Membrane Structure Chapter 11 PDF

CHAPTER ELEVEN 11 Membrane Structure A living cell is a self-reproducing system of molecules held inside a con- THE LIPID BILAYER tainer. That container is the plasma membrane—a protein-studded, fatty film so thin that...

CHAPTER ELEVEN 11 Membrane Structure A living cell is a self-reproducing system of molecules held inside a con- THE LIPID BILAYER tainer. That container is the plasma membrane—a protein-studded, fatty film so thin that it cannot be seen directly in the light microscope. Every cell on Earth uses such a membrane to separate and protect its chemical MEMBRANE PROTEINS components from the outside environment. Without membranes, there would be no cells, and thus no life. The structure of the plasma membrane is simple: it consists of a two-ply sheet of lipid molecules about 5 nm—or 50 atoms—thick, into which pro- teins have been inserted. Its properties, however, are unlike those of any sheet of material we are familiar with in the everyday world. Although it serves as a barrier to prevent the contents of the cell from escaping and mixing with molecules in the surrounding environment (Figure 11−1), the plasma membrane does much more than that. If a cell is to survive and grow, nutrients must pass inward across the plasma mem- brane, and waste products must make their way out. To facilitate this Figure 11–1 Cell membranes act as selective barriers. The plasma membrane plasma separates a cell from its surroundings, membrane enabling the molecular composition of a internal cell to differ from that of its environment. membrane (A) In some bacteria, the plasma membrane is the only membrane. (B) In addition to a plasma membrane, eukaryotic cells also have internal membranes that enclose individual organelles. All cell membranes prevent molecules on one side from freely mixing with those on the other, as indicated (A) BACTERIAL CELL (B) EUKARYOTIC CELL schematically by the colored dots. 366 CHAPTER 11 Membrane Structure 1 receiving information 3 capacity for movement and expansion 2 import and export of small molecules Figure 11–2 The plasma membrane is involved in cell communication, import and export of molecules, and cell growth and motility. (1) Receptor proteins in the plasma membrane enable the cell to receive signals from the environment; (2) channels and transporters in the membrane enable the import and export of small molecules; (3) the flexibility of the membrane and its capacity for expansion allow the cell to grow, change shape, and move. ECB5 e11.02/11.02 exchange, the membrane is penetrated by highly selective channels and transporters—proteins that allow specific, small molecules and ions to be imported and exported. Other proteins in the membrane act as sensors, or receptors, that enable the cell to receive information about changes in its environment and respond to them in appropriate ways. The mechani- cal properties of the plasma membrane are equally impressive. When a cell grows, so does its membrane: this remarkable structure enlarges in area by adding new membrane without ever losing its continuity, and it can deform without tearing, allowing the cell to move or change shape (Figure 11−2). The membrane is also self-healing: if it is pierced, it nei- ther collapses like a balloon nor remains torn; instead, the membrane quickly reseals. nucleus peroxisome endosome As shown in Figure 11–1, many bacteria have only a single membrane— the plasma membrane—whereas eukaryotic cells also contain internal endoplasmic lysosome reticulum membranes that enclose intracellular compartments. The internal mem- branes form various organelles, including the endoplasmic reticulum, Golgi apparatus, endosomes, and mitochondria (Figure 11–3). Although these internal membranes are constructed on the same principles as the plasma membrane, they differ subtly in composition, especially in their resident proteins. Regardless of their location, all cell membranes are composed of lipids and proteins and share a common general structure (Figure 11–4). The lipids are arranged in two closely apposed sheets, forming a lipid bilayer Golgi apparatus (see Figure 11–4B). This lipid bilayer serves as a permeability barrier to transport vesicle most water-soluble molecules, while the proteins embedded within it plasma membrane carry out the other functions of the membrane and give different mem- mitochondrion branes their individual characteristics. Figure 11–3 Internal membranes In this chapter, we consider the structure of biological membranes and form many different compartments the organization of their two main constituents: lipids and proteins. in a eukaryotic cell. Some of the main Although we focus mainly on the plasma membrane, most of the con- membrane-enclosed organelles in a typical animal cell are shown here. Note that cepts we discuss also apply to internal membranes. The functions of cell the nucleus and mitochondria are each membranes, including their role in cell communication, the transport of enclosed by two membranes. small molecules, and energy generation, are considered in later chapters.  The Lipid Bilayer 367 Figure 11–4 A cell membrane consists of a lipid bilayer in which proteins are embedded. (A) An electron micrograph of a plasma membrane of a human red blood lipid cell seen in cross section. In this image, the bilayer (5 nm) proteins that extend from either side of the bilayer form the two closely spaced dark lines indicated by the brackets; the thin, white layer between them is the lipid bilayer. (B) Schematic drawing showing a three- (A) dimensional view of a cell membrane. (A, by permission of E.L. Bearer.) lipid molecule protein (B) molecule THE LIPID BILAYER Because cells are filled with—and surrounded by—water, the structure of cell membranes is determined by the way membrane lipids behave in a watery (aqueous) environment. Lipid molecules are not very soluble in water, although they do dissolve readily in organic solvents such as ben- zene. It was this property that scientists exploited in 1925, when they set out to investigate how lipids are arranged in cell membranes. Using benzene, investigators extracted all the lipids from the plasma membranes of purified red bloodECB5cells. e11.04/11.04 These lipids were then spread out in a film on the surface of a trough filled with water, like an oil slick on a puddle. Using a movable barrier, the researchers then pushed the floating lipids together until they formed a continuous sheet only one molecule thick. When the investigators measured the surface area of this mon- olayer, they found that it occupied twice the area of the original, intact cells. Based on this observation, they deduced that, in an intact cell mem- brane, lipid molecules must double up to form a bilayer—a finding that had a profound influence on cell biology. In this section, we take a closer look at this lipid bilayer, which consti- tutes the fundamental structure of all cell membranes. We consider how lipid bilayers form, how they are maintained, and how their properties establish the general properties of all cell membranes. Membrane Lipids Form Bilayers in Water The lipids found in cell membranes combine two very different proper- ties in a single molecule: each lipid has a hydrophilic (“water-loving”) head and a hydrophobic (“water-fearing”) tail. The most abundant lipids in cell membranes are the phospholipids, which have a phosphate-con- taining, hydrophilic head linked to a pair of hydrophobic, hydrocarbon hydrophilic tails (Figure 11–5). For example, phosphatidylcholine, one of the most head abundant phospholipids in the membranes of animals and plants, has the small molecule choline attached to a phosphate group as its hydrophilic head (Figure 11–6). hydrophobic tails Phospholipids are not the only membrane lipids that are amphipathic, a term used to describe molecules with both hydrophilic and hydrophobic parts. Cholesterol, which is found in animal cell membranes, and gly- colipids, which have sugars as part of their hydrophilic head, are also amphipathic (Figure 11–7). Figure 11–5 Cell membranes are packed with phospholipids. A typical membrane Having both hydrophilic and hydrophobic parts plays a crucial part in driv- phospholipid molecule has a hydrophilic ing lipid molecules to assemble into bilayers in an aqueous environment. head and two hydrophobic tails. ECB5 e11.05/11.05 368 CHAPTER 11 Membrane Structure CH2 N+(CH3)3 CHOLINE polar CH2 (hydrophilic) O head PHOSPHATE _ O P O O head GLYCEROL CH2 CH CH2 O O C O C O 1 2 tails CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 (D) HYDROCARBON TAIL CH2 CH2 CH2 CH2 CH2 CH2 nonpolar double (hydrophobic) CH2 CH bond tails CH2 CH CH2 CH2 HY CH2 CH2 DR CH2 OC CH2 CH2 AR CH2 CH2 BO CH2 CH2 N TA CH2 CH2 I L CH3 CH2 CH3 (A) (B) (C) Figure 11–6 Phosphatidylcholine is the most common phospholipid in cell membranes. It is represented schematically in (A), as a chemical formula in (B), as a space-filling model in (C), and as a symbol in (D). This particular phospholipid is built from five parts: the hydrophilic head, which consists of choline linked to a phosphate group; two hydrocarbon chains, which form the hydrophobic tails; and a molecule of glycerol, which links the head to the tails. Each of the hydrophobic tails is a fatty acid—a hydrocarbon chain with a carboxyl (–COOH) group at one end; glycerol attaches via this carboxyl group, as shown in (B). A kink in one of the hydrocarbon chains occurs where there is a double bond between two carbon atoms. (The “phosphatidyl” part of the name of a phospholipid refers to the phosphate–glycerol–fatty acid portion of the molecule.) ECB5 e11.06/11.06 + NH3 serine H C COO hydrophilic heads CH2 O Gal O P O O OH OH O CH2 CH CH2 CH CH CH2 O O CH3 CH NH C OC O CH C O CH3 CH3 Figure 11–7 Different types of membrane HYDROCARBON TAIL HYDROCARBON TAIL HYDROCARBON TAIL HYDROCARBON TAIL CH lipids are all amphipathic. Each of the three CH2 types shown here has a hydrophilic head and one or two hydrophobic tails. The hydrophilic CH2 hydrocarbon tail head is serine phosphate (shaded blue CH2 and yellow) in phosphatidylserine, an –OH CH group (blue) in cholesterol, and the sugar CH3 CH3 galactose plus an –OH group (both blue) in galactocerebroside. See also Panel 2–4, phosphatidylserine cholesterol galactocerebroside pp. 72–73. (a phospholipid) (a sterol) (a glycolipid)  The Lipid Bilayer 369 hydrogen bonds CH3 _ δ+ C O δ CH3 CH3 O C acetone CH3 _ δ O H H + δ+ δ water acetone in water Figure 11–8 A hydrophilic molecule attracts water molecules. Both acetone and water are polar molecules: thus acetone readily dissolves in water. Polar atoms are shown in red and blue, with δ– indicating a partial negative charge, and δ+ indicating a partial positive charge. Hydrogen bonds (red ) and an electrostatic attraction (yellow) form between acetone and the surrounding water molecules. Nonpolar groups are shown in gray. As discussed in Chapter 2 (see Panel 2–2, pp. 68–69), hydrophilic molecules dissolve readily in water because they contain either charged groups or QUESTION 11–1 uncharged polar groupsECB5that can form electrostatic attractions or hydro- e11.08/11.08 gen bonds with water molecules (Figure 11–8). Hydrophobic molecules, Water molecules are said “to by contrast, are insoluble in water because all—or almost all—of their reorganize into a cagelike structure” atoms are uncharged and nonpolar; they therefore cannot form favora- around hydrophobic compounds (e.g., see Figure 11–9). This ble interactions with water molecules. Instead, they force adjacent water seems paradoxical because water molecules to reorganize into a cagelike structure around them (Figure molecules do not interact with 11–9). Because this cagelike structure is more highly ordered than the the hydrophobic compound. So rest of the water, its formation requires free energy. This energy cost is how could they “know” about its minimized when the hydrophobic molecules cluster together, limiting presence and change their behavior their contacts with the surrounding water molecules. Thus, purely hydro- to interact differently with one phobic molecules, like the fats found in the oils of plant seeds and the another? Discuss this argument adipocytes (fat cells) of animals (Figure 11–10), coalesce into large fat and, in doing so, develop a clear droplets when dispersed in water. concept of what is meant by a “cagelike” structure. How does it compare to ice? Why would this cagelike structure be energetically unfavorable? CH3 HC CH3 CH3 CH3 2-methylpropane HC CH3 Figure 11–9 A hydrophobic molecule CH3 tends to avoid water. Because the _ 2-methylpropane molecule is entirely δ hydrophobic, it cannot form favorable O H H interactions with water. This causes the δ+ δ+ adjacent water molecules to reorganize into a cagelike structure around it, to maximize water 2-methylpropane in water their hydrogen bonds with each other. 370 CHAPTER 11 Membrane Structure triacylglycerol Amphipathic molecules, such as membrane lipids (see Figure 11–7), are subject to two conflicting forces: the hydrophilic head is attracted hydrocarbon tail to water, while the hydrophobic tails shun water and seek to aggregate with other hydrophobic molecules. This conflict is beautifully resolved by the formation of a lipid bilayer—an arrangement that satisfies all parties and is energetically most favorable. The hydrophilic heads face water on both surfaces of the bilayer, while the hydrophobic tails are shielded from the water within the bilayer interior, like the filling in a sandwich (Figure 11–11). C O glycerol The same forces that drive the amphipathic molecules to form a bilayer O help to make the bilayer self-sealing. Any tear in the sheet will create a CH2 CH CH2 free edge that is exposed to water. Because this situation is energetically O O unfavorable, the molecules of the bilayer will spontaneously rearrange to eliminate the free edge. If the tear is small, this spontaneous re- C O C O arrangement will exclude the water molecules and lead to repair of the bilayer, restoring a single continuous sheet. If the tear is large, the sheet may begin to fold in on itself and break up into separate closed vesi- cles. In either case, the overriding principle is that free edges are quickly eliminated. The prohibition on free edges has a profound consequence: the only way an amphipathic sheet can avoid having free edges is to bend and seal, hydrocarbon tails forming a boundary around a closed space (Figure 11–12). Therefore, Figure 11–10 Fat molecules are entirely amphipathic molecules such as phospholipids necessarily assemble into hydrophobic. Unlike phospholipids, self-sealing containers that define closed compartments—from vesicles triacylglycerols, which are the main and organelles to entire cells. This remarkable behavior, fundamental to constituents of animal fats and plant oils, are entirely hydrophobic. Here, the third the creation of a living cell, is essentially a by-product of the nature of hydrophobic tail of the triacylglycerol membrane lipids: hydrophilic at one end and hydrophobic at the other. molecule is drawn facing upward for comparison with the structure of a The Lipid Bilayer Is a Flexible Two-dimensional Fluid phospholipid (see Figure 11–6), although normally it is depicted facing down (see The aqueous environment inside and outside a cell prevents membrane Panel 2–5, pp. 74–75). lipids from escaping from the bilayer, but nothing stops these molecules from moving about and changing places with one another within the plane of the membrane. The lipid bilayer therefore behaves as a two- ECB5 e11.10/11.10 dimensional fluid, a fact that is crucial for membrane function and integrity (Movie 11.1; “laser tweezers” are explained in Movie 11.2). At the same time, the lipid bilayer is also flexible—that is, it is able to bend. Like fluidity, flexibility is important for membrane function, and it water lipid bilayer water (A) (B) 1 nm Figure 11–11 Amphipathic phospholipids form a bilayer in water. (A) Schematic drawing of a phospholipid bilayer in water. (B) Computer simulation showing the phospholipid molecules (red heads and orange tails) and the surrounding water molecules (blue) in a cross section of a lipid bilayer. (B, adapted from R.M. Venable et al., Science 262:223–228, 1993.) ECB5 e11.11/11.11  The Lipid Bilayer 371 sets a lower limit of about 25 nm to the vesicle diameter that cell mem- ENERGETICALLY UNFAVORABLE branes can form. The fluidity of lipid bilayers can be studied using synthetic lipid bilayers, which are easily produced by the spontaneous aggregation of amphi- pathic lipid molecules in water. Pure phospholipids, for example, will in a planar phospholipid bilayer, form closed, spherical vesicles, called liposomes, when added to water; hydrophobic tails (white layer) are exposed to water along these vesicles vary in size from about 25 nm to 1 mm in diameter the edges (Figure 11–13). Using such simple synthetic bilayers, investigators can measure the movements of the lipid molecules in a lipid bilayer. These measure- ments reveal that some types of movement are rare, while others are frequent and rapid. Thus, in synthetic lipid bilayers, phospholipid mole- cules very rarely tumble from one half of the bilayer, or monolayer, to the other. Without proteins to facilitate the process, it is estimated that this formation of a sealed compartment shields event, called “flip-flop,” occurs less than once a month for any individual hydrophobic tails from lipid molecule under conditions similar to those in a cell. On the other water hand, as the result of random thermal motions, lipid molecules continu- ously exchange places with their neighbors within the same monolayer. This exchange leads to rapid lateral diffusion of lipid molecules within ENERGETICALLY FAVORABLE the plane of each monolayer, so that, for example, a lipid in an artifi- cial bilayer may diffuse a length equal to that of an entire bacterial cell Figure 11–12 Phospholipid bilayers (~2 μm) in about one second. spontaneously close in on themselves to form sealed compartments. The closed Similar studies show that individual lipid molecules not only flex their structure is stable because it avoids the hydrocarbon tails, but they also rotate rapidly about their long axis— exposure of the hydrophobic hydrocarbon some reaching speeds of 500 revolutions per second. Studies of whole tails to water, which would be energetically unfavorable. cells—and of isolated cell membranes—indicate that lipid molecules in cell membranes undergo the same movements as they do in synthetic bilayers. The movements of membrane phospholipid molecules are sum- ECB5 e11.12-11.12 marized in Figure 11–14. The Fluidity of a Lipid Bilayer Depends on Its Composition The fluidity of a cell membrane—the ease with which its lipid molecules move within the plane of the bilayer—is important for membrane func- tion and has to be maintained within certain limits. Just how fluid a lipid bilayer is at a given temperature depends on its phospholipid composi- tion and, in particular, on the nature of the hydrocarbon tails: the closer and more regular the packing of the tails, the more viscous and less fluid the bilayer will be. Two major properties of hydrocarbon tails affect how tightly they pack in the bilayer: their length and the number of double bonds they con- (A) 50 nm tain. A shorter chain length reduces the tendency of the hydrocarbon tails to interact with one another and therefore increases the fluidity of the bilayer. The hydrocarbon tails of membrane phospholipids vary in water length between 14 and 24 carbon atoms, with 18 or 20 atoms being the most common. For most phospholipids, one of these hydrocarbon tails contains only single bonds between its adjacent carbon atoms, whereas the other tail includes one or more double bonds (see Figure 11–6). The chain that harbors a double bond does not contain the maximum num- water ber of hydrogen atoms that could, in principle, be attached to its carbon backbone; it is thus said to be unsaturated with respect to hydrogen. The Figure 11–13 Pure phospholipids can form closed, spherical liposomes. (A) An electron micrograph of phospholipid vesicles, or liposomes. (B) A drawing of a small, spherical liposome seen in cross section. (A, courtesy of Jean Lepault.) (B) 25 nm 372 CHAPTER 11 Membrane Structure lateral diffusion hydrocarbon tail with no double bonds has a full complement of hydrogen atoms and is said to be saturated. Each double bond in an unsaturated tail creates a small kink in the tail (see Figure 11–6), which makes it more difficult for the tails to pack against one another. For this reason, lipid flip-flop (rarely occurs) bilayers that contain a large proportion of unsaturated hydrocarbon tails are more fluid than those with lower proportions. In bacterial and yeast cells, which have to adapt to varying temperatures, both the lengths and the degree of saturation of the hydrocarbon tails flexion rotation in the bilayer are adjusted constantly to maintain a membrane with a relatively consistent fluidity: at higher temperatures, for example, the cell Figure 11–14 Membrane phospholipids move within the lipid bilayer. Because of makes membrane lipids with tails that are longer and that contain fewer these motions, the bilayer behaves as a two- double bonds. A similar trick is used in the manufacture of margarine dimensional fluid, in which the individual from vegetable oils. The fats produced by plants are generally unsatu- ECB5 e11.14/11.14 lipid molecules are able to move in their rated and therefore liquid at room temperature, unlike animal fats such own monolayer. Note that lipid molecules as butter or lard, which are generally saturated and therefore solid at do not move spontaneously from one monolayer to the other. room temperature. To produce margarine, vegetable oils are “hydrogen- ated”: this addition of hydrogen removes their double bonds, making the oils more solid and butterlike at room temperature. In animal cells, membrane fluidity is modulated by the inclusion of the sterol cholesterol. This molecule is present in especially large amounts in the plasma membrane, where it constitutes approximately 20% of the lipids in the membrane by weight. With its short and rigid steroid ring structure, cholesterol can fill the spaces between neighboring phospho- lipid molecules left by the kinks in their unsaturated hydrocarbon tails (Figure 11–15). In this way, cholesterol tends to stiffen the bilayer, mak- QUESTION 11–2 ing it less flexible, as well as less permeable. The chemical properties of membrane lipids—and how they affect membrane fluidity—are reviewed Five students in your class always in Movie 11.3 and Movie 11.4. sit together in the front row. This could be because (A) they really like For all cells, membrane fluidity is important for a number of reasons. It each other or (B) nobody else in enables many membrane proteins to diffuse rapidly in the plane of the your class wants to sit next to them. bilayer and to interact with one another, as is crucial, for example, in Which explanation holds for the cell signaling (discussed in Chapter 16). It permits membrane lipids and assembly of a lipid bilayer? Explain. proteins to diffuse from sites where they are inserted into the bilayer after Suppose, instead, that the other their synthesis to other regions of the cell. It ensures that membrane mol- explanation held for lipid molecules. How would the properties of the ecules are distributed evenly between daughter cells when a cell divides. lipid bilayer be different? And, under appropriate conditions, it allows membranes to fuse with one another and mix their molecules (discussed in Chapter 15). If biological phospholipid polar head group cholesterol polar rigid head planar steroid ring cholesterol- 6 nm structure stiffened region nonpolar hydrocarbon more tail fluid (C) region (A) (B) Figure 11–15 Cholesterol tends to stiffen cell membranes. (A) The shape of a cholesterol molecule. The chemical formula of cholesterol is shown in Figure 11–7. (B) How cholesterol fits into the gaps between phospholipid molecules in a lipid bilayer. (C) Space-filling model of the bilayer, with cholesterol molecules in green. Although the nonpolar hydrocarbon tail of cholesterol is shown in green—to visually distinguish it from the hydrocarbon tails of the membrane phospholipids—in reality, the hydrophobic tail of cholesterol is chemically equivalent to the hydrophobic tails of the phospholipids. (C, from H.L. Scott, Curr. Opin. Struct. Biol. 12:495–502, 2002.)  The Lipid Bilayer 373 Figure 11–16 Newly synthesized phospholipids are added to the CYTOSOL cytosolic side of the ER membrane and then redistributed by transporters that transfer them from one half of the lipid bilayer lipid bilayer of to the other. Biosynthetic enzymes bound to the cytosolic monolayer endoplasmic reticulum of the ER membrane (not shown) produce new phospholipids from free fatty acids and insert them into the cytosolic monolayer. Transporters called scramblases then randomly transfer phospholipid ER LUMEN molecules from one monolayer to the other, allowing the membrane to grow as a bilayer in which the two leaflets even out continuously in size and lipid composition. PHOSPHOLIPID SYNTHESIS ADDS TO CYTOSOLIC HALF OF THE BILAYER membranes were not fluid, it is hard to imagine how cells could live, grow, and reproduce. Membrane Assembly Begins in the ER In eukaryotic cells, new phospholipids are manufactured by enzymes bound to the cytosolic surface of the endoplasmic reticulum (ER). Using SCRAMBLASE CATALYZES free fatty acids as substrates (see Panel 2–5, pp. 74–75), these enzymes TRANSFER OF RANDOM deposit the newly made phospholipids exclusively in the cytosolic half of PHOSPHOLIPIDS FROM ONE MONOLAYER TO ANOTHER the bilayer. Despite the unbalanced addition of newly made phospholipids, cell mem- symmetric growth branes manage to grow evenly. So how do new phospholipids make it of both halves to the opposite monolayer? As we saw in Figure 11–14, flip-flops that of bilayer move lipids from one monolayer to the other rarely occur spontaneously. Instead, phospholipid transfers are catalyzed by a scramblase, a type of IN THE ER MEMBRANE, PHOSPHOLIPIDS transporter protein that removes randomly selected phospholipids from ARE RANDOMLY DISTRIBUTED one half of the lipid bilayer and inserts them in the other. (Transporters and their functions are discussed in detail in Chapter 12.) As a result of this scrambling, newly made phospholipids are redistributed equally between each monolayer of the ER membrane (Figure 11–16). Some of this newly assembled membrane will remain in the ER; the rest will be used to supply fresh membrane to other compartments in the cell, ECB5 n11.16a-11.16 including the Golgi apparatus and plasma membrane (see Figure 11–3). GOLGI LUMEN We discuss this dynamic process—in which membranes bud from one organelle and fuse with another—in detail in Chapter 15. lipid bilayer of Golgi apparatus Certain Phospholipids Are Confined to One Side of the Membrane CYTOSOL Most cell membranes are asymmetric: the two halves of the bilayer often include strikingly different sets of phospholipids. But if membranes DELIVERY OF NEW MEMBRANE emerge from the ER with an evenly assorted set of phospholipids, where FROM ER does this asymmetry arise? It begins in the Golgi apparatus. The Golgi membrane contains another family of phospholipid-handling transporters, called flippases. Unlike scramblases, which move random phospholipids from one half of the bilayer to the other, flippases remove specific phospholipids from the side of the bilayer facing the exterior space and flip them into the monolayer that faces the cytosol (Figure 11–17). FLIPPASE CATALYZES TRANSFER OF SPECIFIC PHOSPHOLIPIDS TO Figure 11–17 Flippases help to establish and maintain the CYTOSOLIC MONOLAYER asymmetric distribution of phospholipids characteristic of animal cell membranes. When membranes leave the ER and are incorporated in the Golgi, they encounter a different set of transporters called flippases, which selectively remove phosphatidylserine (light green) and phosphatidylethanolamine (yellow) from the noncytosolic monolayer and flip them to the cytosolic side. This transfer leaves phosphatidylcholine (red ) and sphingomyelin (brown) concentrated in the noncytosolic monolayer. The resulting curvature of the membrane IN THE GOLGI AND OTHER CELL MEMBRANES, PHOSPHOLIPID DISTRIBUTION IS ASYMMETRIC may help drive subsequent vesicle budding. 374 CHAPTER 11 Membrane Structure Figure 11–18 Membranes retain their orientation during transfer noncytosolic face EXTRACELLULAR FLUID between cell compartments. Membranes are transported by a process of vesicle budding and fusing. Here, a vesicle is shown budding from the Golgi apparatus and fusing with the plasma membrane. Note cytosolic face that the orientations of both the membrane lipids and proteins are preserved during the process: the original cytosolic surface of the lipid bilayer (pink ) remains facing the cytosol, and the noncytosolic surface plasma membrane (red ) continues to face away from the cytosol, toward the lumen of the Golgi and the transport vesicle—or toward the extracellular fluid. Similarly, the glycoprotein shown here (blue and green) remains in the same orientation, with its attached sugar facing the noncytosolic side. transport vesicle The action of these flippases—and of similar transporters in the plasma CYTOSOL membrane—initiates and maintains the asymmetric arrangement of phospholipids that is characteristic of the membranes of animal cells. membrane This asymmetry is preserved as membranes bud from one organelle and glycoprotein fuse with another—or with the plasma membrane. This means that all cell membranes have distinct “inside” and “outside” faces: the cytosolic monolayer always faces the cytosol, while the noncytosolic monolayer is exposed to either the cell exterior—in the case of the plasma mem- LUMEN brane—or the interior space (lumen) of an organelle. This conservation of orientation applies not only to the phospholipids that make up the mem- brane, but also to any proteins that might be inserted in the membrane membrane of Golgi apparatus (Figure 11–18). This positioning is very important, as a protein’s orienta- tion within the lipid bilayer is crucial for its function (see Figure 11–20). Among lipids, those that show the most dramatically lopsided distribu- tion in cell membranes are the glycolipids, which are located mainly in the plasma membrane, and only in the noncytosolic half of the bilayer (Figure 11–19). The sugar groups of these membrane lipids face the cell QUESTION 11–3 exterior, where they form part of a continuous coat of carbohydrate that surrounds and protects animal cells. Glycolipid molecules acquire their It seems paradoxical that a sugar groups in the Golgi apparatus, where the enzymes that engineer lipid bilayer can be fluid yet this chemical modification are confined. These enzymes are oriented ECB5 E11.17/11.17 asymmetrical. Explain. such that sugars are added only to lipid molecules in the noncytosolic half of the bilayer. Once a glycolipid molecule has been created in this way, it remains trapped in this monolayer, as there are no flippases that transfer glycolipids to the cytosolic side. Thus, when a glycolipid mol- ecule is finally delivered to the plasma membrane, it displays its sugars to the exterior of the cell. Figure 11–19 Phospholipids and Other lipid molecules show different types of asymmetric distributions, glycolipids are distributed asymmetrically which relate to their specific functions. For example, the inositol phos- in the lipid bilayer of an animal cell pholipids—a minor component of the plasma membrane—have a special plasma membrane. Phosphatidylcholine (red ) and sphingomyelin (brown) are role in relaying signals from the cell surface into the cell interior (dis- concentrated in the noncytosolic monolayer, cussed in Chapter 16); thus they are concentrated in the cytosolic half of whereas phosphatidylserine (light green) the lipid bilayer. and phosphatidylethanolamine (yellow) are found mainly on the cytosolic side. In addition to these phospholipids, phosphatidylinositols (dark green head group), a minor constituent of the plasma EXTRACELLULAR SPACE membrane, are shown in the cytosolic monolayer, where they participate in cell signaling. Glycolipids are drawn with hexagonal blue head groups to represent plasma sugars; these are found exclusively in the membrane noncytosolic monolayer of the membrane. Within the bilayer, cholesterol (green) is distributed almost equally in both monolayers. CYTOSOL  Membrane Proteins 375 TRANSPORTERS AND ANCHORS RECEPTORS ENZYMES Figure 11–20 Plasma membrane CHANNELS proteins have a variety of functions. They transport molecules and ions, act EXTRACELLULAR SPACE as anchors, detect signals, or catalyze reactions. CYTOSOL X Y MEMBRANE PROTEINS Although the lipid bilayer provides the basic structure of all cell mem- branes and serves as a permeability barrier to the hydrophilic molecules on either side of it, most membrane functions are carried out by mem- brane proteins. In animals,ECB5 e11.19/11.19 proteins constitute about 50% of the mass of most plasma membranes, the remainder being lipid plus the relatively small amounts of carbohydrate found on some of the lipids (glycolipids) and many of the proteins (glycoproteins). Because lipid molecules are much smaller than proteins, however, a cell membrane typically contains about 50 times the number of lipid molecules compared to protein mol- ecules (see Figure 11–4B). Membrane proteins serve many functions. Some transport particular nutrients, metabolites, and ions across the lipid bilayer. Others anchor the membrane to macromolecules on either side. Still others function as receptors that detect chemical signals in the cell’s environment and relay them into the cell interior, or work as enzymes to catalyze specific reactions at the membrane (Figure 11–20 and Table 11–1). Each type of cell membrane contains a different set of proteins, reflecting the special- ized functions of the particular membrane. In this section, we discuss the structure of membrane proteins and how they associate with the lipid bilayer. TABLE 11–1 SOME EXAMPLES OF PLASMA MEMBRANE PROTEINS AND THEIR FUNCTIONS Functional Class Protein Example Specific Function Transporters Na+ pump actively pumps Na+ out of cells and K+ in (discussed in Chapter 12) Ion channels K+ leak channel allows K+ ions to leave cells, thereby influencing cell excitability (discussed in Chapter 12) Anchors integrins link intracellular actin filaments to extracellular matrix proteins (discussed in Chapter 20) Receptors platelet-derived binds extracellular PDGF and, as a growth factor consequence, generates intracellular (PDGF) receptor signals that direct the cell to grow and divide (discussed in Chapters 16 and 18) Enzymes adenylyl cyclase catalyzes the production of the small intracellular signaling molecule cyclic AMP in response to extracellular signals (discussed in Chapter 16) 376 CHAPTER 11 Membrane Structure Membrane Proteins Associate with the Lipid Bilayer in Different Ways Although the lipid bilayer has a uniform structure, proteins can interact with a cell membrane in a number of different ways. Many membrane proteins extend through the bilayer, with part of their mass on either side (Figure 11–21A). Like their lipid neighbors, these transmembrane proteins are amphipathic, having both hydrophobic and hydrophilic regions. Their hydrophobic regions lie in the interior of the bilayer, nestled against the hydrophobic tails of the lipid molecules. Their hydrophilic regions are exposed to the aqueous environment on either side of the membrane. Other membrane proteins are located almost entirely in the cytosol and are associated with the cytosolic half of the lipid bilayer by an amphipathic α helix exposed on the surface of the protein (Figure 11–21B). Some proteins lie entirely outside the bilayer, on one side or the other, attached to the membrane by one or more covalently attached lipid groups (Figure 11–21C). Yet other proteins are bound indirectly to one face of the membrane or the other, held in place only by their interactions with other membrane proteins (Figure 11–21D). Proteins that are directly attached to the lipid bilayer—whether they are transmembrane, associated with the lipid monolayer, or lipid-linked—can be removed only by disrupting the bilayer with detergents, as discussed shortly. Such proteins are known as integral membrane proteins. The remaining membrane proteins are classified as peripheral membrane pro- teins; they can be released from the membrane by more gentle extraction procedures that interfere with protein–protein interactions but leave the lipid bilayer intact. (A) (B) (C) (D) MONOLAYER- TRANSMEMBRANE ASSOCIATED LIPID-LINKED PROTEIN-ATTACHED NH2 P P EXTRACELLULAR SPACE lipid bilayer CYTOSOL COOH integral membrane proteins peripheral membrane proteins Figure 11–21 Membrane proteins can associate with the lipid bilayer in different ways. (A) Transmembrane proteins can extend across the bilayer as a single α helix, as multiple α helices, or as a rolled-up β sheet (called a β barrel). (B) Some membrane proteins are anchored to the cytosolic half of the lipid bilayer by an amphipathic α helix. (C) Others are linked to either side of the bilayer solely by a covalently attached lipid molecule (red zigzag lines). (D) Many proteins are attached to the membrane only by relatively weak, noncovalent interactions with other membrane proteins. (A−C) are examples of integral membrane proteins; the proteins shown in (D) are considered peripheral membrane proteins.  Membrane Proteins 377 A Polypeptide Chain Usually Crosses the Lipid Bilayer peptide bonds as an α Helix δ+ _ δ+ R δ R H O H All membrane proteins have a unique orientation in the lipid bilayer, _ H which is essential for their function. For a transmembrane receptor pro- N_ δ+ C δ C N_ δ+ C δ C N δ + C δ C tein, for example, the part of the protein that receives a signal from the H H environment must be on the outside of the cell, whereas the part that O_ H O_ + δ δ R δ passes along the signal must be in the cytosol (see Figure 11–20). This orientation is a consequence of the way in which membrane proteins are synthesized (discussed in Chapter 15). The portions of a transmem- Figure 11–22 The backbone of a brane protein located on either side of the lipid bilayer are connected polypeptide chain is hydrophilic. The by specialized membrane-spanning segments of the polypeptide chain atoms on either side of a peptide bond (red line) are polar and carry partial positive or (see Figure 11–21A). These segments, which run through the hydropho- negativeECB5 E11.21/11.21 charges (δ+ or δ–). These charges bic environment of the interior of the lipid bilayer, are composed largely allow these atoms to hydrogen-bond with of amino acids with hydrophobic side chains. Because these side chains one another when the polypeptide folds cannot form favorable interactions with water molecules, they prefer to into an α helix that spans the lipid bilayer interact with the hydrophobic tails of the lipid molecules, where no water (see Figure 11–23). is present. In contrast to the hydrophobic side chains, however, the peptide bonds that join the successive amino acids in a protein are normally polar, mak- ing the polypeptide backbone itself hydrophilic (Figure 11–22). Because water is absent from the interior of the bilayer, atoms that are part of the polypeptide backbone are thus driven to form hydrogen bonds with one another. Hydrogen-bonding is maximized if the polypeptide chain forms a regular α helix, and so the great majority of the membrane-spanning segments of polypeptide chains traverse the bilayer as α helices (see Figure 4−12). In these membrane-spanning α helices, the hydrophobic side chains are exposed on the outside of the helix, where they contact the hydrophobic lipid tails, while the atoms of the hydrophilic polypep- tide backbone form hydrogen bonds with one another within the helix (Figure 11–23). For many transmembrane proteins, the polypeptide chain crosses the membrane only once (see Figure 11–21A, left). Many of these single- pass transmembrane proteins are receptors for extracellular signals. Other transmembrane proteins function as channels, forming aqueous pores across the lipid bilayer to allow small, water-soluble molecules to cross the membrane. Such channels cannot be formed by proteins with a single transmembrane α helix. Instead, they usually consist of a series of α helices that cross the bilayer a number of times (see Figure 11–21A, center). For many of these multipass transmembrane proteins, one or more of the membrane-spanning regions are amphipathic—formed from hydrophobic amino acid side chain α helices that contain both hydrophobic and hydrophilic amino acid side hydrogen bond chains. These amino acids tend to be arranged so that the hydrophobic side chains fall on one side of the helix, while the hydrophilic side chains are concentrated on the other side. In the hydrophobic environment of the lipid bilayer, α helices of this type pack side by side in a ring, with the hydrophobic side chains exposed to the hydrophobic lipid tails and the hydrophilic side chains forming the lining of a hydrophilic pore Figure 11–23 A transmembrane polypeptide chain usually crosses the lipid bilayer as an α helix. In this segment of a transmembrane protein, the hydrophobic side chains (light green) of the amino acids forming the α helix contact the hydrophobic hydrocarbon tails of the phospholipid molecules, while the hydrophilic parts of the polypeptide backbone form hydrogen bonds with one another (dashed red lines) α helix along the interior of the helix. An α helix containing about 20 amino acids is required to completely traverse a cell membrane. hydrophobic tails of membrane phospholipids 378 CHAPTER 11 Membrane Structure hydrophilic side chains amphipathic Figure 11–24 A transmembrane hydrophilic pore can be formed form an aqueous pore α helix by multiple amphipathic α helices. In this example, five amphipathic transmembrane α helices form a water-filled channel across the lipid bilayer. The hydrophobic amino acid side chains on one side of each helix (green) come in contact with the hydrophobic lipid tails of the lipid bilayer, while the hydrophilic side chains on the opposite side of the helices (red ) form a water-filled pore. lipid bilayer through the membrane (Figure 11–24). How such channels function hydrophobic side chains in the selective transport of small, water-soluble molecules, especially interact with phospholipid tails inorganic ions, is discussed in Chapter 12. Although the α helix is by far the most common form in which a poly- peptide chain crosses a lipid bilayer, the polypeptide chain of some transmembrane proteins crosses the lipid bilayer as a β sheet that is rolled into a cylinder, forming a keglike structure called a β barrel (see Figure 11–21A, right). As expected, the amino acid side chains that face the ECB5 E11.23/11.23 inside of the barrel, and therefore line the aqueous channel, are mostly hydrophilic, while those on the outside of the barrel, which contact the hydrophobic core of the lipid bilayer, are exclusively hydrophobic. A strik- QUESTION 11–4 ing example of a β-barrel structure is found in the porin proteins, which form large, water-filled pores in mitochondrial and bacterial outer mem- Explain why the polypeptide chain branes (Figure 11–25). Mitochondria and some bacteria are surrounded of most transmembrane proteins by a double membrane, and porins allow the passage of small nutrients, crosses the lipid bilayer as an α helix metabolites, and inorganic ions across their outer membranes, while or a β barrel. preventing unwanted larger molecules from crossing. Membrane Proteins Can Be Solubilized in Detergents To understand a protein fully, one needs to know its structure in detail. For membrane proteins, this presents special problems. Most biochemi- cal procedures are designed for studying molecules in aqueous solution. Membrane proteins, however, are built to operate in an environment that is partly aqueous and partly fatty, and taking them out of this environ- ment to study in isolation—while preserving their essential structure—is no easy task. Before an individual protein can be examined in detail, it must be sepa- rated from all the other cell proteins. For most membrane proteins, the first step in this purification process involves solubilizing the membrane with agents that destroy the lipid bilayer by disrupting hydrophobic associations. The most widely used disruptive agents are detergents (Movie 11.5). These small, amphipathic, lipidlike molecules differ from C membrane phospholipids in that they have only a single hydrophobic N tail (Figure 11–26). Because they have one tail, detergent molecules are shaped like cones; in water, these conical molecules tend to aggregate 2 nm into small clusters called micelles, rather than forming a bilayer as do the phospholipids, which—with their two tails—are more cylindrical in Figure 11–25 Porin proteins form water- shape. filled channels in the outer membrane of a bacterium. The protein illustrated is When mixed in great excess with membranes, the hydrophobic ends from E. coli, and it consists of a 16-stranded of detergent molecules interact with the membrane-spanning hydro- β sheet curved around on itself to form a phobic regions of the transmembrane proteins, as well as with the transmembrane water-filled channel. The hydrophobic tails of the phospholipid molecules, thereby disrupting three-dimensional structure was determined the lipid bilayer and separating the proteins from most of the phospho- by x-ray crystallography. Although not shown in the drawing, three porin proteins lipids. Because the other end of the detergent molecule is hydrophilic, associate to form a trimer with three these interactions draw the membrane proteins into the aqueous solu- separate channels. ECB5 e11.24/11.24 tion as protein–detergent complexes; at the same time, the detergent  Membrane Proteins 379 Figure 11–26 SDS and Triton X-100 are two commonly used CH3 detergents. Sodium dodecyl sulfate (SDS) is a strong ionic detergent— CH3 C CH3 that is, it has an ionized (charged) group at its hydrophilic end (blue ). Triton X-100 is a mild nonionic detergent—that is, it has a nonionized CH2 but polar structure at its hydrophilic end (blue ). The hydrophobic CH3 portion of each detergent is shown in red. The bracketed portion of CH3 C CH3 Triton X-100 is repeated about eight times. Strong ionic detergents like CH2 C SDS not only displace lipid molecules from proteins but also unfold the CH2 HC CH proteins (see Panel 4–5, p. 167). HC CH CH2 C CH2 O CH2 CH2 also solubilizes the phospholipids (Figure 11–27). The protein–detergent complexes can then be separated from one another and from the lipid– CH2 CH2 detergent complexes for further analysis. CH2 O CH2 CH2 We Know the Complete Structure of Relatively Few CH2 CH2 Membrane Proteins CH2 O For many years, much of what we knew about the structure of mem- ~8 CH2 CH2 brane proteins was learned by indirect means. The standard method for determining a protein’s three-dimensional structure directly has been O CH2 x-ray crystallography, but this approach requires ordered crystalline O S O O arrays of the molecule. Because membrane proteins have to be puri- O Na + H fied in detergent micelles that are often heterogeneous in size, they are harder to crystallize than the soluble proteins that inhabit the cell cyto- sodium dodecyl sulfate Triton X-100 (SDS) sol or extracellular fluids. Nevertheless, with recent advances in x-ray crystallography, along with powerful new approaches such as cryoelec- tron microscopy, the structures of an increasing number of membrane proteins have now been determined to high resolution (see Panel 4–6, pp. 168–169). One example is bacteriorhodopsin, the structure of which first revealed exactly how α helices cross the lipid bilayer. Bacteriorhodopsin is a small QUESTION 11–5 ECB5 e11.25/11.25 protein found in large amounts in the plasma membrane of Halobacterium halobium, an archaean that lives in salt marshes. Bacteriorhodopsin acts For the two detergents shown as a membrane transport protein that pumps H+ (protons) out of the cell. in Figure 11–26, explain why the Each bacteriorhodopsin molecule contains a single chromophore, a light- blue portions of the molecules are absorbing, nonprotein molecule called retinal, that gives the protein—and hydrophilic and the red portions hydrophobic. Draw a short stretch of a polypeptide chain made up of membrane-spanning three amino acids with hydrophobic hydrophobic region detergent side chains (see Panel 2–6, pp. 76– of protein monomers 77) and apply a similar color scheme. hydrophobic Indicate which portions of your tail polypeptide would form hydrogen + bonds with water. hydrophilic head detergent Figure 11–27 Membrane proteins can membrane protein micelle in lipid bilayer be solubilized by a mild detergent such as Triton X-100. The detergent molecules (gold ) are shown as both monomers and micelles, the form in which these molecules tend to aggregate in water. The detergent disrupts the lipid bilayer and interacts with + the membrane-spanning hydrophobic portion of the protein (dark green). These actions bring the proteins into solution as protein–detergent complexes. As illustrated, water-soluble complexes water-soluble mixed the phospholipids in the membrane are also of transmembrane protein lipid–detergent micelles solubilized by the detergents, forming lipid– and detergent detergent micelles. 380 CHAPTER 11 Membrane Structure Figure 11–28 Bacteriorhodopsin acts as a proton pump. The polypeptide chain of H+ this small protein (about 250 amino acids in length) crosses the lipid bilayer as seven α NH2 helices. The location of the retinal (purple) EXTRACELLULAR and the probable pathway taken by protons SPACE during the light-activated pumping cycle (red arrows) are highlighted. Strategically placed polar amino acid side chains— retinal shown in red, yellow, and blue—guide the movement of the proton (H+) across the bilayer, allowing it to avoid contact with the lipid environment. The retinal is then lipid bilayer regenerated by taking up a H+ from the cytosol, returning the protein to its original conformation—a cycle shown in Movie 11.6. Retinal is also used to detect light in our own eyes, where it is attached to a protein with a structure very similar to that of bacteriorhodopsin. CYTOSOL (Adapted from H. Luecke et al., Science transmembrane 286:5438 255–260, 1999.) helices HOOC H+ the entire organism—a deep purple color. When retinal, which is cova- ECB5 e11.27/11.27 lently attached to one of bacteriorhodopsin’s transmembrane α helices, absorbs a photon of light, it changes shape. This shape change causes the surrounding helices to undergo a series of small conformational changes, which pump one proton from the retinal to the outside of the organism (Figure 11–28). In the presence of sunlight, thousands of bacteriorhodopsin mol- ecules pump H+ out of the cell, generating a concentration gradient of H+ across the plasma membrane. The cell uses this proton gradient to store energy and convert it into ATP, as we discuss in detail in Chapter 14. Bacteriorhodopsin is a pump, a class of transmembrane protein that actively moves small organic molecules and inorganic ions into and out of cells. We will discuss the action of other important transmembrane pumps in Chapter 12. The Plasma Membrane Is Reinforced by the Underlying Cell Cortex A cell membrane by itself is extremely thin and fragile. It would require nearly 10,000 cell membranes laid on top of one another to achieve the thickness of this paper. Most cell membranes are therefore strengthened and supported by a framework of proteins, attached to the membrane via transmembrane proteins. For plants, yeasts, and bacteria, the cell’s shape and mechanical properties are conferred by a rigid cell wall—a fibrous layer of proteins, sugars, and other macromolecules that encases the plasma membrane. By contrast, the plasma membrane of animal cells is stabilized by a meshwork of filamentous proteins, called the cell cortex, that is attached to the underside of the membrane. The cortex of the human red blood cell has a relatively simple and regular structure and has been especially well studied. Red blood cells are small and have a distinctive flattened shape (Figure 11−29A). The main com- ponent of their cortex is the dimeric protein spectrin, a long, thin, flexible rod about 100 nm in length. Spectrin forms a lattice that provides sup- port for the plasma membrane and maintains the cell’s biconcave shape. The spectrin network is connected to the membrane through intracellular  Membrane Proteins 381 spectrin dimer actin attachment proteins (A) 5 µm (B) transmembrane proteins 100 nm Figure 11–29 A cortex made largely of spectrin gives human red blood cells their characteristic shape. (A) Scanning electron micrograph showing human red blood cells, which have a flattened, biconcave shape. These cells lack a nucleus and other intracellular organelles. (B) In the cortex of a red blood cell, spectrin dimers (red) are linked end-to-end to form longer tetramers. The spectrin tetramers, together with a smaller number of actin molecules, are linked together into a mesh. This network is attached to the plasma membrane by the binding of at least two types of attachment proteins (shown here in yellow and blue) to two kinds of transmembrane proteins (shown here in green and brown). (A, courtesy of Bernadette Chailley.) ECB5 e11.28-29/11.28 attachment proteins that link spectrin to specific transmembrane pro- teins (Figure 11−29B and Movie 11.7). The importance of this meshwork QUESTION 11–6 is seen in mice and humans that, due to genetic alterations, produce a form of spectrin with an abnormal structure. These individuals are ane- Look carefully at the transmembrane mic: they have fewer red blood cells than normal. The red cells they do proteins shown in Figure 11−29B. have are spherical instead of flattened and are abnormally fragile. What can you say about their mobility in the membrane? Proteins similar to spectrin, and to its associated attachment proteins, are present in the cortex of most animal cells. But the cortex in these cells is especially rich in actin and the motor protein myosin, and it is much more complex than that of red blood cells. Whereas red blood cells need their cortex mainly to provide mechanical strength as they are pumped through blood vessels, other cells also use their cortex to selectively take up materials from their environment, to change their shape, and to move, as we discuss in Chapter 17. In addition, cells also use their cortex to restrain the diffusion of proteins within the plasma membrane, as we see next. A Cell Can Restrict the Movement of Its Membrane Proteins Because a membrane is a two-dimensional fluid, many of its proteins, like its lipids, can move freely within the plane of the bilayer. This lateral diffusion was initially demonstrated by experimentally fusing a mouse cell with a human cell to form a double-sized hybrid cell and then moni- toring the distribution of certain mouse and human plasma membrane proteins. At first, the mouse and human proteins are confined to their own halves of the newly formed hybrid cell, but within half an hour or so the two sets of proteins become evenly mixed over the entire cell sur- face (Figure 11–30). We describe some other techniques for studying the movement of membrane proteins in How We Know, pp. 384–385. The picture of a cell membrane as a sea of lipid in which all proteins float freely is too simple, however. Cells have ways of confining particular proteins to localized areas within the bilayer, thereby creating function- ally specialized regions, or membrane domains, on the surface of the cell or organelle. 382 CHAPTER 11 Membrane Structure Figure 11–30 Formation of mouse–human