Membrane Carriers

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CHAPTER 9 Membrane Carriers

Carriers are integral membrane proteins that use electrochemical gradients to move select chemical substrates across lipid bilayers (Fig. 9-1). Transport by well-characterized carriers depends on a conformational change to move each substrate. Typically, the carriers work step by step, more like enzymes than channels. Channels simply provide a selective pore for transport, and they generally transport at much higher rates (see Chapter 10). Common substrates for carriers are ions and small soluble organic molecules, but in some cases, substrates are lipid soluble.

Figure 9-1 primary and secondary transport reactions. An ATP-driven pump produces a gradient of an ion, such as Na⁺, across a membrane. The triangles represent gradients of Na⁺ (purple), glucose (green), sugar (blue), and Ca²⁺ (light blue) across the membrane. This ion gradient drives secondary transport reactions mediated by carriers. Uniporters allow an ion or other solute to move across the membrane down its concentration gradient. Symporters and antiporters couple transport of an ion (Na⁺ in this example) down its concentration gradient with the transport of a solute (glucose or Ca²⁺ in these examples) up its concentration gradient. Antiporters carry out these reactions in succession, picking up Na⁺ outside, reorienting, dissociating Na⁺ inside, picking up Ca²⁺ inside, reorienting, and releasing Ca²⁺ outside.

Like pumps and channels, carriers are found in all membranes, wherever cells need to exchange molecules for metabolism or extrude wastes. Carriers are also known as facilitators or porters.

Carriers that transport a single substrate across a membrane down its concentra-tion gradient are called uniporters. Remarkably, many carriers also transport substrates up concentration gradients, provided that their passage through the carrier and across the membrane is coupled to the transport of another substrate down its electrochemical gradient. Glucose provides good examples of both downhill and uphill movement through different carriers. The GLUT1 uniporter allows glucose to move down its concentration gradient from plasma into red blood cells. On the other hand, the SGLT1 carrier uses a gradient of Na⁺ established by the Na⁺K⁺−ATPase pump to move glucose up its concentration gradient into intestinal cells. It is called a symporter, since glucose and Na⁺ move in the same direction. Another class of carriers called antiporters move a substrate in the opposite direction to the ion gradient driving the reaction. All carrier-mediated reactions are reversible, so substrates can move in either direction across the membrane, depending on the polarity of the driving forces.

When a carrier uses an ion gradient to provide the energy to transport a substrate, it is said to catalyze a secondary reaction. In this sense, pumps catalyze primary transport reactions, using energy from ATP hydrolysis, electron transport, or absorption of light to create ion gradients (see Chapter 8). Coupling an ion gradient created by pumps to drive transport by a carrier is called a chemiosmotic cycle (see Fig. 11-1).

Diversity of Carrier Proteins

Biological experimentation and exploration of genomes have revealed more than a hundred families of carriers, many of which can be grouped into superfamilies. The major facilitator superfamily (MFS) is the focus of this chapter, since it includes about one third of all known carrier proteins, including many of the best-characterized carriers. Thousands of MFS genes in all branches of the phylogenetic tree are likely to have arisen from a common ancestor. Two thirds of known carriers have other origins and structures but have converged on mechanical solutions for solute transport similar to MFS carriers. No one knows how many structurally distinct groups exist in nature. Box 9-1 illustrates a small selection of carrier families with different evolutionary origins and structures.

BOX 9-1 Crystal Structures of Diverse Carrier Proteins

Four crystal structures (Fig. 9-2) illustrate the diversity of carrier proteins. They differ in evolutionary origins and structures, but all function as carriers. They converged toward common mechanisms implemented by different structures. Conformational changes are believed to contribute to transport in all cases, so their mechanisms will be better understood when structures of additional conformations of each protein are available.

• MFS carriers consist of single polypeptides that form 10 to 14 (usually 12) transmembrane helices (Fig. 9-2B). Substrates bind in a pocket among these helices. A conformational change exposes this binding site on either side of the membrane so that substrates can bind and dissociate. These carriers are active as monomers.

• Mitochondrial adenine nucleotide carriers transport ATP and ADP across the membranes of mitochondria and chloroplasts. Their genes were formed by a threefold duplication and divergence of a sequence that encodes a pair of transmembrane helices. These six transmembrane helices form a cup exposing a nucleotide binding site near the middle of the membrane bilayer. These carriers are believed to operate in pairs to enable cooperative binding of ATP and ADP on opposite sides of the inner mitochondrial membrane. Once bound, the nucleotides are transported down their concentration gradients.

• Multidrug transporters help bacteria to live in hostile environments by extruding a wide variety of toxic hydrophobic chemicals. Substrates include bile salts, dyes, detergents, and lipid-soluble antibiotics. When overexpressed these carriers can make bacteria resistant to antibiotics. The protein is a homotrimer of huge subunits, each with 12 transmembrane helices. Large domains above the membrane help to span the periplasmic space. Substrates that are soluble in the membrane diffuse into a hydrophobic binding site in the center of the carrier and are transported out of the cell by a mechanism that depends on a proton gradient across the plasma membrane.

• Glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft after a nerve impulse and transport glutamate into bacteria. The protein is a trimer of identical subunits, each composed of eight complex transmembrane helices. Each subunit has two α-helical hairpin loops that partially cross the bilayer and bind a glutamate. Each glutamate transported across the membrane is accompanied by three Na⁺ and one H⁺ moving in the same direction and one k⁺ moving in the opposite direction. Transport may involve transient fluctuations that open and close a gate near the bound glutamate, but the details are not known.

Structure of MFS Carrier Proteins

Crystal structures of two MFS carrier proteins from Escherichia coli (Fig. 9-3A-B) confirmed much of what had been learned about their organization from less direct methods. GlpT is a glycerol-3-phosphate–phosphate antiporter. LacY, the lactose permease, is a lactose-proton symporter. Both proteins consist of 12 transmembrane α-helices. The sequences and structures of the two halves of each protein are homologous, so it is believed that the original gene was created by duplication of an ancestral gene, which coded for a six-helix protein that formed functional dimers. As the MFS gene family grew during evolution, the ancient gene duplication and fusion process had two advantages. First, it allowed the two halves of each gene to diversify separately to increase specificity for a wide variety of substrates. Second, a single polypeptide simplifies assembly of a functional carrier, as two half-sized subunits do not have to find each other. If the two halves of a 12-helix MFS carrier are expressed in the same cell, they can assemble functional carriers, but less efficiently than the intact protein.

Figure 9-3 structures and transport reactions of two mfs carrier proteins from e. coli. A, LacY, a proton-lactose symporter. The red space-filling model is bound lactose. (PDB file: 1PV7.)B, GlpT, a glycerol-3-phosphate (G₃P)–phosphate antiporter. Bot-tom, Transport reactions carried out by postulated reorientation of transmembrane helices. Only 6 of the 12 transmembrane helices of each carrier are shown.

(References: Abramson J, Smirnova I, Kasho V, et al: Structure and mechanism of the lactose permease of E. coli. Science 301:610–615, 2003; Huang Y, Lemieux MJ, Song J, et al: Structure and mechanism of the glycerol-3-phosphate transporter from E. coli. Science 301:616–620, 2003.)

Both carriers bind substrates in the center of a cluster of transmembrane helices. LacY achieves specificity for lactose by providing geometrically favorable hydrogen bonds and hydrophobic interactions for the substrate. GlpT has a pair of conserved arginines near the middle of the bilayer that are required to bind phosphate.

MFS carriers are believed to alternate between two conformations: one with the substrate-binding site(s) open to the cytoplasmic side of the membrane, as in the crystals, and another with the substrate-binding site(s) exposed on the opposite side of the membrane. The architecture of both proteins is compatible with the proposed conformational change, but structures of the proteins in the alternate conformation must be understood before the mechanism can be established. When open to the periplasmic side of the membrane, GlpT binds glycerol-3-phosphate preferentially, since its affinity is higher than that of phosphate. Bound substrates may facilitate interconversion of the two conformations. When exposed to the cytoplasm, glycerol-3-phosphate dissociates and is replaced by phosphate, which is present at a higher concentration in the cytoplasm. Transport of lactose by LacY is coupled to transport of a proton. A glutamic acid is a likely candidate for proton binding, but it is not yet clear why the conformation of the protein that is open on the periplasmic side of the membrane favors binding of lactose plus a proton.

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