Information on anionic transporters

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CHAPTER 1:

General Introduction

General Introduction Background

Nitrate is the main nitrogen source for most bacteria, fungi, algae, yeast and plants. NrtA is the protein responsible for high affinity nitrate transport. The functional role of its amino acids is not well understood. An analysis of structure and function of NrtA is particularly important since it can provide information on anionic transporters.

Further analysis of this protein responsible for the transport of nitrate is appropriate due to the increased use of nitrate in agriculture as the main component of fertilizers and its subsequent drastic effects on the environment. Hence, the importance of extending our understanding of the structure and function of this protein.

Nitrate Contamination

Nitrate is the nitrogen source for most bacteria, fungi, algae, yeast and higher plants; however it can pass through soil and contaminate ground water. Pollutants such as nitrate are not always removed from the water by filtering of through the soil and the pores of rocks. Other sources are water from lakes, streams, ponds and sewage treatment systems. The impurities of water are normally filtered out through the soil and rocks, but not all soils and rocks are good filters. Soils all have different drainage characteristics, thus soils with a higher amount of sand and gravel are going to filter liquids down to the aquifer at a faster rate than soils comprised of tougher particles.

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In order to increase the supply of food we rely more than ever in the use of artificial fertilizers to boost the natural nitrogen found in soil. Agriculture is the largest contributor of nitrogen pollution to groundwater. Nitrogen is essential to the chemistry of life and sometimes its destruction. Excess nitrogen is washed off by rainfall or irrigation then leaches from soil into groundwater. Nitrates from livestock waste, septic systems, automobile exhaust and other fossil-fuel combustion add to this overload. Excess amounts of nitrate can pollute supplies of groundwater by travelling through the soil, carried by rain or irrigation water into groundwater supplies such as shallow wells, wells in sandy soil or wells that are improperly constructed or maintained.

Nitrate contamination can cause health problems for infants as it interferes with their blood's ability to transport oxygen. This causes methemoglobinemia, an oxygen deficiency known as the "blue baby syndrome" since the most common symptom of nitrate poisoning is bluish skin colouring, especially around the eyes and mouth (Morales et al. 1995). Nitrate contamination can also cause algae bloom, eutrophication of sea water thus turning the water darker affecting the tourism industry. Nitrate pollution of groundwater is a significant problem in Europe (Table 1.1).

The concentration of nitrate in drinking ground water is rising dramatically in many countries in the world (Bogardi and Kuzelka 1990) due to the lack of proper sewage treatment, wastewater from certain industries, atmosphere natural fixation, precipitation and mainly by the increasingly large amount of fertilizers used by farmers (Beevers 1976). Nitrogen sources such as nitrate in high concentrations have a significant ecological, agricultural and medical importance. One example of an ecological problem is eutrophication in coastal areas harming the fishing and tourism industry (Beevers 1976).

Nitrate uptake has an important role in nitrogen metabolism and has been widely studied at a physiological and molecular level (Orsel et al. 2002a). It is assumed that the uptake of nitrate is dependent upon the development of a carrier mechanism which facilitates nitrate entry into the tissue (Beevers 1976). The transport of nitrate into cells has been largely studies using different microorganisms models such as Neurospora crassa (Schloemer et al. 1974), Aspergillus nidulans formally known as Emericella nidulans (Unkles et al. 1991), the algae Chlamydomonas reinhardtii (Rexach et al. 2002) and the yeast Hansenula polymorpha (Navarro et al. 2003) as well as Arabidopsis thaliana a diploid higher plant (Filleur and Daniel-Vedele 1999).

Nitrate Assimilation

Nitrate assimilation is the two step reduction of nitrate to ammonium also one of the two major biological processes done by most bacteria, yeast, filamentous fungi and plants. The rate-controlling and regulated step in the process of nitrate assimilation seems to be the conversion of nitrate to nitrite, catalysed by the enzyme nitrate reductase (Figure 1.1). Nitrate arising from chemical fertilisers and industrial wastes is also a major factor contributing to nitrate contamination.

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The control of nitrate assimilation can be important from the standpoint of both agricultural productivity and water resource management. Thereby solving the ecological, economical and medical issues related to ground water contamination of nitrate. Although the nitrate assimilation pathway of A. nidulans is not commercially important it can however act as an applicable model for comparison with higher plants.

Nitrate Transport Systems

Previous work has shown that nitrate provides the main source of nitrogen for the growth and yield of plants (Zhou and Miller 2000) other studies indicate that nitrate entry into cells requires an active transport system (King et al. 1992). Nevertheless, the main aspects of function, structure and the regulation of nitrate transport is not clearly understood. There are two nitrate transport systems which are the High Affinity Transport System (HATS) and the Low Affinity Transport System (LATS) both constitutively and inducible expressed and subject to negative feedback regulation.

A constitutive system is expressed in the absence of nitrate and its activity is increased dramatically by nitrate treatment, whereas the nitrate inducible system is negatively feedback-regulated by products of nitrate uptake (Forde 2000). HATS is saturable system has been categorized into two genetically separate transport systems distinguished by whether they are substrate induced (iHATS) or constitutively active (cHATS). HATS operates at external nitrate concentration < than 1mmolL-1 nitrate (Williams and Miller 2001). LATS possibly has a signalling role it acknowledge the entrance of nitrate into the cell to induce the expression of the transporter and assimilatory genes, and presumably plays a physiological role in the nitrate uptake only above a certain threshold as LATS operates linearly at nitrate concentration > than 1mmolL-1 nitrate (Lea 2001) (Crawford and Glass 1998).

Nitrate transporters belong to two different families: NRT1 previously PTR/PTO (Peptide transporter and Proton-dependent oligopeptide transport, respectively) low affinity nitrate transporters and NRT2, a high affinity nitrate transporter, previously NNP (Nitrate-Nitrite Porter) (Crawford 1995). The NRT1 family constitutes of a peptide transporter superfamily, transporting nitrate or histidine with efficiency (Rexach et al. 1999). The NRT2 has family members in Gram-positive and Gram-negative bacteria and various eukaryotes such as yeast, fungi, algae and plants, these large proteins (395 to 547 residues long) catalyses nitrate uptake (nitrate permeases) or nitrite efflux (nitrite permeases) and the energy-coupling mechanism is not well understood (Pao et al. 1998).

The AtNRT1.1 (formerly called CHL1) gene was the first cloned nitrate transporter gene from a higher plant on the basis of its function. It was the first attempt to characterize proteins and genes involved in the nitrate transport. The Arabidopsis gene was isolated by T-DNA tagging of a chlorate resistant mutant. AtNRT1.1 mutants were first isolated in early 1970s for screening of chlorate resistant mutants. The product of the AtNRT1.1 gene is a typical hydrophobic membrane transport protein. Functional analysis of the properties of the expressed protein in Xenopus oocytes indicated that the AtNRT1.1 gene encoded a nitrate-inducible form of LATS that had not previously been identified. Following the identification of HATS in the fungus A. nidulans encoded by the nrtA gene, sequences of a barley root cDNA library were amplified. Two cDNA clones (BCH1-2) were isolated that encoded nitrate-inducible HATS transporter proteins of molecular mass of approximately 55 kDa, with 12 TMs (Lea 2001) (Unkles et al. 1991).

The NRT2 family constitutes of nitrate and nitrite transporters, is one of the 17 members of the Major Facilitator Superfamily (MFS) protein group, has prokaryotes and eukaryotes members, and has an important role on nitrate and nitrite influx (Forde 2000). The vast majority of the NRT2 genes possess a nitrate inducible expression (Rexach et al. 1999).

The first eukaryote member o the NRT2 family to be cloned was the nrtA (formely crnA) gene from the lower eukaryotic A. nidulans (Unkles et al. 1991). The identification of the two nitrate transport families NRT1 and NRT2 shed some light into the understanding of the nitrate uptake system. NRT1 and NRT2 are two gene families involved in high and low affinity nitrate transport systems in plants, however NRT1 is not present in fungi (Crawford 1995).

NRT1 and NRT2 have conserved genes amongst plants, fungi and algae (Yokoyama et al. 2001). NRT1 and NRT2 genes have been cloned from a wide range of plant species including barley (Hordeum vulgore) (Trueman et al. 1996) Soybean (Glycine max) (Amarasinghe et al. 1998) (Yokoyama et al. 2001), Nicotiana plumbaginifolia (Quesada et al. 1997) and A. thaliana (Filleur and Daniel-Vedele 1999). The plant and algal NRT2 gene products are approximately 30% identical to their fungal homologues and are predicted to possess a similar 12 transmembrane domain (TM) structure (Forde 2000) (Orsel et al. 2002a) (Tsay et al. 2007).

Nitrate and Nitrite Reductases

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Nitrate assimilation by bacteria, fungi, algae, some yeast and higher plants is a sequential reduction of nitrate to nitrite and nitrite to ammonium by the enzymatic action of nitrate reductase (NR) and nitrite reductase (NiR) (Figure 1.1). Nitrate reductase is able to convert nitrate to nitrite because of the presence of reduced pyrimidine nucleotide (Campbell 1999). Nitrite reductase converts nitrite into ammonium and NiR expression is synchronized with NR (Desikan et al. 2002). The regulation of fungal nitrate uptake is regulated by the induction of nitrate (Gojon et al. 1998) and the feedback repression due to glutamine (Unkles et al. 2004a and references therein).

Nitrate Reductase is fundamental for nitrate uptake activity in the cells of fungi but according to Unkles et al. (2004b) NR activity is not required by plants cells. They state fundamental differences between the yeast Pichia pastoris or A. nidulans, a lower eukaryotic fungus and A. thaliana a higher eukaryotic plant. Using mutant organisms devoid of NR activity they demonstrated that P. pastoris, a unicellular fungus and A. nidulans, a filamentous fungus failed to accumulate nitrate, whereas in A. thaliana, a higher plant there were significant uptake of nitrate.

In a similar study, Gojon et al. (1998) investigated the physiological consequences for NO3- utilization by NR under-expression and over-expression using transgenic Nicotiana tabacum and N. plumbaginifolia. They suggested that nitrate reductase has a fundamental role in nitrate uptake and metabolism as the level of NR expression markedly affected the NO3- reduction efficiency in detached leaves and intact plants.

Nitrate also acts as a signal control of most morphological e.g. in lateral root formation and metabolic processes (Unkles et al. 2004a) (Zhang and Forde 2000). Unkles et al. (2001) found that the active nitrate reductase is required for nitrate uptake by growing a mutant strain and comparing with a wild type using different nitrate concentrations. The characterization was done using the radiotracer 13N and the results indicated that the genes involved in nitrate uptake maybe induced by extracellular sensing of nitrate without necessarily entering the cell.

The use of A. nidulans as a model organism

Fungi plays a crucial role in the earth ecosystems, they are responsible for almost all degradation of plant material and recycling nitrogen also the remineralization of organic matter. A. nidulans is of considerable biological interest that's why is a leading experimental system used to help unravel many fundamental cellular processes.

A. nidulans is an ascomycete, homothallic being able to self-fertilize and form fruiting bodies in the absence of a sexual partner, also is a filamentous fungi that lives on decaying vegetation where the nature and amounts of nitrogen sources changes frequently (Figure 1.2). The Broad Institute have completed the genome sequence of A. nidulans in 2005 which represented a major breakthrough in the study of Aspergillus, providing the foundation for comparative and functional genomics studies.

A. nidulans it is an important research organism for studying eukaryotic cell biology for over 50 years (Osmani and Mirabito 2004) also it has been used on a wide range of subjects such as recombination, DNA repair, mutation, cell cycle control, pathogenesis, and metabolism (Martinelli and Kinghorn 1994) (Galagan et al. 2005). Sexual crossing is a massively important evolutionary mechanism and A. nidulans is one of the few species in its genus able to form sexual spores through meiosis, allowing crossing of strains in the laboratory. Given the tremendous diversity of breeding systems there has been much interest in the genetics of fungi as a research organism. Fungi include species with heterothallism where two defined sexes of opposite mating type occur, also groups such as yeasts where silent cassettes of genetic information allow interchange of sexual identity, pathogens in which same-sex mating can occur and lichen-forming fungi in which the sexual act may be prolonged for months or even years. Furthermore, many fungi are homothallic whilst retaining the ability to outcross (Dyer 2008).

Why use A. nidulans?

  • is easy to grow in the lab,
  • haploid so mutants can be readily isolated,
  • has a good genetic system,
  • can utilize many nitrogen sources
  • great potential in Biotechnology
  • well worked out genetic map of its genome
  • has a long history of use for the study of a wide range of subjects including classic genetics, cell biology and pathogenesis

Membrane Proteins

Membrane protein lies at the interface between the inside of the cell and its environment and are responsible for the influx or efflux transport of a variety array of molecules such as ions, sugars and drugs. They act as a highly selective permeability barrier. Membrane proteins are essential components of cellular organisms, allowing them to communicate with their surroundings by mediating and controlling the interactions bridging the outside and inside of cells and organelles.

A membrane consists of a bilayer of lipids, the majority being phospholipids. Its amphiphilic, the interior is hydrophobic with the fatty acids side chain of each lipid molecule located inwards and the polar phosphate side chains located at the surface of the membrane (Lee 2004). The core of integral membrane proteins is hydrophilic, allowing the passage of water-soluble molecules, and the surface is hydrophobic, allowing interaction with the interior (Lodish et al. 2000).

Integral membrane proteins come in two basic architectures: a-helix bundles and, less commonly ß-barrels. The lipid-facing surface of integral membrane proteins is composed of a central hydrophobic belt flanked by two aromatic girdles. In the helix-bundle proteins, non-translocated loops are enriched in positively charged residues Lys and Arg compared to translocated loops. Helix-bundle membrane proteins are built from transmembrane a-helices, interfacial helices lying flat on the membrane, loops and globular domains (Elofsson and Heijne 2007).

ß-barrel Integral Membrane Proteins

The integral membrane proteins with ß-barrel structures are known from outer membranes of bacteria, mitochondria, and chloroplasts. The ß-barrel is characterized by the number of antiparallel ß-barrel and by the shear number,which is a measure for the inclination angle of the ß-strands against the barrel axis (Figure 1.3).

Monomers such as OmpA, FhuA, OmpG (Conlan et al. 2000), dimers as OmPlA and trimers such as OmpF, PhoE are all known 3D ß-barrel membrane proteins that has a wide range of different functions. They can be non-specific diffusion pores (OmpA, OmpC, OmpF), specific pores (LamB, ScrY), active transporters (FhuA, FepA, BtuB), enzymes such as proteases (OmpT), lipases (OmPlA), acyltransferases (PagP), or, like TolC are involved in solute efflux (Koronakis et al. 2000).

This is an illustration of the outer membrane ion channel OmpG of Escherichia coli. OmpG porin is unique, as it appears to function as a monomer. The structure shows a 14-stranded ß-barrel with a relatively simple architecture (Liang and Tamm 2007).

Examples of ß-barrel proteins are OmpA, a small ion channel (Arora et al. 2000); OmpT, a protease; NalP, an auto transporter; FadL, a long chain fatty acid transporter; PhoE, a diffusion pore; ScrY, a sucrose specific porin; OmPlA, a phospholipase. FhuA and BtuB are active transporters for ferrichrom iron and vitamin B12 uptake, respectively.

The outer membrane proteins (OMP) of mitochondria are predicted to form similar TM ß-barrels. Examples are the VDAC channels, out of which more than a dozen have been sequenced (Kleinschmidt 2006). Also soluble bacterial toxins that can insert into membranes, such as a-hemolysine from Staphylococcus aureus (Song et al. 1996), perfringolysine O from Clostridium perfringens (Heuck et al. 2000) also form ß-barrels, but these are oligomeric (Shepard et al. 1998).

OMP family of bacteria are composed of ß-barrels with even numbers of ß-strands. OMP is a family of highly conserved bacterial proteins that promote bacterial adhesion to and entry into mammalian cells. These homologous outer membrane proteases, known as omptins are implicated in the virulence of several pathogenic Gram-negative bacteria (Schulz 2000) (Marsh and Pali 2006).

All examples below are membrane proteins of known 3D structures obtained by diffraction or Nuclear Magnetic Resonance (NMR). OmpA is a 19 kDa (177 residues) protein A of E. coli an intensely studied example in the field of membrane protein folding. Data from NMR dynamic experiments revealed a gradient of conformational flexibility in the structure that may contribute to the membrane channel function of this protein (Arora et al. 2001). It consists of large water-filled cavities with no pore and the structure supports the concept that all outer membrane proteins consist of ß-strands (Pautsch and Schulz 2000).

Vogt and Schulz (1999) improved the crystal quality of OmpX by conciliating mutational studies with x-ray crystallography. They presented a structure of OmpX containing the mutation H100N and the results revealed that the core of the barrel consists of an extended hydrogen-bonding network of highly conserved residues.

Vandeputte-Rutten et al. (2001) identified a putative binding site for lipopolysaccharide, a molecule that is essential for OmpT activity. Based on active site residues they proposed a proteolytic mechanism, involving an H-N dyad and an N-N couple that activate a putative nucleophilic water molecule.

Snijder et al. (1999) reported X-ray structures of monomeric and dimeric OmPLA from E.coli which provided detailed information of activation by dimerization of a membrane protein. Dimerization results in functional oxyanion holes and substrate-binding pockets.

Porins provide diffusion channels for salts and small organic molecules in the outer membrane of bacteria. Porins form aqueous channels that aid the diffusion of small hydrophilic molecules across the outer membrane of Gram-negative bacteria (Schirmer 1998).

Zeth et al. (2000) showed the strong selectivity of Omp32 an anion-selective porin from Comamonas acidovorans. The Omp32 structure selectivity is conferred by a positive potential created by R which creates a charge filter in the constriction zone and a positive surface potential at the external and periplasmic sides. OmpF creates an electrostatic field across the channel originating from a surplus of negative charges creating cation selectivity (Cowan et al. 1995).

The crystal structures of the matrix porin and phosphoporin explained at the molecular level shed light to the functional characteristics of PhoE aided by alterations of known mutations. Where, the charge of residues side chain distribution affects ion selectivity (Cowan et al. 1992).

Trimeric maltoporin (LamB protein) facilitates the diffusion of maltodextrins across the outer membrane of Gram-negative bacteria. Six conserved aromatic residues line the channel and form a path from the vestibule to the periplasmic outlet (Schirmer et al. 1995). The X-ray structure of a sucrose-specific porin ScrY from Salmonella typhimurium has been determined and the higher permeability for sucrose of ScrY as compared to maltoporin, LamB is mainly accounted for by differences in their pore-lining residues (Forst et al. 1998).

Integral outer membrane receptors for iron chelates and vitamin B12 carry out specific ligand transport against a concentration gradient. The crystal structure of FepA and its active transport and the blockage of the pore suggest that the N-terminal domain must undergo a conformational rearrangement to allow ligand transport into the periplasm (Buchanan et al. 1999).

Chimento et al. (2003) have solved crystal structures of BtuB, the outer membrane cobalamin transporter from E. coli, in the absence and presence of vitamin B12. Calcium has been implicated as a necessary factor for the high-affinity binding of vitamin B12 to BtuB. The absence of a channel and the peripheral binding of R135 imply that BtuB serves to bind the colicin, and that the coiled-coil delivers the colicin to a neighbouring outer membrane protein for translocation, thus forming a colicin translocon. The translocator was concluded to be OmpF from the occlusion of OmpF channels by colicin E3 (Kurisu et al. 2003).

FhuA is the receptor for ferrichrome-iron in E. coli, mediate the active transport of ferric siderophores across the outer membrane of Gram-negative bacteria. In contrast to the typical trimeric arrangement found in porins, FhuA is monomeric. Upon binding of ferrichrome-iron, conformational changes are transduced to the periplasmic pocket of FhuA, signaling the ligand-loaded status of the receptor. Sequence homologies and mutagenesis data are used to propose a structural mechanism for transport across the outer membrane (Ferguson et al. 1998).

The neisserial surface protein A (NspA) from Neisseria meningitidis is a promising vaccine candidate because it is highly conserved among meningococcal strains and induces bactericidal antibodies. The four loops at the extracellular side of the NspA molecule contains mainly hydrophobic residues suggesting that the protein might function in the binding of hydrophobic ligands, such as lipids (Vandeputte-Rutten et al. 2003).

The bacterial outer membrane enzyme PagP transfers a palmitate chain from a phospholipid to lipid A. Three residues located at extracellular loops near the membrane interface critical for enzymatic activity have been studied. Findings indicated that an emerging paradigm for outer membrane enzymes is related in providing an adaptive response toward disturbances in the outer membrane (Hwang et al. 2002).

Acyl group specificity was modulated by a mutation of G88 lining the bottom of the hydrophobic pocket, thus confirming the hydrocarbon ruler mechanism for palmitate recognition. A striking structural similarity between PagP and the lipocalins imply an evolutionary link between these proteins (Ahn et al. 2004).

α-helix Integral Membrane Proteins

The a-helix integral membrane protein is the major category of transmembrane proteins (Figure 1.4). They are present in the inner membranes of bacterial cells or the plasma membrane of eukaryotes and sometimes in the outer membranes. The main a-helical channels are voltage-gated ion channels (VIC), CorA, aquaporins and chloride ion channels (CIC).

The voltage-gated ion channels (VIC) which, are class a of transmembrane ion channels that are activated by changes in electrical potential difference near the channel. They are found in a wide range of bacteria, archaea, eukaryotes and viruses; composed of several subunits with a central pore through which ions can travel down their electrochemical gradients. The channels tend to be ion-specific. Potassium channels are the most widely distributed type of ion channel and are found in virtually all living organisms. They form potassium-selective pores that span cell membranes. Furthermore, potassium channels are found in most cell types and control a wide variety of cell functions (Gulbis et al. 1999).

CorA is a large and diverse family with sequenced members in Gram-positive and Gram-negative bacteria, blue-green bacteria, archaea, plants, animals, yeast, slime molds, Guillardia theta and Plasmodium. The molecular mechanisms of Mg2+ uptake from the environment has been elucidated by a variety of studies. In bacteria Mg2+ is supplied by the CorA protein and, where the CorA protein is absent, by the MgtE protein. In yeast the initial uptake is via the Alr1p and Alr2p proteins, but at this stage the only internal Mg2+ distributing protein identified is Mrs2p. Many transporters have been identified within the protozoa XntAp, in metazoa, Mrs2p and MgtE homologues, in plants, a family of Mrs2p homologues has been identified along with another novel protein, AtMHX (Lunin et al. 2006) (Payandeh et al. 2008).

Aquaporins are proteins embedded in the cell membrane that regulate the flow of water. Aquaporins are integral membrane proteins from a larger family of major intrinsic proteins (MIP) that form pores in the membrane of biological cells. There have been many studies involving this class of membrane proteins. The influx of arsenite and antimonite via the Fps1 protein into yeast cells is well documented (Wysocki et al. 2001). Similarly, these compounds are taken up via aquaporins in Leishmania (Gourbal et al. 2004). Moreover, AQP6 of renal epithelia have been reported to transport anions at low pH (Yasui et al. 1999). Demonstration of the involvement of the cyanobacterial channel protein in copper homeostasis suggests that it may transport Cu2+. Finally, Yang et al. (2005) showed that arsenite exits the Mesorhizobium meliloti cell by downhill movement through AqpS.

Chloride channels are a superfamily of ion channels consisting of approximately 13 members. Chloride channels display a variety of important physiological and cellular roles that include regulation of pH, volume homeostasis, organic solute transport, cell migration, proliferation and differentiation. Based on sequence homology the chloride channels can be subdivided into a number of groups. This family of ion channels contains 10 or 12 TMs. Each protein forms a single pore. It has been shown that some members of this family form homodimers. In terms of primary structure, they are unrelated to known cation channels or other types of anion channels. The ClC family is a large family consisting of hundreds of sequenced proteins derived from Gram-negative and Gram-positive bacteria, archaea, and all kinds of eukaryotes. These proteins are essentially ubiquitous, although they are not encoded within the genomes of several prokaryotes with small genomes (Matulef and Maduke 2007).

Crystallographic structure of the aquaporin 1 (AQP1) channel (Sui et al. 2001).

All examples below are membrane proteins of known 3D structures obtained by diffraction or NMR. Pore-forming toxins (PFT) are a class of potent virulence factors that convert from a soluble form to a membrane-integrated pore. They exhibit their toxic effect either by destruction of the membrane permeability barrier or by delivery of toxic components through the pores.

Among the PFT group, the bacterial PFT are some of the most dangerous toxins, such as diphtheria and anthrax. Examples of eukaryotic PFT are perforin and the membrane-attack complex, proteins of the immune system. It has two types of membrane integration, either by a-helical or ß-sheet elements. The crystal structure shows the large extent of interdependent conformational changes indicating a sequential mechanism for membrane insertion and pore formation (Mueller et al. 2009).

Extracellular polysaccharides (EPS) are secreted polymers produced by bacteria. Some polymers have limited association with the cell surface, whereas others are attached to the cell surface forming a structural layer. The crystal structure of Wza, an integral outer membrane lipoprotein is a novel a -helical barrel was presented by Dong et al. (2006). This work provided insight into the export of other large polar molecules such as DNA and proteins.

The cell wall of Corynebacterium glutamicum contains a mycolic acid layer, which is a protective non-polar barrier similar to the outer membrane of Gram-negative bacteria. The exchange of material across this barrier requires porins such as Porin B (PorB). The 16 crystal structures vary greatly with respect to the 29 residues in the N- and C-terminal extensions. A a-helical porin in a bacterial outer envelope is unusual due to the fact that all presently known structures of such porins consist of ß-barrels (Ziegler et al. 2008).

Electrochemical Potential-Driven Transporters

There are two families of membrane proteins found unibiquitously in all living organisms which are ABC an ATP-Binding Cassette and MFS the Major Facilitator Superfamily. ABC is multicompound family with primary active transporters capable of transporting molecules by ATP hydrolysis. MFS transporters also called the uniporter-symporter-antiporter family are single polypeptide secondary carriers which transport small solutes by chemiosmotic ion gradients (Pao et al. 1998). Examples of porters such asuniporters-symporters-antiporters are mitochondrial carrier (MC) proteins, AcrB a multidrug efflux transporter, Sodium/proton antiporter 1 NhaA, proton glutamate symporter, Neurotransmitter:Sodium Symporter (NSS) and MFS, Glycerol-3-phosphate transporter, GlpT; Lactose permease, LacY and the multidrug transporter EmrD a hydrophobic uncoupler H+ antiporter.

Mitochondrial carrier proteins, permeases of the MC family (the human SLC25 family) possess six transmembrane a-helical spanners (Bamber et al. 2007). Functional and structural roles for residues in the transmembrane domains of MC have been proposed by Cappello et al. (2007). Resistance-nodulation-cell division (multidrug efflux transporter AcrB), Dicarboxylate/amino acid:cation symporter (proton glutamate symporter). The members of the DAACS family catalyze Na+ and/or H+ symport. The bacterial members are of about 450 amino acyl residues while the mammalian proteins are of about 550 residues in length. These proteins possess between 10-12 transmembrane hydrophobic domains per polypeptide chain (Gendreau et al. 2004).

The CPA1 family is a large family of proteins derived from Gram-positive and Gram-negative bacteria, blue-green bacteria, archaea, yeast, plants and animals. Transporters from eukaryotes have been functionally characterized, and all of these catalyze Na+:H+ exchange such as the monovalent cation/proton antiporter; sodium/proton antiporter 1 NhaA (Waditee et al. 2001).

The Neurotransmitter Sodium Symporter (NSS) family catalyzes the uptake of a variety of neurotransmitters, amino acids, osmolytes and related nitrogenous substances by a solute, Na+ symport mechanism. Sometimes Cl- is co-transported, and some exhibit a K+ dependency. The human dopamine transporter probably co-transports the positively charged or zwitterionic dopamine species with 2 Na+ and 1 Cl- (Quick et al. 2006). Other examples are the MFS multidrug transporter EmrD, a hydrophobic uncoupler H+ antiporter, the glycerol-3-phosphate transporter, GlpT and lactose permease, LacY (discussed on sector 1.8 Major Facilitator Superfamily).

Why Study Membrane Proteins?

Membrane protein plays crucial roles in many cellular processes; in particular they play key roles in signalling, transport across cell membranes and energy transduction. They are essential mediators in a diverse array of cellular processes. They are a used as a potential novel drug targets due to their fundamental role in diseases. Given their crucial physiological, highly specialized functions and biomedical perspective importance it is imperative that this class of proteins becomes better understood.

Any insights gained into the diversity of membrane protein world should be an important focus on future research. It has becoming increasingly clear that despite the importance there has been little progress in determining the structure and function mechanisms of membrane proteins. The rapid increase in high-resolution structural data for membrane proteins means that in the future both benchmarking and development of novel prediction methods will be based on structural data reinforced by biochemical analysis. These are exciting times in membrane protein research. Much of the development is driven by the increasing flow of new structures, inspiring new hypotheses concerning protein structure and function thus providing a rich background for studies of protein and its amino acids interactions (Elofsson and Heijne 2007).

Considerable research is ongoing in the area of membrane protein structure and function. Site directed mutagenesis is a powerful experimental tool by introducing specific mutations into a gene then expressed the altered protein to study the relationship between amino acid sequence and protein structure and function. Von Heijne (2006) membrane topology review is of particular importance, since it illustrates the new era defined by in numerous studies and recent advances in high resolution membrane protein structures.

The number of high resolution structures of integral membrane proteins is growing exponentially which is very encouraging but it remains an understudied territory. There have been several studies in membrane protein structure determination Green et al. (2000) mutagenesis study revealed that a particular TM played a crucial role on the conformation of LacY (Lagerstedt et al. 2004) (Hirai et al. 2004) (Yang et al. 2005) to (Alisio and Muecklen 2004) which a 3D model was developed that is consistent with the results of numerous mutagenesis studies.

A theoretical 3D model was generated by point mutations in a X. oocyte expression system. Effects of these residues on p-aminohippurate (PAH) and cidofovir transport were assessed using Organic anion transporters (OATs). In this investigation they found out that five aromatic amino acids were required for transport of hydrophilic substrates. In addition, they found out that only a few aromatic residues conserved between rOAT3 and hOAT1 surround the hOAT1 binding site. They suggested a possible structural difference in the binding site of OAT1 and OAT3 that may impact substrate specificity (Perry et al. 2006). Hong et al. (2007) showed that two aromatic residues were critically involved in the stability of the transporter thus playing critical roles in the substrate binding of hOAT1, and two other aromatic residues were essential for maintaining the stability of the transporter.

Sheldon et al. (2003) showed evidence that supports the idea that the residue K355 in TM11 of OxlT facilitates binding of the anionic substrate, oxalate. Zhou et al. (2004) kinetic analysis of estrone sulfate transport in the human organic anion transporter hOAT4 expressing parental CHO (Chinese hamster ovary cells) and mutant CHO cells defective in the different steps of glycosylation processing indicated that these mutant cells had significantly lower binding affinity for its substrates compared with that expressed in parental CHO cells. The low binding affinity of hOAT4 in CHO-Lec1 cells contributed to the low transport activity observed in these cells.

There have been various studies on the structure and function of membrane protein, yet it remains an under-explored territory. This continuing progress however, would be better achieved with technical advances in crystallization and expression. One of the major problems encountered in membrane protein research is attributed mainly to the difficulty in solubilising these hydrophobic proteins. This class of proteins still is under-represented and future studies are essential in the understanding of functional and structural characteristics of membrane proteins (Tan et al. 2008).

Currently, the use of protein alignments as tools is complemented by a growing resource of genetic and genomic resources, available for a wide range of species as reviewed by Volff (2005), ranging from expressed sequence tags to genetic maps and whole genome sequencing projects. Based on the crystal structures, several residues of LacY and GlpT important for substrate binding have been identified in a variety of studies. Protonation and binding of substrates induce conformational changes, thereby allowing the transport in and out of the membrane (Bruser and Sanders 2003).

The overall importance of membrane proteins is shown by the fact that as much as 30% of the genome of most living organisms encodes such proteins. Additionally, membrane proteins are crucial players in the cell and take centre stage in processes ranging from basic small-molecule transport to sophisticated signalling pathways. They have been used for drug targets and it has been estimated that more than half of all drugs currently on the market are directed against membrane proteins (Elofsson and Heijne 2007).

3D structures of approximately 160 different integral membrane proteins are currently determined at atomic resolution by X-ray crystallography or NMR spectroscopy, despite difficulties with extraction and crystallization. In addition, structures of many water-soluble domains of integral membrane proteins are available in the Protein Data Bank. Their membrane-anchoring a-helices have been removed to facilitate the extraction and crystallization.

The structure alone, however, does not tell the whole story of how a protein works. Within the contact area, a subset of residues may contribute to the majority of binding energy, through hydrogen bonds, salt bridges, dipole-dipole interactions and hydrophobic interactions (Morrison and Weiss 2001). Biochemical methods such as mutagenesis and kinetic energy of proteins have been particularly useful for the identification of important residues in proteins.

Major Facilitator Superfamily

Membrane protein transport is responsible for the crucial maintenance of a selective cellular environment. The largest subset of secondary transporter protein family is the MFS (TC 2.A.1.). Compounds such as ions, nucleosides, sugars, sugar-phosphates, drugs, amino acids, esters, metabolites and hydrophilic solutes are transported by MFS permeases by electrochemical gradients into substrate gradient. This family is found ubiquitously in bacteria, archaea and eukarya.

A typical MFS protein is 400 to 600 amino acids in length and has 12-14 TMs divided into two halves of a 6 a-helix, each connected by long central loop (Pao et al. 1998) (Figure 1.5). Among the different families of transporter only two occur ubiquitously in all classifications of organisms which are ABC and MFS. The MFS transporters are single-polypeptide secondary carriers capable only of transporting small solutes in response to chemiosmotic ion gradients (Walmsley et al. 1998) with 58 distinct members and more than 15,000 sequences identified to date (Law et al. 2008).

Secondary structure of a typical MFS protein with its 12 TMs, cytoplasmic N- and C-termini and a long cytoplasmic loop between TM6 and TM7 (Pelis et al. 2006).

The MFS family contains members of direct medical and pharmaceutical significance that function as uniporters, symporters or antiporters. In addition their solute specificity is also diverse. In order to transport substrates across the membrane there are conformational changes postulated in switching one opening of the hydrophilic cavity from the periplasm to the cytoplasm, thereby allowing the transport of substrate across the membrane. This is an alternating access mechanism that involves a switch type movement of the two halves of the protein. This substrate-translocation transport mechanism is called alternating-access which operates via a single binding site; one substrate binding pocket has access from one side of the membrane at a time (Bruser and Sanders 2003).

Membrane Protein Classification Code

All proteins have been classified according to the Transporter Classification (TC) System which has been approved by the International Union of Biochemistry and Molecular Biology (IUBMB). Each functionally dissimilar protein is classified by the letters TC followed by a five digit code. The first digit is a number which refers to the class of transport, the second digit is a letter which refers to the subclass and in the case of primary active transporters refers to the energy source used to drive the transport, the third digit which is a number refers to the family or superfamily, the fourth also a number refers to a phylogenetic cluster within the family and the fifth digit is a number which refers to the substrate specificities. Further information on genome analysis, multiple alignments, phylogenetic trees and other analysis can be found at URL:[http://www.tcdb.org/] (Saier et al. 2006).

Main MFS Family Classes

Sugar Porter (SP)

The sugar porter (TC 2.A.1.1) is the largest MFS member; it contains 133proteins with 12 TMs. Sugar porter proteins vary in relation to sequence and function. These proteins are derived from bacteria, archaea, eukaryotic protists, fungi, yeasts, animals, and plants. They function by uniport, solute:solute antiport, and/or solute:cation symport, depending on the organisms and conditions. Uniporters exhibit no polarity but can usually catalyze both uniport and antiport depending on whether a substrate is present on the trans side of the membrane. Symporters function with inwardly-direct polarity in the presence of a membrane potential but many of these proteins have also been shown to catalyze antiport when a substrate is present on the trans side of the membrane.

Substrates catalysed are specifically sugars such as galactose, arabinose, xylose, and glucose in bacteria; galactose, quinate, myoinositol, lactose, maltose, and alpha -glucosides in yeasts and fungi; hexoses in trypanosomes and plants; and sugars as well as organic cations and neurotransmitters in animals (Goswitz and Brooker 1995) (Henderson 1991) (Olson and Pessin 1996).

Previous work by Will and Tanner (1996) showed evidence of an improved transport mechanism. They have constructed various chimeras and by heterologous expression in Schizosaccharomyces pombe, they replaced the first part of the external loop 1 of the HUPI symporter by the corresponding portion of HUP2 of Chlorella, they obtained decreased Km values for D-galactose which yielded a chimera better apt to transport the substrate.

The bacterial sugar porters are usually smaller than the eukaryotic ones as the larger sizes of the eukaryotic proteins are due to large hydrophilic N and/or C termini or specific inter-TMs loops. The hydrophilic regions of the eukaryotic proteins may play roles in regulation or in cytoskeletal attachment, and they are frequently subject to phosphorylation by ATP-dependent protein kinases. A representative well-characterized example of this family is the arabinose:H+ symport permease AraE of E. coli (Naftalin et al. 2007).

Drug Efflux: H+ Antiporter DHA1

All MFS proteins that catalyze drug efflux are from three subfamilies: the drug:H+ anti-porter families DHA1, DHA2 and DHA3 (Pao et al. 1998) (Sa-Correia et al. 2008) (Kumar and Schweizer 2005). The DHA1 drug efflux family (TC 2.A.1.2) consists of 12 proteins with 12 TMs and is found in prokaryotes and eukaryotes. All functionally catalyze drug efflux in which are multiple drug resistance (MDR) pumps such as anti-arrhythmic and anti-malarial drug quinidine: QDR1-3, AQR1, DTR1, TPO1 and TPO4.

The involvement of QDR1, QDR2 and QDR3 in the efflux of quinidine out of the yeast cell has been shown in drug-efflux assays. AQR1 catalyse amino acid excretion, DTR1 is a pro-spore membrane bisformyldityrosine transporter, TPO1 catalyses the uptake of polyamines at alkaline pH and excretion at acid pH and TPO4 catalyses quinidine and cycloheximide. These transporters might have specific physiological substrates, whereas drugs would be transported opportunistically.

Members of the DHA1 family export sugars, polyamines, uncouplers, monoamines, acetylcholine, paraquat and methylglyoxal. Other proteins which are able to catalyze drug efflux are the putative drug-specific pumps from Gram-positive bacteria and hypothetical or uncharacterized proteins from Gram-negative bacteria, yeasts, and fungi (Paulsen et al. 1996).

Drug Eflux: H+ Antiporter DHA2

The DHA2 drug efflux family (TC 2.A.1.3) (formerly known as the DHA14 family) consists of 35members with 14 TMs in which have been shown to be MDR pumps, drug-specific efflux pumps, hypothetical or uncharacterized proteins (Gbelska et al. 2006). Like the DHA1 family, functionally characterized members of the DHA2 family exhibit specificities only for drugs, although the range of drugs transported is highly significant.

Members belonging to the DHA2 family exhibit more restricted substrate specificity as substrates transported include bile salts and dyes. DHA2 family members are found in a wider range of organisms in comparison with the DHA1 family. DHA2 MDR pumps are found in animals as well as in yeasts and a variety of Gram-negative and Gram-positive bacteria. Uncharacterized members of this family include a wider range of organisms such as humans and archaea (Paulsen et al. 1996).

DHA2 family members include multidrug exporters, such as the staphylococcal multidrug exporter QacA, and substrate-specific exporters, such as the Gram-positive tetracycline transporters TetA. QacA mediates resistance to a wide array of monovalent or divalent cationic, lipophilic, antimicrobial compounds (Brown and Skurray 2001). Tetracycline efflux proteins belong to the major facilitator superfamily. Efflux proteins are membrane-associated proteins that recognise and export tetracycline from the cell. They are found in both Gram-positive and Gram-negative bacteria. There are at least 22 different tetracycline efflux proteins, grouped according to sequence similarity. Since the DHA2 family drug export proteins are important mediators of drug resistance in various pathogenic bacteria, there is a current need for the development of new anti-microbial compounds.

Drug Eflux: H+ Antiporter DHA3

The DHA3 drug efflux family (TC 2.A.1.21) consists of 8proteins with 12 TMs. Members of the DHA3 family are only found in prokaryotes, and are known to efflux antibiotics, including macrolides and tetracycline. Tetracycline efflux pumps are found in both Gram-negative and Gram-positive bacteria. Most of them confer resistance to tetracycline, but not to minocycline or glycylcyclines. However, some Gram-negative Tet proteins confer resistance to both tetracycline and minocycline, but not to glycylcyclines (Tamura et al. 2001) (Yin et al. 2006). Member of this family also includes the macrolide (erythromycin; oleando-mycin; azithromycin) efflux, MefA of Streptococcus pyogenes (Kumar and Schweize 2005).

Organophosphate: Inorganic Phosphate Antiporter (OPA)

The OPA family (TC 2.A.1.4) is composed of 7 proteins with 12 TMs which are derived from prokaryotes and eukaryotes. It is responsible for the transport of sugar phosphates, glycerol phosphate, phosphoglycerates and phosphoenolpyruvate. Members of the OPA family include the UhpC protein which regulates the transport of hexose phosphate synthesis being a rare example of an MFS member which does not serve a primary transport function. It belongs to the phosphorelay system UhpB-UhpC-UhpA (Wright and Kadner 2001).

The predominant mechanism of transport catalyzed by permeases of the OPA family under normal physiological conditions appears to be antiport of an organophosphate ester for inorganic phosphate (Maloney 1992). Other members are also capable of catalyzing substrate: H+ symport. The best-characterized members of the family are UhpT and GlpT, both of E.coli, for which detailed topological and 3D models have been presented (See sector 1.8.2 for GlpT).

The OPA family includes several proteins from the worm, Caenorhabditis elegans and the fruit fly, Drosophila melanogaster. Other members of this family are PgtP of S. typhimurium a P-glycerate:Pi antiporter, G3PP and GSD1b of Homo sapiens a microsomal glucose-6-P:Pi antiporter (Chen et al. 2008) and Hpt of Chlamydia pneumoniae (Schwoppe et al. 2002).

Oligosaccharide: H+ Symporter (OHS)

The current OHS family (TC 2.A.1.5) consists of 7 proteins with 12 TMs, three of which are ß-galactoside permeases from closely related Gram-negative bacteria. LacY of E.coli is the best characterized member of this family and also the most extensively studied permease in the MFS (Abramson et al. 2003) (See sector 1.8.2).

The other members of the OHS family are closely related to the sugar transporter family by being specific for the transport of trisaccharide and raffinose, RafB from E. coli (Van Camp et al. 2007); a a,ß non-reducing glucoside-fructoside, sucrose, CscB permease also from E. coli (Peng et al. 2009); a-galactoside melibiose, MelY from Enterobacter cloacae (Shinnick et al. 2003). All transported with the concomitant uptake of hydrogen ions (proton symporters).

Metabolite: H+ Symporter (MHS)

The MHS family (TC 2.A.1.6) includes 16proteins with varying specificities and 12 TMs. Those of known transport function recognize citrate, CitA of Klebsiella pneumoniae (Lewis et al. 2004); taurine, ectoine, pipecolate, proline-betaine, N,N-dimethylglycine, carnitine, and 1-carboxymethyl-pyridinium, a -ketoglutarate, KgtP of E. coli; (Seol and Shatkin 1992); ProP of E. coli subject to osmotic activation (MacMillan et al. 1999); dicarboxylates, PcaT of Pseudomonas putida (Culham et al. 1993); 4-Methyl-o-phthalate MopB of Burkholderia cepacia (Saint and Romas 1996); shikimate, ShiA of E. coli(Whipp et al. 1998); The citrate/tricarballylate: H+ symporter TcuC of Salmonella enterica serovar (Lewis et al. 2004); acetate/monochloroacetate permease, Deh4p of B. cepacia (Yu et al. 2007).

The a -ketoglutarate: H+ symport permease of E.coli (KgtP) is probably the best-characterized member of the MHS family. An experimentally documented 12 TMs topological model has been proposed for this permease (Saier and Paulsen 2001). Metabolites transported by members of the MHS family have little in common, except that they all possess at least one carboxyl group. Several protein members of the MHS family are specific for Krebs cycle intermediates. All are from bacteria, characterized members of the MHS family and function by proton symport.

Fucose: H+ Symporter (FHS)

The FHS (TC 2.A.1.7) is a small family with 7 members containing 12 TMs responsible for the transport of sugars by proton transport. Although the proteins of the small FGHS family also catalyze sugars by H+ symport, they are not related to the proteins of the sugar porter family. FGHS family members are all derived from bacteria and despite being small they all exhibit a surprising degree of sequence diversion.

Members of this family include fucose permease of E.coli, FucP (Gunn et al. 1995); a galactose/glucose permease of Brucella abortus, Ggp (Essenberg et al. 1997) both which transport sugars of the galacto configuration (Paulsen et al. 1998); a glucose/mannose permease of Bacilus subtilis GlcP, like the E.coli FucP protein, which is a sugar:proton symporter. NaGLT1 of Rattus norvegicus, a Na+-dependent glucose (methyl a-glucoside) transporter (Horiba et al. 2003); 2-Deoxy-D-ribose porter, DeoP from S. typhimurium (Christensen et al. 2003); the putative sucrose permease, ScrT of Shewanella frigidimarina (Reid and Abratt 2005) and the Na+ dependent sugar transporter, HP1174 of Helicobacter pylori which transports glucose, galactose, mannose and 2-deoxyglucose (Psakis et al. 2009).

NRT2 family (formally NNP Nitrate-Nitrite Porter)

The NRT2 family (TC2.A.1.8) contains 13 proteins with 12 TMs which are derived from a variety of Gram-negative and Gram-positive bacteria as well as various eukaryotes including yeasts (YNT1), fungi (NrtA), algae (Nar3), and higher plants (NRT2.1-2.7). Irrespective of the organism, the nitrate permeases of the NRT2 family take up their substrate while the nitrite permeases apparently extrude theirs both transport their substrate by a symport proton (H+) mechanism that is driven by the pH gradients across membranes.

Well-characterized members of the family are the NarK nitrite extrusion system involved in anaerobic nitrate-dependent respiration in E.coli (Rowe et al. 1994) (Goddard et al. 2008); the NrtA nitrate uptake permease of A. nidulans (Unkles et al. 1991), NasA of B. subtilis (Ogawa et al. 1995); NrtP of Synechococcus elongates (Aichi et al. 2006); NarU of E. coli (Clegg et al. 2006) and NRT2.1-2.7 (Wirth et al. 2007).

Phosphate: H+ Symporter (PHS)

The PHS family (TC 2.A.1.9) it constitutes of 11 proteins with 12 TMs which share similarity in function, sequence and size, larger than most bacterial MFS proteins also is an unusual family as includes members from yeasts, fungi and plants but none from bacteria and other eukaryotes. Two widely studied proteins from this old family are the Pho84 inorganic phosphate transporter of S. cerevisiae (Bun-Ya et al. 1991) (Jensen et al. 2003); the GvPT phosphate transporter of Glomus versiforme (Harrison and Buuren 1995) and PHT1-6 phosphate transporter of Oryza sativa (Liu et al. 2008) (Ai et al. 2009).

Nucleoside: H+ Symporter (NHS)

The NHS family (TC 2.A.1.10) has 2 main proteins with 12 TMs from E. coli which exhibits a 50% identical identity; a nucleoside:proton symporter, NupG, a xanthosine permease. NupG has been examined structurally and is the better characterized of the two proteins (Patching et al. 2005). The similar identity high percentage arose by a gene duplication event that occurred relatively recently in evolutionary time despite sharing similar identity these two proteins differ dramatically in specificity. The other protein is XapB of E. coli, a xanthosine porter (Norholm and Dandanell 2001).

Formate Antiporter (OFA)

OFA family (TC 2.A.1.11) is composed of 5 proteins with 12 TMs, a small but diverse family since is present in the bacterial, archaeal, and eukaryotic kingdoms. A widely studied example is the oxalate:formate antiporter from Oxalobacter formigenes, which provided the basis for naming the OFA family (Abe et al. 1996) (Ye et al. 2001), OxlT exchanges formate for oxalate across the cytoplasmic membrane of the organism O. formigenes. The OFA protein, OxlT has been purified, reconstituted in an artificial membrane system and studied structurally (Heymann et al. 2001) (Hirai et al. 2002).

Sialate: H+ Symporter (SHS)

The SHS family (TC 2.A.1.12) possesses 2 proteins with 14 TMs which are the sialic acid porter NanT of E. coli (Verheijen et al. 1999) and the lactate/pyruvate: H+ symporter Jen1 (YKL217w) of S. cerevisiae (Akita et al. 2000) a lactate transporter, required for uptake of lactate and pyruvate.

Monocarboxylate porter (MCP)

The MCP family (TC 2.A.1.13) consists of 13 proteins with 12 TMs, derived exclusively from yeasts and animals and all are from eukaryotes. Most of these proteins are derived from various animal sources including 3 from C. elegans. However, S. cerevisiae possesses four paralogs. These permeases appear to be energized by proton symport. Monocarboxylates transported by these permeases include lactate, pyruvate, and mevalonate with inwardly-directed polarity (Pao et al. 1998).

Mct1 of H. sapiens (Becker et al. 2005) transports methionine hydroxy analogue 2-hydroxy (4-methylthio) butanate (Martin-Venegas et al. 2007) (Becker and Deitmer 2008); Tat1 of R. norvegicus, is a low affinity aromatic amino acid transporter which, also transports N-methyl amino acids (Meredith and Christian 2008); MCT8 of Mus musculus, the thyroid hormone transporter, which transports L- and D-isomers of thyroxine (T4), 3,3',5-triiodothyronine (T3) and 3,3'-diiodothyronine (Friesema et al. 2003) (Jansen et al. 2008); Mch5 of S. cerevisiae, a high affinity riboflavin-regulated riboflavin transporter (Reihl and Stolz 2005) and Mct2 of H. sapiens a monocarboxylate transporter responsible for ?-hydroxybutyrate uptake (Wang and Morris 2007).

Anion: Cation Symporter (ACS)

The ACS family (TC 2.A.1.14) is a large family with 40 protein members containing 12 TMs. All of the recognized substrates of the ACS family permeases are either organic or inorganic anions. They accumulate their substrates in symport with either Na+ or H+, depending on the system. Among the organic anions transported are glucarate, hexuronates, phthalate, allantoate, and probably tartrate. Proteins of the ACS family are widely distributed in nature. They are found in both Gram-negative and Gram-positive bacteria and in both the animal and fungal eukaryotic kingdoms.

Some examples are the GudT of B. subtilis a glucarate porter; ExuT of E. coli a hexuronate transporter; TtuB of Agrobacterium vitis a putative tartrate transporter (Mancini et al. 2000); Dal5 of S. cerevisiae a dipeptide, allantoate, ureidosuccinate, allantoin transporter (Cai et al. 2007); Pht1 of P. putida a phthalate transporter; Npt1 of M. musculus a Na:Pi symporters; DgoT (YidT) of E. coli a galactonate transporter (Mancini et al. 2000); OphD of B. cepacia a phthalate transporter; HpaX of Salmonella dublin a putative p-hydroxyphenylacetate transporter (Chang and Zylstra 1999); Sialin of H. sapiens a lysosomal sialate transporter (Verheijen et al. 1999); Tna1 of S. cerevisiae a nicotinate permease (Klebl et al. 2000); Vht1 of S. cerevisiae a biotin H+ symporters (Weider et al. 2006); BNPI of R. norvegicus a Na+ symporter which transports glutamate, phosphate, chloride, amongst other substrates (Mimura et al. 2002) and YhaU of E. coli a D-galactarate H+ symporter (Fujisawa et al. 2004).

Other examples are the OATv1 of Sus scrofa it's a voltage-driven but Na+-independent organic anion transporter, which transports p-aminohippurate; probably transports organic anions and it may catalyze excretion of various drugs, xenobiotics, and their metabolites (Jutabha et al. 2003); VGLUT2 of R. norvegicus an anion transporter which transports glutamate in a ??-dependent fashion requiring Cl- but phosphate by a Na+-dependent mechanism via a different pathway (Juge et al. 2006); Liz1 of S. pombe a pantothenate H+ symporters (Stolz et al. 2004); Fen2 of S. cerevisiae a pantothenate H+ symporters (Stolz and Sauer 1999); Vht1 of S. pombe , a plasma membrane, high affinity vitamin H transporter 1 (H+ biotin symporter) (Stolz 2003); Yct1 of S. cerevisiae a endoplasmic reticular cysteine transporter (Kaur and Bachhawat 2007); SLC17A9 of H. sapiens a vesicular purine nucleotide (ADP, ATP, GTP) transporter (Sawada et al. 2008); ANTR1 of A. thaliana, a chloroplast thylakoid Na+:phosphate symporter (Pavon et al. 2008); VGLUT3 of M. musculus, a vesicular glutamate transporter (Schaefer et al. 2002).

Aromatic Acid: H+ Symporter (AAHS)

The AAHS family (TC 2.A.1.15) members have 12 TMs, are derived exclusively from Gram-negative bacteria, show uniform sizes and transports a variety of aromatic acids as well as cis,cis-muconate. PcaK, a 4-Hydroxybenzoate/protocatachuate porter of P. putida is the founding member of the AAHS family (Ditty and Harwood 2002) also is an unusual member as it also mediates chemotaxis of P. putida to 4-HBA allowing the bacteria to swim up concentration gradients of its substrates (Nichols and Harwood 1997). This is the only documented case where an MFS protein apparently serves as a chemoreceptor.

One of the AAHS proteins, BenK transports benzoate and two additional putative benzoate H+ symporters (BenE) have been sequenced. One is the functionally characterized BenE protein of Acinetobacter calcoaceticus, and the other is a closely related protein from E.coli. These two proteins both contain a single region that exhibits limited sequence similarity to family 15porters, as might be expected on the basis of the specificity of the A.calcoaceticus protein (Collier et al. 1997) (D'Argenio et al. 1999).

Other examples are TfdK of Ralstonia eutropha 2,4-Dichlorophenoxyacetate porter; MucK of A.calcoaceticus ADP1 cis,cis-muconate porter; VanK of A.calcoaceticus ADP1 putative vanillate porter; MhpT of E. coli, a 3-Hydroxyphenyl propionate porter (Chaudhry et al. 2007); YceI of B. subtilis, the putative niacin uptake porter (Rodionov et al. 2008); Orf1 of Nocardioides sp. probable 1-hydroxy-2-naphthoate transporter (Iwabuchi and Harayama 1997) and MmlH of R. eutropha a probable 4-methylmuconolactone transporter (Erb et al. 1998).

Siderophore-Iron Transporter (SIT) Family

The SIT family (TC 2.A.1.16) consists of 4 proteins with 12 TMs, derived exclusively from the yeast S. cerevisiae, Sit1 a siderophore-iron (ferrioxamine): H+ sym- porter (Lesuisse et al. 1998); Enb1, the ferric enterobactin: H+ symporters (Heymann et al. 2000); Taf1, the ferric triacetylfusarinine C: H+ symporter (Heymann et al. 1999) and Arn1p, the ferrichrome:H+ symporters (Moore et al. 2003).

Cyanate Porter (CP) Family

The CP family (TC 2.A.1.17) is composed of 3 proteins, two from E. coli and one from B. subtilis which are small proteins with 12TMs. The substrate of one of these proteins CynX of E.coli is believed to be cyanate (NCO-). The other two members, from E.coli, Orf 393 and from B. subtilis, YycB are strikingly divergent in sequence but not in size (Sung and Fuchs 1989) (Pao et al. 1998).

Polypol Porter (PP) Family

The PP family (TC 2.A.1.18) is composed of 2 proteins with 12TMs. These proteins are DalT of K. pneumoniae a D-Arabinitol transporter and RbtT also from K. pneumoniae, a ribitol transporter both H+ symporters (Heuel et al. 1997).

Organic Cation Transporter (OCT) Family

The OCT family (TC 2.A.1.19) is composed of 19 proteins with 12TMs. Members of this family are Oct1 of R. norvegicus, an organic cation, basolateral multivalent and potential-sensitive transporter responsible for the uptake of cationic drugs, xenobiotics, vitamins, neuro-transmitters amongst other substrates. Oct1 of A. thaliana transports L-carnitine expressed in vascular tissues of various organs and at sites of lateral root formation (Lelandais-Briere et al. 2007). Oct2 of S. scrofa exhibits the properties of an ion channel transporter responsible for the uptake of dopamine, noradrenaline, adrenaline and 5-hydroxytryptamine also transports ochratoxin (Rizwan et al. 2007), cisplatin and oxaliplatin (Yonezama et al. 2006). Oct2 of R. norvegicus, is the rat kidney basolateral potential-driven symporter, transports tetraethylammonium and many other organic cations (Sweet and Pritchard 1999). Oct3 of R. norvegicus transport substrates such as neurotoxin 1-methyl-4-phenylpyridinium and monoamine neuro- transmitters such as dopamine (Khomenko et al. 2008). Other examples are OctN1 and OctN2 of H. sapiens, a