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Source: Data from Marr, A.G. & Ingraham, J.L. (1962) Effect of temperature on the composition of fatty acids in Escherichia coli. J. Bacteriol. 84, 1260.

Source: Data from Marr, A.G. & Ingraham, J.L. (1962) Effect of temperature on the composition of fatty acids in Escherichia coli. J. Bacteriol. 84, 1260.

*The exact fatty acid composition depends not only on growth temperature but on growth stage and growth medium composition.

tRatios calculated as the total percentage of 16:1 plus 18:1 divided by the total percentage of 14:0 plus 16:0. Hydroxymyristic acid was omitted from this calculation.

(a) Uncatalyzed transverse ("flip-flop") diffusion

(a) Uncatalyzed transverse ("flip-flop") diffusion

(b) Transverse diffusion catalyzed by flippase

Flip Flop And Lateral Diffusion

(b) Transverse diffusion catalyzed by flippase

(c) Uncatalyzed lateral diffusion
Flip Flop And Lateral Diffusion

FIGURE 11-16 Motion of single phospholipids in a bilayer. (a) Movement from one leaflet to the other is very slow, unless (b) catalyzed by a flippase; in contrast, lateral diffusion within the leaflet (c) is very rapid and requires no protein catalysis.

FIGURE 11-16 Motion of single phospholipids in a bilayer. (a) Movement from one leaflet to the other is very slow, unless (b) catalyzed by a flippase; in contrast, lateral diffusion within the leaflet (c) is very rapid and requires no protein catalysis.

aqueous environment and move into the hydrophobic interior of the bilayer, a process with a large, positive free-energy change. There are, however, situations in which such movement is essential. For example, during synthesis of the bacterial plasma membrane, phospho-lipids are produced on the inside surface of the membrane and must undergo flip-flop diffusion to enter the outer leaflet of the bilayer. Similar transbilayer diffusion must also take place in eukaryotic cells as membrane lipids synthesized in one organelle move from the inner to the outer leaflet and into other organelles. A family of proteins, the flippases (Fig. 11-16b), facilitates flipflop diffusion, providing a transmembrane path that is energetically more favorable and much faster than the uncatalyzed movement.

Lipids and Proteins Diffuse Laterally in the Bilayer

Individual lipid molecules can move laterally in the plane of the membrane by changing places with neighboring lipid molecules (Fig. 11-16c). A molecule in one monolayer, or leaflet, of the lipid bilayer—the outer leaflet of the erythrocyte plasma membrane, for example—can diffuse laterally so fast that it circumnavigates the ery-throcyte in seconds. This rapid lateral diffusion within the plane of the bilayer tends to randomize the positions of individual molecules in a few seconds.

Lateral diffusion can be shown experimentally by attaching fluorescent probes to the head groups of lipids and using fluorescence microscopy to follow the probes over time (Fig. 11-17). In one technique, a small region (5 ^m2) of a cell surface with fluorescence-tagged lipids is bleached by intense laser radiation so that the irradiated patch no longer fluoresces when viewed in the much dimmer light of the fluorescence microscope. However, within milliseconds, the region recovers its fluorescence as unbleached lipid molecules diffuse into the bleached patch and bleached lipid molecules diffuse away from it. The rate of /luorescence recovery after photobleaching, or FRAP, is a measure of the rate of lateral diffusion of the lipids. Using the FRAP technique,

FIGURE 11-17 Measurement of lateral diffusion rates of lipids by fluorescence recovery after photobleaching (FRAP). The lipids in the outer leaflet of the plasma membrane are labeled by reaction with a membrane-impermeant fluorescent probe (red), so the surface is uniformly labeled when viewed with a fluorescence microscope. A small area is bleached by irradiation with an intense laser beam, leaving that area nonfluorescent. With the passage of time, labeled lipid molecules diffuse into the bleached region, and it again becomes fluorescent. From the time course of fluorescence return to this area, the diffusion coefficient for the labeled lipid is determined. The rates are typically high; a lipid moving at this speed could circumnavigate E. coli in one second. (The FRAP method can also be used to measure the lateral diffusion of membrane proteins.)

researchers have shown that some membrane lipids diffuse laterally by up to 1 ^m/s.

Another technique, single particle tracking, allows one to follow the movement of a single lipid molecule in the plasma membrane on a much shorter time scale. Results from these studies confirm the rapid lateral diffusion within small, discrete regions of the cell sur-

Fluorescent probe on lipids

React cell with fluorescent probe to label lipids

React cell with fluorescent probe to label lipids

View surface with fluorescence microscope

Intense laser beam bleaches small area

Intense laser beam bleaches small area

With time, unbleached phospholipids diffuse into bleached area
Measure rate of fluorescence return
1 |m

face and show that movement from one such region to a nearby region is inhibited; lipids behave as though corralled by fences that they can occasionally jump (Fig. 11-18).

Many membrane proteins seem to be afloat in a sea of lipids. Like membrane lipids, these proteins are free to diffuse laterally in the plane of the bilayer and are in

FIGURE 11-19 Restricted motion of the erythrocyte chloride-bicarbonate exchanger and glycophorin. The proteins span the membrane and are tethered to spectrin, a cytoskeletal protein, by another protein, ankyrin, limiting their lateral mobilities. Ankyrin is anchored in the membrane by a covalently bound palmitoyl side chain (see Fig. 11-14). Spectrin, a long, filamentous protein, is cross-linked at junctional complexes containing actin. A network of cross-linked spectrin molecules attached to the cytoplasmic face of the plasma membrane stabilizes the membrane against deformation. This network of anchored membrane proteins may be the "corral" suggested by the experiment shown in Figure 11-18; the lipid tracks shown here are confined to subregions defined by the tethered membrane proteins.

FIGURE 11-18 Hop diffusion of individual lipid molecules. The motion of a single fluorescent lipid molecule in a cell surface is recorded on video by fluorescence microscopy, with a time resolution of 25 ^s (equivalent to 40,000 frames/s). The track shown here represents a molecule followed for 56 ms (a total of 2,250 frames); the trace begins in the purple area and continues through blue, green, and orange. The pattern of movement indicates rapid diffusion within a confined region (about 250 nm in diameter, shown by a single color), with occasional hops into an adjoining region. This finding suggests that the lipids are corralled by molecular fences that they occasionally jump.

constant motion, as shown by the FRAP technique with fluorescence-tagged surface proteins. Some membrane proteins associate to form large aggregates ("patches") on the surface of a cell or organelle in which individual protein molecules do not move relative to one another; for example, acetylcholine receptors (see Fig. 11-51) form dense patches on neuron plasma membranes at synapses. Other membrane proteins are anchored to internal structures that prevent their free diffusion. In the erythrocyte membrane, both glycophorin and the chloride-bicarbonate exchanger (p. 395) are tethered to spectrin, a filamentous cytoskeletal protein (Fig. 11-19). One possible explanation for the pattern of lateral diffusion of lipid molecules shown in Figure 11-18 is that membrane proteins immobilized by their association with spectrin are the "fences" that define the regions of relatively unrestricted lipid motion.

Sphingolipids and Cholesterol Cluster Together in Membrane Rafts

We have seen that diffusion of membrane lipids from one bilayer leaflet into the other is very slow unless catalyzed, and that the different lipid species of the plasma membrane are asymmetrically distributed in the two leaflets of the bilayer (Fig. 11-5). Even within a single leaflet, the lipid distribution is not random. Glycosphin-golipids (cerebrosides and gangliosides), which typically contain long-chain saturated fatty acids, form transient clusters in the outer leaflet that largely exclude glycero-phospholipids, which typically contain one unsaturated fatty acyl group and a shorter saturated fatty acyl group. The long, saturated acyl groups of sphingolipids can form more compact, more stable associations with the long ring system of cholesterol than can the shorter, often unsaturated, chains of phospholipids. The cholesterol-sphingolipid microdomains in the outer monolayer of the plasma membrane, visible with atomic-force microscopy (Box 11-1), are slightly thicker and more ordered (less fluid) than neighboring microdomains rich in phospholipids (Fig. 11-20) and are more difficult

Chloride-bicarbonate , Glycophorin exchange proteins / Outside

Plasma membrane

Chloride-bicarbonate , Glycophorin exchange proteins / Outside

FIGURE 11-19 Restricted motion of the erythrocyte chloride-bicarbonate exchanger and glycophorin. The proteins span the membrane and are tethered to spectrin, a cytoskeletal protein, by another protein, ankyrin, limiting their lateral mobilities. Ankyrin is anchored in the membrane by a covalently bound palmitoyl side chain (see Fig. 11-14). Spectrin, a long, filamentous protein, is cross-linked at junctional complexes containing actin. A network of cross-linked spectrin molecules attached to the cytoplasmic face of the plasma membrane stabilizes the membrane against deformation. This network of anchored membrane proteins may be the "corral" suggested by the experiment shown in Figure 11-18; the lipid tracks shown here are confined to subregions defined by the tethered membrane proteins.

Plasma membrane

Ankyrin-

Spectrin-

Path of single lipid molecule

Junctional complex (actin)

Inside

Ankyrin-

Spectrin-

Path of single lipid molecule

Junctional complex (actin)

Inside

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