3.2 The Cell Membrane and Transport

How does a cell distinguish “itself” from what is outside? The structure that defines the existence of a cell is the cell membrane (Fig. 3.5). Overall, the membrane contains the cytoplasm within the external medium, mediating exchange between the two. The cell membrane consists of a phospholipid bilayer containing lipid-soluble proteins. It behaves as a two-dimensional fluid, within which proteins and lipids can diffuse. The proteins form about half the mass of the membrane and provide specific functions, such as nutrient transport. For review of elementary cell structure, see eAppendix 2.

FIGURE 3.5 Bacterial cell membrane. The cell membrane consists of a phospholipid bilayer, with hydrophobic fatty acid chains directed inward, away from water. The bilayer contains stiffening agents such as hopanoids. Half the membrane volume consists of proteins.

Membrane Lipids

Most membrane lipids are phospholipids. A phospholipid possesses a charged phosphoryl “head” that contacts the water interface, as well as a hydrophobic “tail” of fatty acids packed within the bilayer. Lipid biosynthesis is a key process that is vulnerable to antibiotics. For example, the bacterial enzyme enoyl reductase, which synthesizes fatty acids (discussed in Chapter 15) is the target of triclosan, a common antibacterial additive in detergents and cosmetics.

A typical phospholipid consists of glycerol with ester links to two fatty acids and a phosphoryl polar head group, which at neutral pH is deprotonated (negatively charged) (Fig. 3.6). This kind of phospholipid is called a phosphatidate. The negatively charged head group of the phosphatidate can contain various organic groups, such as glycerol to form phosphatidylglycerol (Fig. 3.6A). In other lipids, the polar head group has a side chain with positive charge. The positive charge commonly resides on an amine group, such as ethanolamine in phosphatidylethanolamine (Fig. 3.6B). Phospholipids with positive charge or with mixed charges are concentrated in portions of the membrane that interact with DNA, which has negative charge.

FIGURE 3.6 Phospholipids. A. Phosphatidylglycerol consists of glycerol with ester links to two fatty acids, and a phosphoryl group linked to a terminal glycerol. B. Phosphatidylethanolamine contains a glycerol linked to two fatty acids, and a phosphoryl group with a terminal ethanolamine. The ethanolamine carries a positive charge.

In the bilayer, all phospholipids face each other tail to tail, keeping their hydrophobic side chains away from the water inside and outside the cell. The two layers of phospholipids in the bilayer are called leaflets. One leaflet of phospholipids faces the cell interior; the other faces the exterior. As a whole, the phospholipid bilayer imparts fluidity and gives the membrane a consistent thickness (about 8 nm).

Membrane Proteins

Membrane proteins serve functions such as transport, communication with the environment, and structural support.

  • Structural support. Some membrane proteins anchor together different layers of the cell envelope (discussed in Section 3.3). Other proteins attach the membrane to the cytoskeleton, or form the base of structures extending out from the cell, such as flagella.
  • Detection of environmental signals. In Vibrio cholerae, the causative agent of cholera, the membrane protein ToxR detects acidity and elevated temperature—signs that the bacteria is in the host’s digestive tract. The ToxR domain facing the cytoplasm then binds to a DNA sequence, activating expression of cholera toxin.
  • Secretion of virulence factors and communication signals. Membrane protein complexes export toxins and cell signals across the envelope. For example, symbiotic nitrogen-fixing rhizobia require membrane proteins NodI and NodJ to transport nodulation signals out to the host plant roots, inducing the plant to form root nodules containing the bacteria.
  • Ion transport and energy storage. Transport proteins manage ion flux between the cell and the exterior. Ion transport generates gradients that store energy.

An example of a bacterial membrane protein is the leucine transporter LeuT (Fig. 3.7). LeuT drives uptake of leucine, coupled to a gradient of sodium ions. The protein complex was purified for X-ray diffraction from Aquifex aeolicus, a thermophile whose heat-stable proteins form durable crystals. Remarkably, LeuT is homologous (shares common ancestry) with a human neuron protein that transports neurotransmitters. Thus, this bacterial protein serves as a model for study of neuron function.

FIGURE 3.7 A cell membrane–embedded transport protein: the LeuT sodium/leucine cotransporter of Aquifex bacteria. The protein complex carries leucine across the cell membrane into the cytoplasm, coupled to sodium ion influx. (PDB code: 3F3E) Inset: Aquifex aeolicus grows at 96°C in hot springs. Source: Karl Stetter and Reinhardt Rachel, U. Regensburg, Germany.

Proteins embedded in a membrane require a hydrophobic portion that is soluble within the membrane. Typically, several hydrophobic alpha helices thread back and forth through the membrane. Other peptide regions extend outside the membrane, containing charged and polar amino acids that interact favorably with water. Figure 3.7 shows the LeuT charge distribution. Hydrophobic amino acid residues (white) make the protein soluble in the membrane, while portions with negative charge (red) and positive charge (blue) lock the protein in.

Transport across the Cell Membrane

The cell membrane acts as a barrier to keep water-soluble proteins and other cell components within the cytoplasm. But how do nutrients from outside get into the cell—and how do secreted products such as toxins get out? Specific membrane proteins transport molecules across the membrane between the cytoplasm and the outside. Selective transport is essential for cell survival; it means the ability to acquire scarce nutrients, exclude waste, and transmit signals to neighbor cells.

Passive diffusion. Small uncharged molecules, such as O2, CO2, and water, easily permeate the membrane. Some molecules, such as ethanol, also disrupt the membrane—an action that can make such molecules toxic to cells. By contrast, large, strongly polar molecules such as sugars, and charged molecules such as amino acids, generally cannot penetrate the hydrophobic interior of the membrane, and thus require transport by specific proteins. Water molecules permeate the membrane, but their rate of passage is increased by protein channels called aquaporins.

Osmosis. Most cells maintain a concentration of total solutes (molecules in solution) that is higher inside the cell than outside. As a result, the internal concentration of water is lower than the concentration outside the cell. Because water can cross the membrane but charged solutes cannot, water tends to diffuse across the membrane into the cell, causing the expansion of cell volume, in a process called osmosis. The resulting pressure on the cell membrane is called osmotic pressure (see figure in Appendix 1). Osmotic pressure will cause a cell to burst, or lyse, in the absence of a countering pressure such as that provided by the cell wall. That is how bacteria are killed by penicillin, which disrupts cell wall synthesis.

Membrane-permeant weak acids and bases. A special case of movement across cell membranes is that of membrane-permeant weak acids and weak bases (Fig. 3.8), which exist in equilibrium between charged and uncharged forms:

Weak acid: HA ⇌ H+ + A

Weak base: B + H2O ⇌ BH+ + OH

FIGURE 3.8 Common drugs are membrane-permeant weak acids and bases. In its charged form (A or BH+), each drug is soluble in the bloodstream. The uncharged form (HA or B) is hydrophobic and penetrates the cell membrane.

Membrane-permeant weak acids and weak bases cross the membrane in their uncharged form: HA (weak acid) or B (weak base). On the other side, entering the aqueous cytoplasm, the acid dissociates (HA to A and H+) or the base reassociates with H+ (B to BH+). In effect, membrane-permeant acids conduct acid (H+) across the membrane, causing acid stress; similarly, membrane-permeant bases conduct OH across the membrane, causing alkali stress. If the H+ concentration (acidity) outside the cell is greater than inside, it will drive weak acids into the cell.

Many key substances in cellular metabolism are membrane-permeant weak acids and bases, such as acetic acid. Most pharmaceutical drugs—therapeutic agents delivered to our tissues via the bloodstream—are weak acids or bases whose uncharged forms exist at sufficiently low concentration to cross the membrane without disrupting it. Examples of weak acids that deprotonate (acquiring negative charge) at neutral pH include aspirin (acetylsalicylic acid) and penicillin (Fig. 3.8A). Examples of weak bases that protonate (acquiring positive charge) at neutral pH include Prozac (fluoxetine) and tetracycline (Fig. 3.8B).

Transmembrane ion gradients. Molecules that carry a fixed charge, such as hydrogen and sodium ions (H+ and Na+), cannot cross the phospholipid bilayer. Such ions usually exist in very different concentrations inside and outside the cell. An ion gradient (ratio of ion concentrations) across the cell membrane can store energy for nutrition or to drive the transport of other molecules. Inorganic ions require transport through specific transport proteins, or transporters. So, too, do organic molecules that carry a charge at cytoplasmic pH, such as amino acids and vitamins. Transport may be passive or active. In passive transport, molecules accumulate or dissipate along their concentration gradient. Active transport—that is, transport from lower to higher concentration—requires cells to spend energy. A transporter protein obtains energy for active transport by cotransport of another substance down its gradient from higher to lower concentration, or by coupling transport to a chemical reaction (discussed in Chapter 4).

Membrane Lipid Diversity

Membranes require a uniform thickness and stability to maintain structural integrity and function. So why do individual membrane lipids differ in structure? Different environments favor different forms of membrane lipids. For example, lipid structure helps determine whether an organism can grow in a hot spring, or whether it can colonize human lungs.

Environmental stress. Starvation stress increases bacterial production of lipids with an unusual type of phosphoryl head group. Cardiolipin, or diphosphatidylglycerol, is actually a double phospholipid linked by a glycerol (Fig. 3.9A). Cardiolipin concentration increases in bacteria grown to starvation or stationary phase (discussed in Chapter 4). Within a cell, cardiolipin does not diffuse at random; it concentrates in patches called “domains” near the cell poles. The polar localization of cardiolipin was demonstrated by fluorescence microscopy, in which a cardiolipin-specific fluorophore localized to the poles of E. coli (Fig. 3.9B). The “wedge” shape of cardiolipin, with its narrow head group and wide fatty acid group, is thought to form concave domains of lipid that stabilize the curve of the polar membrane. Cardiolipin may enhance the formation of smaller cells during starvation. How does this benefit the cell? At the cell pole (Fig. 3.9C), cardiolipin binds certain environmental stress proteins, such as a protein that transports osmoprotectants when the cell is under osmotic stress. Thus, a phospholipid can have specific functions associated with specific membrane proteins.

FIGURE 3.9 Cardiolipin localizes to the poles. A. Cardiolipin is a double phospholipid joined by a third glycerol. B. A space-filling model of cardiolipin shows its triangular shape. C. Cardiolipin localizes to the bacterial cell poles, as shown by microscopy with a cardiolipin-specific fluorophore.

FIGURE 3.10 Phospholipid side chains.

The fatty acid component of phospholipids also varies. The most common bacterial fatty acids are hydrogenated chains of varying length, typically between 6 and 22 carbons. But some fatty acid chains are partly unsaturated (possess one or more carbon-carbon double bonds). Most unsaturated bonds in membranes are cis, meaning that both alkyl chains are on the same side of the bond, so the unsaturated chain has a “kink,” as in the cis form of oleic acid (Fig. 3.10). Because the kinked chains do not pack as closely as the straight hydrocarbon chains do, the membrane is more “fluid.” This is why, at room temperature, unsaturated vegetable oils are fluid, whereas highly saturated butterfat is solid. The enhanced fluidity of a kinked phospholipid improves the function of the membrane at low temperature; hence, bacteria can respond to cold and heat by increasing or decreasing their synthesis of unsaturated phospholipids.

Another interesting structural variation is cyclization of part of the chain to form a stiff planar ring with decreased fluidity. The double bond of unsaturated fatty acids can incorporate a carbon from S-adenosyl-L-methionine to form a three-membered ring, generating a cyclopropane fatty acid (Fig. 3.10). Bacteria convert unsaturated fatty acids to cyclopropane during starvation and acid stress, conditions under which membranes require stiffening. Cyclopropane conversion is an important factor in the pathogenesis of Mycobacterium tuberculosis and in the acid resistance of food-borne toxigenic E. coli.

FIGURE 3.11 Hopanoids add strength to membranes. Hopanoids limit the motion of phospholipid tails, thus stiffening the membrane.

FIGURE 3.12 Terpene-derived lipids of archaea. In archaea, the hydrocarbon chains are ether-linked to glycerol, and every fourth carbon has a methyl branch. In some archaea, the tails of the two facing lipids of the bilayer are fused, forming tetraethers; thus, the entire membrane consists of a monolayer.

In addition to phospholipids, membranes include planar molecules that fill gaps between hydrocarbon chains. These stiff, planar molecules reinforce the membrane, much as steel rods reinforce concrete. In eukaryotic membranes, the reinforcing agents are sterols, such as cholesterol. In some bacteria, the same function is filled by pentacyclic (five-ring) hydrocarbon derivatives called hopanoids, or hopanes (Fig. 3.11). Like cholesterol, hopanoids fit between the fatty acid side chains of membranes and limit their motion, thus stiffening the membrane. Hopanoids appear in geological sediments, where they indicate ancient bacterial decomposition; they provide useful data for petroleum exploration.

Archaea have unique membrane lipids. The membrane lipids of archaea differ fundamentally from those of bacteria and of eukaryotes. All archaeal phospholipids replace the ester link between glycerol and fatty acid with an ether link, C–O–C (Fig. 3.12). Ethers are much more stable than esters, which hydrolyze easily in water. This is one reason why some archaea can grow at higher temperatures than all other forms of life. Another modification is that archaeal hydrocarbon chains are branched terpenoids, polymeric structures derived from isoprene, in which every fourth carbon extends a methyl branch. The branches strengthen the membrane by limiting movement of the hydrocarbon chains.

The most extreme hyperthermophiles, which live beneath the ocean at 110°C, have terpenoid chains linked at the tails, forming a tetraether monolayer. In some species, the terpenoids cyclize to form cyclopentane rings. These planar rings stiffen the membrane under stress to an even greater extent than the cyclopropyl chains of bacteria. For more on archaeal cells, see Chapter 19.

Interestingly, both hopanoids and cholesterol are synthesized from the same precursor molecules as the unique lipids of archaea (Fig. 3.13). It may be that hopanoids and cholesterol persist in bacteria and eukaryotes as derivatives of lipids that were once possessed by a common ancestor of all three domains.

FIGURE 3.13 Synthesis of terpene-derived lipids. Archaea synthesize their membrane lipids from isoprene chains, to form terpenoids such as squalene. In bacteria and eukaryotes, squalene is converted to cholesterol; and in some bacteria, squalene cyclizes to form hopanes (hopanoids).

To Summarize
  • The cell membrane consists of a phospholipid bilayer containing hydrophobic membrane proteins. Membrane proteins serve diverse functions, including transport, cell defense, and cell communication.
  • Small uncharged molecules, such as oxygen, can penetrate the cell membrane by diffusion.
  • Membrane-permeant weak acids and weak bases exist partly in an uncharged form that can diffuse across the membrane and increase or decrease, respectively, the H+ concentration within the cell.
  • Polar molecules and charged molecules require membrane proteins to mediate transport. Such facilitated transport can be active or passive.
  • Ion gradients generated by membrane pumps store energy for cell functions. Active transport (up the concentration gradient) requires input of energy, whereas passive transport (down a gradient) does not.
  • Diverse fatty acids are found in different microbial species and in microbes adapted to different environments.
  • Archaeal membranes have ether-linked terpenoids, which confer increased stability at high temperature and extreme acidity. Some archaea have diglycerol tetraethers, which form a monolayer.