The process by which a carrier protein transfers a solute molecule across the lipid bilayer resembles an enzyme-substrate reaction, and in many ways carriers behave like enzymes. In contrast to ordinary enzyme-substrate reactions, however, the transported solute is not covalently modified by the carrier protein, but instead is delivered unchanged to the other side of the membrane.
Each type of carrier protein has one or more specific binding sites for its solute (substrate). It transfers the solute across the lipid bilayer by undergoing reversible conformational changes that alternately expose the solute-binding site first on one side of the membrane and then on the other. A schematic model of how such a carrier protein is thought to operate is shown in . When the carrier is saturated (that is, when all solute-binding sites are occupied), the rate of transport is maximal. This rate, referred to as Vmax, is characteristic of the specific carrier and reflects the rate with which the carrier can flip between its two conformational states. In addition, each transporter protein has a characteristic binding constant for its solute, Km, equal to the concentration of solute when the transport rate is half its maximum value (). As with enzymes, the binding of solute can be blocked specifically by either competitive inhibitors (which compete for the same binding site and may or may not be transported by the carrier) or noncompetitive inhibitors (which bind elsewhere and specifically alter the structure of the carrier).
A model of how a conformational change in a carrier protein could mediate the passive transport of a solute. The carrier protein shown can exist in two conformational states: in state A, the binding sites for solute are exposed on the outside of the lipid (more...)
The kinetics of simple diffusion and carrier-mediated diffusion. Whereas the rate of the former is always proportional to the solute concentration, the rate of the latter reaches a maximum (Vmax) when the carrier protein is saturated. The solute concentration (more...)
As we discuss below, it requires only a relatively minor modification of the model shown in to link the carrier protein to a source of energy in order to pump a solute uphill against its electrochemical gradient. Cells carry out such active transport in three main ways ():
Three ways of driving active transport. The actively transported molecule is shown in yellow, and the energy source is shown in red.1.
Coupled carriers couple the uphill transport of one solute across the membrane to the downhill transport of another.2.
ATP-driven pumps couple uphill transport to the hydrolysis of ATP.3.
Light-driven pumps, which are found mainly in bacterial cells, couple uphill transport to an input of energy from light, as with bacterio-rhodopsin (discussed in Chapter 10).
Amino acid sequence comparisons suggest that, in many cases, there are strong similarities in molecular design between carrier proteins that mediate active transport and those that mediate passive transport. Some bacterial carriers, for example, which use the energy stored in the H+ gradient across the plasma membrane to drive the active uptake of various sugars, are structurally similar to the carriers that mediate passive glucose transport into most animal cells. This suggests an evolutionary relationship between various carrier proteins; and, given the importance of small metabolites and sugars as an energy source, it is not surprising that the superfamily of carriers is an ancient one.
We begin our discussion of active transport by considering carrier proteins that are driven by ion gradients. These proteins have a crucial role in the transport of small metabolites across membranes in all cells. We then discuss ATP-driven pumps, including the Na+ pump that is found in the plasma membrane of almost all cells.
Active Transport Can Be Driven by Ion Gradients
Some carrier proteins simply transport a single solute from one side of the membrane to the other at a rate determined as above by V max and Km; they are called uniporters. Others, with more complex kinetics, function as coupled carriers, in which the transfer of one solute strictly depends on the transport of a second. Coupled transport involves either the simultaneous transfer of a second solute in the same direction, performed by symporters, or the transfer of a second solute in the opposite direction, performed by antiporters ().
Three types of carrier-mediated transport. This schematic diagram shows carrier proteins functioning as uniporters, symporters, and antiporters.
The tight coupling between the transport of two solutes allows these carriers to harvest the energy stored in the electrochemical gradient of one solute, typically an ion, to transport the other. In this way, the free energy released during the movement of an inorganic ion down an electrochemical gradient is used as the driving force to pump other solutes uphill, against their electrochemical gradient. This principle can work in either direction; some coupled carriers function as symporters, others as antiporters. In the plasma membrane of animal cells, Na+ is the usual co-transported ion whose electrochemical gradient provides a large driving force for the active transport of a second molecule. The Na+ that enters the cell during transport is subsequently pumped out by an ATP-driven Na+ pump in the plasma membrane (as we discuss later), which, by maintaining the Na+ gradient, indirectly drives the transport. (For this reason ion-driven carriers are said to mediate secondary active transport, whereas ATP-driven carriers are said to mediate primary active transport.) Intestinal and kidney epithelial cells, for example, contain a variety of symport systems that are driven by the Na+ gradient across the plasma membrane; each system is specific for importing a small group of related sugars or amino acids into the cell. In these systems, the solute and Na+ bind to different sites on a carrier protein. Because the Na+ tends to move into the cell down its electrochemical gradient, the sugar or amino acid is, in a sense, dragged into the cell with it. The greater the electrochemical gradient for Na+, the greater the rate of solute entry; conversely, if the Na+ concentration in the extracellular fluid is reduced, solute transport decreases ().
One way in which a glucose carrier can be driven by a Na+ gradient. As in the model shown in Figure 11-6, the carrier oscillates between two alternate states, A and B. In the A state, the protein is open to the extracellular space; in the B state, it (more...)
In bacteria and yeasts, as well as in many membrane-enclosed organelles of animal cells, most active transport systems driven by ion gradients depend on H+ rather than Na+ gradients, reflecting the predominance of H+ pumps and the virtual absence of Na+ pumps in these membranes. The active transport of many sugars and amino acids into bacterial cells, for example, is driven by the electrochemical H+ gradient across the plasma membrane. One well-studied H+ -driven symport is lactose permease, which transports lactose across the plasma membrane of E. coli. Although the folded structure of the permease is unknown, biophysical studies and extensive analyses of mutant proteins have led to a detailed model of how the symport works. The permease consists of 12 loosely packed transmembrane α helices. During the transport cycle, some of the helices undergo sliding motions that cause them to tilt. These motions alternately open and close a crevice between the helices, exposing the binding sites for the solutes lactose and H+, first on one side of the membrane and then on the other ().
A model for the molecular mechanism of action of the bacterial lactose permease. (A) A view from the cytosol of the proposed arrangement of the 12 predicted transmembrane helices in the membrane. The loops that connect the helices on either side of the (more...)
Na+ -driven Carrier Proteins in the Plasma Membrane Regulate Cytosolic pH
The structure and function of most macromolecules are greatly influenced by pH, and most proteins operate optimally at a particular pH. Lysosomal enzymes, for example, function best at the low pH (~5) found in lysosomes, whereas cytosolic enzymes function best at the close to neutral pH (~7.2) found in the cytosol. It is therefore crucial that cells be able to control the pH of their intracellular compartments.
Most cells have one or more types of Na+ -driven antiporters in their plasma membrane that help to maintain the cytosolic pH (pHi), at about 7.2. These proteins use the energy stored in the Na+ gradient to pump out excess H+, which either leaks in or is produced in the cell by acid-forming reactions. Two mechanisms are used: either H+ is directly transported out of the cell or HCO3- is brought into the cell to neutralize H+ in the cytosol (according to the reaction HCO3- + H+ H2O + CO2). One of the antiporters that uses the first mechanism is a Na+-H+ exchanger, which couples an influx of Na+ to an efflux of H+. Another, which uses a combination of the two mechanisms, is a Na+-driven Cl--HCO3- exchanger that couples an influx of Na+ and HCO3- to an efflux of Cl- and H+ (so that NaHCO3 comes in and HCl goes out). The Na+-driven Cl--HCO3- exchanger is twice as effective as the Na+-H+ exchanger, in the sense that it pumps out one H+ and neutralizes another for each Na+ that enters the cell. If HCO3- is available, as is usually the case, this antiporter is the most important carrier protein regulating pHi. Both exchangers are regulated by pHi and increase their activity as the pH in the cytosol falls.
An Na+-independent Cl--HCO3- exchanger also has an important role in pHi regulation. Like the Na+-dependent transporters, the Cl--HCO3- exchanger is regulated by pHi, but the movement of HCO3-, in this case, is normally out of the cell, down its electrochemical gradient. The rate of HCO3- efflux and Cl- influx increases as pHi rises, thereby decreasing pHi whenever the cytosol becomes too alkaline. The Cl--HCO3- exchanger is similar to the band 3 protein in the membrane of red blood cells discussed in Chapter 10. In red blood cells, band 3 protein facilitates the quick discharge of CO2 as the cells pass through capillaries in the lung.
ATP-driven H+ pumps are also used to control the pH of many intracellular compartments. As discussed in Chapter 13, the low pH in lysosomes, as well as in endosomes and secretory vesicles, is maintained by such H+ pumps, which use the energy of ATP hydrolysis to pump H+ into these organelles from the cytosol.
An Asymmetric Distribution of Carrier Proteins in Epithelial Cells Underlies the Transcellular Transport of Solutes
In epithelial cells, such as those involved in absorbing nutrients from the gut, carrier proteins are distributed nonuniformly in the plasma membrane and thereby contribute to the transcellular transport of absorbed solutes. As shown in , Na+ -linked symporters located in the apical (absorptive) domain of the plasma membrane actively transport nutrients into the cell, building up substantial concentration gradients for these solutes across the plasma membrane. Na+ -independent transport proteins in the basal and lateral (basolateral) domain allow the nutrients to leave the cell passively down these concentration gradients.
Transcellular transport. The transcellular transport of glucose across an intestinal epithelial cell depends on the nonuniform distribution of transport proteins in the cell's plasma membrane. The process shown here results in the transport of glucose (more...)
In many of these epithelial cells, the plasma membrane area is greatly increased by the formation of thousands of microvilli, which extend as thin, fingerlike projections from the apical surface of each cell (see ). Such microvilli can increase the total absorptive area of a cell by as much as 25-fold, thereby enhancing its transport capabilities.
Although, as we have seen, ion gradients have a crucial role in driving many essential transport processes in cells, the ion pumps that use the energy of ATP hydrolysis are responsible mainly for establishing and maintaining these gradients, as we next discuss.
The Plasma Membrane Na+ -K+ Pump Is an ATPase
The concentration of K+ is typically 10 to 20 times higher inside cells than outside, whereas the reverse is true of Na+ (see Table 11-1, p. 616). These concentration differences are maintained by a Na + -K + pump, or Na + pump, found in the plasma membrane of virtually all animal cells. The pump operates as an antiporter, actively pumping Na+ out of the cell against its steep electrochemical gradient and pumping K+ in. Because the pump hydrolyzes ATP to pump Na+ out and K+ in, it is also known as a Na + -K + ATPase ().
The Na+-K+pump. This carrier protein actively pumps Na+ out of and K+ into a cell against their electrochemical gradients. For every molecule of ATP hydrolyzed inside the cell, three Na+ are pumped out and two K+ are pumped in. The specific inhibitor (more...)
We have seen that the Na+ gradient produced by the pump drives the transport of most nutrients into animal cells and also has a crucial role in regulating cytosolic pH. As we discuss below, the pump also regulates cell volume through its osmotic effects; indeed, it keeps many animal cells from bursting. Almost one-third of the energy requirement of a typical animal cell is consumed in fueling this pump. In electrically active nerve cells, which, as we shall see, repeatedly gain small amounts of Na+ and lose small amounts of K+ during the propagation of nerve impulses, this number approaches two-thirds of the cell's energy requirement.
An essential characteristic of the Na+ -K+ pump is that the transport cycle depends on autophosphorylation of the protein. The terminal phosphate group of ATP is transferred to an aspartic acid residue of the pump and is subsequently removed, as explained in . Different states of the pump are thus distinguished by the presence or absence of the phosphate group. Ion pumps that phosphorylate themselves in this way are called P-type transport ATPases. They constitute a family of structurally and functionally related proteins, which includes a variety of Ca2+ pumps and H+ pumps, as we discuss below.
A model of the pumping cycle of the Na+ -K+ pump. (1) The binding of Na+ and (2) the subsequent phosphorylation by ATP of the cytoplasmic face of the pump induce the protein to undergo a conformational change that (3) transfers the Na+ across the membrane (more...)
Like any enzyme, the Na+ -K+ pump can be driven in reverse, in this case to produce ATP. When the Na+ and K+ gradients are experimentally increased to such an extent that the energy stored in their electrochemical gradients is greater than the chemical energy of ATP hydrolysis, these ions move down their electrochemical gradients and ATP is synthesized from ADP and phosphate by the Na+ -K+ pump. Thus, the phosphorylated form of the pump (step 2 in ) can relax by either donating its phosphate to ADP (step 2 to step 1) or changing its conformation (step 2 to step 3). Whether the overall change in free-energy is used to synthesize ATP or to pump Na+ out of the cell depends on the relative concentrations of ATP, ADP, and phosphate, as well as on the electrochemical gradients for Na+ and K+.
Some Ca2+ and H+ Pumps Are Also P-type Transport ATPases
In addition to the Na+ -K+ pump, the P-type transport ATPase family includes Ca 2+ pumps that remove Ca2+ from the cytosol after signaling events and the H + -K + pumps that secrete acid from specialized epithelial cells in the lining of the stomach. The Ca2+ pumps are especially important. Eucaryotic cells maintain very low concentrations of free Ca2+ in their cytosol (~10-7 M) in the face of very much higher extracellular Ca2+ concentrations (~10-3 M). Even a small influx of Ca2+ significantly increases the concentration of free Ca2+ in the cytosol, and the flow of Ca2+ down its steep concentration gradient in response to extracellular signals is one means of transmitting these signals rapidly across the plasma membrane (discussed in Chapter 15). The maintenance of a steep Ca2+ gradient is therefore important to the cell. The Ca2+ gradient is maintained by Ca2+ transporters in the plasma membrane that actively move Ca2+ out of the cell. One of these is a P-type Ca2+ ATPase; the other is an antiporter (called a Na + -Ca 2+ exchanger)that is driven by the Na+ electrochemical gradient (see ).
The best-understood P-type transport ATPase is the Ca 2+ pump, or Ca 2+ ATPase, in the sarcoplasmic reticulum membrane of skeletal muscle cells. The sarcoplasmic reticulum is a specialized type of endoplasmic reticulum that forms a network of tubular sacs in the muscle cell cytosol and serves as an intracellular store of Ca2+. (When an action potential depolarizes the muscle cell membrane, Ca2+ is released from the sarcoplasmic reticulum through the Ca2+ -release channels into the cytosol, stimulating the muscle to contract, as discussed in Chapter 16.) The Ca2+ pump, which accounts for about 90% of the membrane protein of the organelle, is responsible for moving Ca2+ from the cytosol back into the sarcoplasmic reticulum. The endoplasmic reticulum of nonmuscle cells contains a similar Ca2+ pump, but in smaller quantities.
The three-dimensional structure of the sarcoplasmic reticulum Ca2+ pump has been determined at high resolution. This structure and the analysis of a related fungal H+ pump have provided the first views of P-type transport ATPases, which are all thought to have similar structures. They contain 10 transmembrane α helices, three of which line a central channel that spans the lipid bilayer. In the unphosphorylated state of the Ca2+ pump, two of these helices are disrupted and form a cavity that is accessible from the cytosolic side of the membrane and binds two Ca2+ ions. The binding of ATP to a binding site on the same side of the membrane and the subsequent phosphorylation of an adjacent domain lead to a drastic rearrangement of the transmembrane helices. The rearrangement disrupts the Ca2+ -binding site and releases the Ca2+ ions on the other side of the membrane, into the lumen of the sarcoplasmic reticulum ().
A model of how the sarcoplasmic reticulum Ca2+ pump moves Ca2+. The structure of the unphosphorylated Ca2+ -bound state (left) is based on the X-ray crystallographic structure of the pump. The structure of the phosphorylated, Ca2+ -free state (right) (more...)
The Na+ -K+ Pump Is Required to Maintain Osmotic Balance and Stabilize Cell Volume
Since the Na+ -K+ pump drives three positively charged ions out of the cell for every two it pumps in, it is electrogenic. It drives a net current across the membrane, tending to create an electrical potential, with the cell's inside negative relative to the outside. This electrogenic effect of the pump, however, seldom contributes more than 10% to the membrane potential. The remaining 90%, as we discuss later, depends on the Na+ -K + pump only indirectly.
On the other hand, the Na+ -K+ pump does have a direct and crucial role in regulating cell volume. It controls the solute concentration inside the cell, thereby regulating the osmolarity (or tonicity) that can make a cell swell or shrink (). Because the plasma membrane is weakly permeable to water, water moves slowly into or out of cells down its concentration gradient, a process called osmosis. If cells are placed in a hypotonic solution (that is, a solution having a low solute concentration and therefore a high water concentration), there is a net movement of water into the cells, causing them to swell and burst (lyse). Conversely, if cells are placed in a hypertonic solution, they shrink (see ). Many animal cells also contain specialized water channels in their plasma membrane to facilitate osmotic water flow called aquaporins.
Response of a human red blood cell to changes in osmolarity of the extracellular fluid. The cell swells or shrinks as water moves into or out of the cell down its concentration gradient.
The importance of the Na+ -K+ pump in controlling cell volume is indicated by the observation that many animal cells swell, and often burst, if they are treated with ouabain, which inhibits the Na+ -K+ pump. As explained in Panel 11-1, cells contain a high concentration of solutes, including numerous negatively charged organic molecules that are confined inside the cell (the so-called fixed anions)and their accompanying cations that are required for charge balance. This tends to create a large osmotic gradient that, unless balanced, would tend to pull water into the cell. For animal cells this effect is counteracted by an opposite osmotic gradient due to a high concentration of inorganic ions-chiefly Na+ and Cl-in the extracellular fluid. The Na+ -K+ pump maintains osmotic balance by pumping out the Na+ that leaks in down its steep electrochemical gradient. The Cl- is kept out by the membrane potential.
Intracellular Water Balance: the Problem and Its Solution.
There are, of course, other ways for a cell to cope with its osmotic problems. Plant cells and many bacteria are prevented from bursting by the semirigid cell wall that surrounds their plasma membrane. In amoebae the excess water that flows in osmotically is collected in contractile vacuoles, which periodically discharge their contents to the exterior (see Panel 11-1). Bacteria have also evolved strategies that allow them to lose ions, and even macromolecules, quickly when subjected to an osmotic shock. But for most animal cells, the Na+ -K+ pump is crucial.
Membrane-bound Enzymes That Synthesize ATP Are Transport ATPases Working in Reverse
The plasma membrane of bacteria, the inner membrane of mitochondria, and the thylakoid membrane of chloroplasts all contain transport ATPases. These, however, belong to the family of F-type ATPases and are structurally very different from P-type ATPases. F-type ATPases are turbine-like structures, constructed from multiple different protein subunits. We shall discuss them in detail in Chapter 14.
The F-type ATPases are known as ATP synthases because they normally work in reverse: instead of ATP hydrolysis driving ion transport, H+ gradients across their membranes drive the synthesis of ATP from ADP and phosphate. The H+ gradients are generated either during the electron-transport steps of oxidative phosphorylation (in aerobic bacteria and mitochondria), during photosynthesis (in chloroplasts), or by the light-activated H+ pump (bacteriorhodopsin) in Halobacterium.
The ATP synthases can, like the P-type transport ATPases, work in either direction: when the electrochemical gradient across their membrane drops below a threshold value, they will hydrolyze ATP and pump H+ across the membrane. Structurally related to the F-type ATPases is a distinct family of V-type ATPases.Certain organelles, such as lysosomes, synaptic vesicles, and plant vacuoles, contain V-type ATPases that pump H+ into the organelles and hence are responsible for acidification of their interiors. Thus, V-type ATPases normally pump proteins rather than synthesize ATP.
ABC Transporters Constitute the Largest Family of Membrane Transport Proteins
The last type of carrier protein that we discuss is a family of transport ATPases that are of great clinical importance, even though their normal functions in eucaryotic cells are only just beginning to be discovered. The first of these proteins to be characterized was found in bacteria. We have already mentioned that the plasma membranes of all bacteria contain carrier proteins that use the H+ gradient across the membrane to pump a variety of nutrients into the cell. Many also have transport ATPases that use the energy of ATP hydrolysis to import certain small molecules. In bacteria such as E. coli,which have double membranes (), the transport ATPases are located in the inner membrane, and an auxiliary mechanism exists to capture the nutrients and deliver them to the transporters ().
A small section of the double membrane of an E. coli bacterium. The inner membrane is the cell's plasma membrane. Between the inner and outer lipid bilayer membranes is a highly porous, rigid peptidoglycan, composed of protein and polysaccharide, that (more...)
The auxiliary transport system associated with transport ATPases in bacteria with double membranes. The solute diffuses through channel-forming proteins (porins) in the outer membrane and binds to a periplasmic substrate-binding protein. As a result, (more...)
The transport ATPases in the bacterial plasma membrane belong to the largest and most diverse family of transport proteins known. It is called the ABC transporter superfamily because each member contains two highly conserved ATP-binding cassettes (). ATP binding leads to dimerization of the two ATP-binding domains, and ATP hydrolysis leads to their dissociation. These structural changes in the cytosolic domains are thought to be transmitted to the transmembrane segments, driving cycles of conformational changes that alternately expose substrate-binding sites to one or the other side of the membrane. In this way, ABC transporters use ATP binding and hydrolysis to transport molecules across the bilayer.
A typical ABC transporter. (A) A topology diagram. (B) A hypothetical arrangement of the polypeptide chain in the membrane. The transporter consists of four domains: two highly hydrophobic domains, each with six putative membrane-spanning segments that (more...)
In E. coli, 78 genes (an amazing 5% of the bacterium's genes) encode ABC transporters, and animal cells contain many more. Although each is thought to be specific for a particular substrate or class of substrates, the variety of substrates transported by this superfamily is great and includes amino acids, sugars, inorganic ions, polysaccharides, peptides, and even proteins. Whereas bacterial ABC transporters are used for both import and export, those described in eucaryotes mostly seem specialized for export. ABC transporters also catalyze the flipping of lipids from one face of the lipid bilayer to the other, and thus have an important role in membrane biogenesis and maintenance, as discussed in Chapter 12. When the substrates are lipids or overall hydrophobic molecules, the binding sites for them must be exposed on the surface of the transporter that is in contact with the hydrophobic interior of the lipid bilayer.
Indeed, the first eucaryotic ABC transporters identified were discovered because of their ability to pump hydrophobic drugs out of the cytosol. One of these is the multidrug resistance (MDR) protein, whose overexpression in human cancer cells can make the cells simultaneously resistant to a variety of chemically unrelated cytotoxic drugs that are widely used in cancer chemotherapy. Treatment with any one of these drugs can result in the selection of cells that overexpress the MDR transport protein. The transporter pumps the drugs out of the cell, thereby reducing their toxicity and conferring resistance to a wide variety of therapeutic agents. Some studies indicate that up to 40% of human cancers develop multidrug resistance, making it a major hurdle in the battle against cancer.
A related and equally sinister phenomenon occurs in the protist Plasmodium falciparum, which causes malaria. More than 200 million people are infected with this parasite, which remains a major cause of human death, killing more than a million people every year. The control of malaria is hampered by the development of resistance to the antimalarial drug chloroquine, and resistant P. falciparum have been shown to have amplified a gene encoding an ABC transporter that pumps out the chloroquine.
In yeasts, an ABC transporter is responsible for exporting a mating pheromone (which is a peptide 12 amino acids long) across the yeast cell plasma membrane. In most vertebrate cells, an ABC transporter in the endoplasmic reticulum (ER) membrane actively transports a wide variety of peptides, produced by protein degradation, from the cytosol into the ER. This is the first step in a pathway of great importance in the surveillance of cells by the immune system (discussed in Chapter 24). The transported protein fragments, having entered the ER, are eventually carried to the cell surface, where they are displayed for scrutiny by cytotoxic T lymphocytes, which kill the cell if the fragments seem foreign (as they do if they derive from a virus or other microorganisms lurking in the cytosol).
Yet another member of the ABC family has been discovered through studies of the common genetic disease cystic fibrosis. This disease is caused by a mutation in a gene encoding an ABC transporter that functions as a regulator of a Cl- channel in the plasma membrane of epithelial cells. One in 27 white persons carries a mutant gene encoding this protein; and, in 1 in 2500, both copies of the gene are mutant, causing the disease. It is still uncertain how the ABC transporter acts to regulate Cl-conductance across the membrane.
Carrier proteins bind specific solutes and transfer them across the lipid bilayer by undergoing conformational changes that expose the solute-binding site sequentially on one side of the membrane and then on the other. Some carrier proteins simply transport a single solute downhill, whereas others can act as pumps to transport a solute uphill against its electrochemical gradient, using energy provided by ATP hydrolysis, by a downhill flow of another solute (such as Na+ or H+), or by light to drive the requisite series of conformational changes in an orderly manner. Carrier proteins belong to a small number of families. Each family comprises proteins of similar amino acid sequence that are thought to have evolved from a common ancestral protein and to operate by a similar mechanism. The family of P-type transport ATPases, which includes the ubiquitous Na+ -K+ pump, is an important example; each of these ATPases sequentially phosphorylates and dephosphorylates itself during the pumping cycle. The superfamily of ABC transporters is the largest family of membrane transport proteins and is especially important clinically. It includes proteins that are responsible for cystic fibrosis, as well as for drug resistance in cancer cells and in malaria-causing parasites.