to determine what mechanism is used by the cell to maintain this asymmetry. Several studies have indicated that a transmembrane protein that has been nicknamed a “flippase” is likely to be partially responsible for maintaining phospholipid asymmetry in the bilayer. It has been hypothesized that the flippase enzyme would act by “flipping” a phospholipid from the outer leaflet of the membrane to the inner leaflet. Specific transport of some types of phospholipids but not others would create the asymmetric distribution that has been observed.

The investigators sonicated (disrupted with sound waves) solutions of a single kind of phospholipid in order to form phospholipid vesicles. The vesicles were then added to red blood cells. The phospholipids in the vesicle migrated from the vesicle to the red blood cells. The investigators then used a microscope to observe the shape of the red blood cells, which normally have a biconcave disk shape. If excess phospholipids are added to the outer membrane leaflet, the red blood cells become echinocytic or “spiky”. But if the added phospholipids are acted upon by the flippase enzyme, they are flipped from the outer leaflet to the inner leaflet of the membrane. This results in an excess of phospholipids in the inner leaflet of the membrane. As a result, the red blood cell surface is stomatocytic (covered with “craters”). By observing which phospholipids could be flipped and which could not, the investigators were able to ascertain the properties of the flippase enzyme. In this case, we examine some of these experiments and use our observations to describe the flippase enzyme in more detail. The experimental design is shown in Figure 13.1.

In order to quantitate their results, the investigators used a numbering system that is diagramed in

Figure 13.2. Biconcave red blood cell disks were given a score of zero. If the red blood cell became echinocytic, it was given a score from +1 to +5, indicating an increasing number of “spikes.” Similarly, stomatocytic cells were given scores from -1 to -4, indicating an increasing number of “craters”. In each experiment, a field of 100 cells was observed, each cell was assigned numbers, and the numbers were averaged to yield a value called the morphological index (MI). Echinocytic cells have an excess amount

Figure 13.2: The morphological index (MI) scale for assessing red blood cell shape change.

of phospholipid in the outer leaflet of the membrane and have positive MI values. Stomatocytic cells have an excess amount of phospholipid in the inner leaflet in the membrane and have negative MI values. In this manner, the ability of the flippase enzyme to translocate phospholipids from the outer leaflet to the inner leaflet could be ascertained.

Figure 13.3: Structures of phospholipids used in shape change experiments.

These studies are important because it is known that the asymmetric distribution of lipids just described is found in healthy cells. Under some circumstances, however, the membrane phospholipids may become “scrambled” and the asymmetry will be lost. This occurs in cells about to undergo a process called apoptosis, or programmed cell death. An understanding of the apoptotic process has implications for the development of therapeutic drugs that might encourage cells like cancer cells to undergo apoptotic death. This case also illustrates how it is possible to study the properties of a protein without purifying it first.
Questions

Figure 13.4: Shape changes induced by incubating red blood cells with phospholipid vesicles. (Based on Daleke and Heustis, 1985.)

1.Experiments with phospholipid vesicles were carried out as shown in Figure 13.1. First, phospholipid vesicles containing one specific type of phospholipid were incubated with red blood cells at 4◦C (at this temperature the flippase is inactive). Then the temperature was warmed up to 37◦C to activate the flippase. The ability of the flippase to translocate three different types of phospholipids was measured. The phospholipids tested were dilauroylphosphatidylserine (DLPS), dilauroylphosphatidylethanolamine (DLPE) and dilauroylphosphatidylcholine (DLPC). The structures of these lipids are shown in Figure 13.3. The results are shown in Figure 13.4.

  1. What type(s) of phospholipids is(are) preferentially translocated by the flippase? Explain.
  2. Given the distribution of lipids in the membrane, what can you say about the distribution of charge on each side of the membrane?

2. The ability of the flippase to translocate DLPS and lyso-PS was compared (the structure of lyso-PS is shown in Figure 13.5). The results are shown in Figure 13.6. What does this tell you about the structural requirement for translocation activity?

Figure 13.5: Structure of lyso-PS.

Figure 13.6: Shape changes in red blood cells induced by lyso PS (based on Daleke and Heustis, 1985).

Figure 13.7: Effect of ATP depletion on red blood cell shape change experiments (based on Daleke and Heustis, 1985).

3. Additional experiments were carried out to ascertain the involvement of ATP. Previous experiments have shown that incubating red blood cells with iodoacetamide and inosine causes ATP levels to decrease by 60%. Experiments similar to those diagramed in Figure 13.1 were carried out, but one batch of red cells was pre-treated with iodoacetamide and inosine for 30 minutes first, then phospholipid vesicles containing purified dimyristoylphosphatidylserine (DMPS) were added. The control batch of red blood cells was not treated with iodoacetamide and inosine. The results are shown in Figure 13.7. What is your interpretation of these results?

4. Experiments were carried out to determine which amino acid side chains in the flippase enzyme were essential to its translocation ability. Red blood cells were pretreated with diamide, a reagent that modifies sulfhydryl groups as shown in Figure 13.8. After the pretreatment, the shape change experiments were carried out by measuring the ability of the red cells to translocate DMPS. The results are shown in Figure 13.9. What is your interpretation of these results?

Figure 13.9: Effect of diamide treatment on DMPS-induced red blood cell shape change. (Based on Daleke and Heustis, 1985.)

Figure 13.8: Reaction of sulfhydryl groups with diamide.

5. In the next series of experiments, red blood cells were treated with a mixture of an ionophore and a chelating agent (EDTA). The ionophore “pokes holes” in the membrane, so that ions leak out. The chelating agent binds the ions so they can’t go back inside the cell. Following this treatment, the shape change experiments were carried out using DMPS. After 1.5 hours, Mg2+ ions were added back to the red blood cells. The results are shown in Figure 13.10. What is your interpretation of these results?

Figure 13.10: Effect of ion depletion on red blood cell shape change experiments (based on Daleke and Heustis, 1985).

6. The experiments such as the ones described here give a good picture of the characteristics of an enzyme that has yet to be completely purified. Write a paragraph that summarizes the characteristics of the flippase enzyme. Include in your description an explanation of how the flippase functions to maintain phospholipid asymmetry.

Reference

Daleke, D. L., and Heustis, W. H. (1985) Biochemistry24, pp. 5406-5416.

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