Eukaryotic major facilitator superfamily transporter modeling based on the prokaryotic GlpT crystal structure (Review)

The major facilitator superfamily (MFS) of transporters represents the largest family of secondary active transporters and has a diverse range of substrates. With structural information for four MFS transporters, we can see a strong structural commonality suggesting, as predicted, a common architecture for MFS transporters. The rate for crystal structure determination of MFS transporters is slow, making modeling of both prokaryotic and eukaryotic transporters more enticing. In this review, models of eukaryotic transporters Glut1, G6PT, OCT1, OCT2 and Pho84, based on the crystal structures of the prokaryotic GlpT, based on the crystal structure of LacY are discussed. The techniques used to generate the different models are compared. In addition, the validity of these models and the strategy of using prokaryotic crystal structures to model eukaryotic proteins are discussed. For comparison, E. coli GlpT was modeled based on the E. coli LacY structure and compared to the crystal structure of GlpT demonstrating that experimental evidence is essential for accurate modeling of membrane proteins.     

Purification and properties of the Escherichia coli nucleoside transporter NupG,a paradigm for a major facilitator transporter sub-family

NupG from Escherichia coli is the archetype of a family of nucleoside transporters found in several eubacterial groups and has distant homologues in eukaryotes, including man. To facilitate investigation of its molecular mechanism, we developed methods for expressing an oligohistidine-tagged form of NupG both at high levels (>20% of the inner membrane protein) in E. coli and in Xenopus laevis oocytes. In E. coli recombinant NupG transported purine (adenosine) and pyrimidine (uridine) nucleosides with apparent K_m values of ～20–30 μM and transport was energized primarily by the membrane potential component of the proton motive force. Competition experiments in E. coli and measurements of uptake in oocytes confirmed that NupG was a broad-specificity transporter of purine and pyrimidine nucleosides. Importantly, using high-level expression in E. coli and magic-angle spinning cross-polarization solid-state nuclear magnetic resonance, we have for the first time been able directly to measure the binding of the permeant ([1′-¹³C]uridine) to the protein and to assess its relative mobility within the binding site, under non-energized conditions. Purification of over-expressed NupG to near homogeneity by metal chelate affinity chromatography, with retention of transport function in reconstitution assays, was also achieved. Fourier transform infrared and circular dichroism spectroscopy provided further evidence that the purified protein retained its 3D conformation and was predominantly α-helical in nature, consistent with a proposed structure containing 12 transmembrane helices. These findings open the way to elucidating the molecular mechanism of transport in this key family of membrane transporters.     

Simulations of substrate transport in the multidrug transporter EmrD

EmrD is a multidrug resistance (MDR) transporter from Escherichia coli, which is involved in the efflux of amphipathic compounds from the cytoplasm, and the first MDR member of the major facilitator superfamily to be crystallized. Molecular dynamics simulation of EmrD in a phospholipid bilayer was used to characterize the conformational dynamics of the protein. Motions that support a previously proposed lateral diffusion pathway for substrate from the cytoplasmic membrane leaflet into the EmrD central cavity were observed. In addition, the translocation pathway of meta-chloro carbonylcyanide phenylhydrazone (CCCP) was probed using both standard and steered molecular dynamics simulation. In particular, interactions of a few specific residues with CCCP have been identified. Finally, a large motion of two residues, Val 45 and Leu 233, was observed with the passage of CCCP into the periplasmic space, placing a lower bound on the extent of opening required at this end of the protein for substrate transport. Overall, our simulations probe details of the transport pathway, motions of EmrD at an atomic level of detail, and offer new insights into the functioning of MDR transporters.     

Alternating access mechanism in the POT family of oligopeptide transporters

Short chain peptides are actively transported across membranes as an efficient route for dietary protein absorption and for maintaining cellular homeostasis. In mammals, peptide transport occurs via PepT1 and PepT2, which belong to the proton‐dependent oligopeptide transporter, or POT family. The recent crystal structure of a bacterial POT transporter confirmed that they belong to the major facilitator superfamily of secondary active transporters. Despite the functional characterization of POT family members in bacteria, fungi and mammals, a detailed model for peptide recognition and transport remains unavailable. In this study, we report the 3.3‐Å resolution crystal structure and functional characterization of a POT family transporter from the bacterium Streptococcus thermophilus. Crystallized in an inward open conformation the structure identifies a hinge‐like movement within the C‐terminal half of the transporter that facilitates opening of an intracellular gate controlling access to a central peptide‐binding site. Our associated functional data support a model for peptide transport that highlights the importance of salt bridge interactions in orchestrating alternating access within the POT family.     

CgMFS1, a Major Facilitator Superfamily Transporter,Is Required for Sugar Transport,Oxidative Stress Resistance,and Pathogenicity of Colletotrichum gloeosporioides from Hevea brasiliensis

Colletotrichum gloeosporioides is the main causal agent of anthracnose in various plant species. Determining the molecular mechanisms underlying the pathogenicity and fungicide resistance of C. gloeosporioides could help build new strategies for disease control. The major facilitator superfamily (MFS) has multiple roles in the transport of a diverse range of substrates. In the present study, an MFS protein CgMFS1 was characterized in C. gloeosporioides. This protein contains seven transmembrane domains, and its predicted 3D structure is highly similar to the reported hexose transporters. To investigate the biological functions of CgMFS1, the gene knock-out mutant ΔCgMFS1 was constructed. A colony growth assay showed that the mutant was remarkably decreased in vegetative growth in minimal medium supplemented with monosaccharides and oligosaccharides as the sole carbon sources, whereas it showed a similar growth rate and colony morphology as wild types when using soluble starch as the carbon source. A stress assay revealed that CgMFS1 is involved in oxidative stress but not in the fungicide resistance of C. gloeosporioides. Furthermore, its pathogenicity was significantly impaired in the mutant, although its appressorium formation was not affected. Our results demonstrate that CgMFS1 is required for sugar transport, resistance to oxidative stress, and the pathogenicity of Colletotrichum gloeosporioides from Hevea brasiliensis.     

Identification of a major glucose transporter in Flavobacterium johnsoniae: Inhibition of F. johnsoniae colony spreading by glucose uptake

Many members of the phylum Bacteroidetes, such as Flavobacterium johnsoniae, can glide over a solid surface: an ability called gliding motility. It can be usually observed on agar plates as thin, flat, spreading colonies with irregular, feathery edges; this phenomenon is called colony spreading. Colony spreading of F. johnsoniae on 1.5% agar plates containing poor nutrients is dose‐dependently inhibited by addition of D‐glucose, as previously reported. Accordingly, here, we created mutants (by transposon mutagenesis) that partially suppressed glucose‐mediated inhibition of colony spreading. Among the isolates, we found that one had a transposon insertion in Fjoh_4565, tentatively named mfsA, which encodes a major facilitator superfamily (MFS) transporter previously shown to be required for growth on glucose, N‐acetyl‐glucosamine, and chitin. We constructed an mfsA deletion mutant and found that the mutant showed no glucose‐mediated acceleration of growth or glucose uptake. The mfsA gene complemented the phenotype of a glucose‐negative Escherichia coli. These results suggest that the mfsA gene encodes the sole MFS transporter of glucose in F. johnsoniae and that glucose uptake is partially required for the glucose‐mediated inhibition of F. johnsoniae colony spreading.

     

Functional interactions between the subunits of the lactose transporter from Streptococcus thermophilus

Although the quaternary state has been assessed in detail for only a few members of the major facilitator superfamily (MFS), it is clear that multiple oligomeric states are represented within the MFS. One of its members, the lactose transporter LacS from Streptococcus thermophilus assumes a dimeric structure in the membrane and in vitro analysis showed functional interactions between both subunits when proton motive force ((Delta)p)-driven transport was assayed. To study the interactions in further detail, a covalent dimer was constructed consisting of in tandem fused LacS subunits. These covalent dimers, composed of active and completely inactive subunits, were expressed in Escherichia coli, and initial rates of (Delta)p-driven lactose uptake and lactose counterflow were determined. We now show that also in vivo, both subunits interact functionally; that is, partial complementation of the inactive subunit was observed for both transport modes. Thus, both subunits interact functionally in (Delta)p-driven uptake and in counterflow transport. In addition, analysis of in tandem fused LacS subunits containing one regulatory LacS-IIA domain showed that regulation is primarily an intramolecular event.     

Identification and substrate specificity of a ferrichrome-type siderophore transporter (Arn1p) in Saccharomyces cerevisiae

Genes encoding transporters for heterologous siderophores have been identified in Saccharomyces cerevisiae, of which SIT1, TAF1, and ENB1 encode the transporters for ferrioxamines, ferric triacetylfusarinine C and ferric enterobactin, respectively. In the present communication we have shown that a further gene encoding a member of the major facilitator superfamily, ARN1 (YHL040c), is involved in the transport of a specific class of ferrichromes, possessing anhydromevalonyl residues linked to N(delta)-ornithine (ARN). Ferrirubin and ferrirhodin, which both are produced by filamentous fungi, are the most common representatives of this class of ferrichromes. A strain possessing a disruption in the ARN1 gene was unable to transport ferrirubin, ferrirhodin and also ferrichrome A, indicating that the encoded transporter recognizes anhydromevalonyl and the structurally-related methylglutaconyl side-chains surrounding the iron center. Ferrichromes possessing short-chain ornithine-N(delta)-acetyl residues such as ferrichrome, ferricrocin and ferrichrysin, were excluded by the Arn1 transporter. Substitution of the iron-surrounding N-acyl chains of ferrichromes by propionyl residues had no effect, whereas substitution by butyryl residues led to recognition by the Arn1 transporter. This would indicate that a chain length of four C-atoms is sufficient to allow binding. Using different asperchromes (B1, D1) we also found that a minimal number of two anhydromevalonyl residues is sufficient for recognition by Arn1p. Contrary to the iron-surrounding N-acyl residues, the peptide backbone of ferrichromes was not an important determinant for the Arn1 transporter.     

Using cellular fitness to map the structure and function of a major facilitator superfamily effluxer

The major facilitator superfamily (MFS) effluxers are prominent mediators of antimicrobial resistance. The biochemical characterization of MFS proteins is hindered by their complex membrane environment that makes in vitro biochemical analysis challenging. Since the physicochemical properties of proteins drive the fitness of an organism, we posed the question of whether we could reverse that relationship and derive meaningful biochemical parameters for a single protein simply from fitness changes it confers under varying strengths of selection. Here, we present a physiological model that uses cellular fitness as a proxy to predict the biochemical properties of the MFS tetracycline efflux pump, TetB, and a family of single amino acid variants. We determined two lumped biochemical parameters roughly describing K_m and V_max for TetB and variants. Including in vivo protein levels into our model allowed for more specified prediction of pump parameters relating to substrate binding affinity and pumping efficiency for TetB and variants. We further demonstrated the general utility of our model by solely using fitness to assay a library of tet(B) variants and estimate their biochemical properties.     

Projection structure of the bacterial oxalate transporter OxlT at 3.4A resolution

OxlT is a bacterial transporter protein with 12 transmembrane segments that belongs to the Major Facilitator Superfamily of transporters. It facilitates the exchange of oxalate and formate across the membrane of the Gram-negative bacterium Oxalobacter formigenes. From an electron crystallographic analysis of two-dimensional, tube-like crystals of OxlT, we have previously determined the three-dimensional structure of this transporter at 6.5 A resolution. Here, we report conditions to obtain crystalline, two-dimensional sheets of OxlT with diameters exceeding 2 microm. Images of the crystalline sheets were recorded at liquid nitrogen temperatures on a transmission electron microscope equipped with a field-emission gun, operated at 300 kV. Computed optical diffraction patterns from the best images display measurable reflections to about 3.4A, and electron diffraction patterns show spots to about 3.2 A resolution in the best cases. As in the case of the tube-like crystals, the new crystalline sheets also belong to the p22(1)2(1) symmetry group. However, the unit cell dimensions of 102.7A x 67.3 A are significantly smaller in one direction than those previously observed with the tube-like crystals that display unit cell dimensions of 100.3A x 79.0 A. Different regions of OxlT are involved in intermolecular contacts in the two types of crystals, and the improved resolution of the sheet crystals appears to be mainly attributable to this tighter packing of the monomers within the unit cell.     

Cloning,sequencing and analysis of the pucC genes from Rubrivivax gelatinosus strain 151 and Rhodopseudomonas acidophila strain 10050

The pucC genes of Rubrivivax gelatinosus strain 151 and Rhodopseudomonas acidophila strain 10050 have been identified, cloned and sequenced. In Rubrivivax gelatinosus the arrangement of the pucC gene with regard to the pucBA genes was shown to differ from that found in other species of photosynthetic bacteria. The Rhodopseudomonas acidophila pucC was found downstream of four new pucBA gene pairs, bringing the sequenced pucBA pairs to a total of eight in this strain. The predicted PucC protein sequences were compared to those of PucC from other species and showed high similarity. Similarity was also seen to more distantly related proteins LhaA and orf428 of Rhodobacter capsulatus, orf G115 of Rhodospirillum rubrum and `orf428' from Synechocystis sp. PCC6803. An analysis of the predicted secondary structure of these proteins is given, and their structural similarity to proteins in the Major Facilitator Superfamily is discussed with regard to their possible function. This revised version was published online in June 2006 with corrections to the Cover Date.     

Use of molecular modelling to probe the mechanism of the nucleoside transporter NupG

Abstract

Nucleosides play key roles in biology as precursors for salvage pathways of nucleotide synthesis. Prokaryotes import nucleosides across the cytoplasmic membrane by proton- or sodium-driven transporters belonging to the Concentrative Nucleoside Transporter (CNT) family or the Nucleoside:H⁺ Symporter (NHS) family of the Major Facilitator Superfamily. The high resolution structure of a CNT from Vibrio cholerae has recently been determined, but no similar structural information is available for the NHS family. To gain a better understanding of the molecular mechanism of nucleoside transport, in the present study the structures of two conformations of the archetypical NHS transporter NupG from Escherichia coli were modelled on the inward- and outward-facing conformations of the lactose transporter LacY from E. coli, a member of the Oligosaccharide:H⁺ Symporter (OHS) family. Sequence alignment of these distantly related proteins (～ 10% sequence identity), was facilitated by comparison of the patterns of residue conservation within the NHS and OHS families. Despite the low sequence similarity, the accessibilities of endogenous and introduced cysteine residues to thiol reagents were found to be consistent with the predictions of the models, supporting their validity. For example C358, located within the predicted nucleoside binding site, was shown to be responsible for the sensitivity of NupG to inhibition by p-chloromercuribenzene sulphonate. Functional analysis of mutants in residues predicted by the models to be involved in the translocation mechanism, including Q261, E264 and N228, supported the hypothesis that they play important roles, and suggested that the transport mechanisms of NupG and LacY, while different, share common features.     

A gene of the major facilitator superfamily encodes a transporter for enterobactin (Enb1p) in Saccharomyces cerevisiae

While in fungi iron transport via hydroxamate siderophores has been amply proven, iron transport via enterobactin is largely unknown. Enterobactin is a catecholate-type siderophore produced by several enterobacterial genera grown in severe iron deprivation. By using the KanMX disruption module in vector pUG6 in a fet3 background of Saccharomyces cerevisiae we were able to disrupt the gene YOL158c Sce of the major facilitator super family (MFS) which has been previously described as a gene encoding a membrane transporter of unknown function. Contrary to the parental strain, the disruptant was unable to utilize ferric enterobactin in growth promotion tests and in transport assays using ⁵⁵Fe-enterobactin. All other siderophore transport properties remained unaffected. The results are evidence that in S. cerevisiae the YOL158c Sce gene of the major facilitator super family, now designated ENB1, encodes a transporter protein (Enb1p), which specifically recognizes and transports enterobactin.     

Structural and functional insights into Spns2-mediated transport of sphingosine-1-phosphate

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Regulation of GmNRT2 expression and nitrate transport activity in roots of soybean (Glycine max)

A full-length cDNA, GmNRT2, encoding a putative high-affinity nitrate transporter was isolated from a Glycine max (L.) root cDNA library and sequenced. The deduced GmNRT2 protein is 530 amino acids in length and contains 12 putative membrane-spanning domains and a long, hydrophilic C-terminal domain. GmNRT2 is related to high-affinity nitrate transporters in the eukaryotes Chlamydomonas reinhardtii and Aspergillus nidulans, and to putative high-affinity nitrate transporters in barley and tobacco. Southern blot analysis indicated that GmNRT2 is part of a small, multigene family in soybean. Expression of GmNRT2 in roots was regulated by the type of nitrogen source provided to plants: GmNRT2 mRNA levels were barely detectable in ammonium-grown plants, higher in nitrogen-deprived plants, and highest in nitrate-grown plants. Induction of GmNRT2 mRNA levels in roots occurred within 1 h after exposure of plants to nitrate. Nitrate induction of GmNRT2 mRNA levels was accompanied by a fourfold increase in net nitrate uptake by soybean roots at 100 μM external nitrate. The molecular and physiological evidence indicates that GmNRT2 is probably a high-affinity nitrate transporter involved in nitrate uptake by soybean roots. Received: 22 November 1997 / Accepted: 26 January 1998     

Atomic resolution structure of the E. coli YajR transporter YAM domain

YajR is an Escherichia coli transporter that belongs to the major facilitator superfamily. Unlike most MFS transporters, YajR contains a carboxyl terminal, cytosolic domain of 67 amino acid residues termed YAM domain. Although it is speculated that the function of this small soluble domain is to regulate the conformational change of the 12-helix transmembrane domain, its precise regulatory role remains unclear. Here, we report the crystal structure of the YAM domain at 1.07-Å resolution, along with its structure determined using nuclear magnetic resonance. Detailed analysis of the high resolution structure revealed a symmetrical dimer in which a belt of well-ordered poly-pentagonal water molecules is embedded. A mutagenesis experiment and a thermal stability assay were used to analyze the putative role of this dimerization in response to changes in halogen concentration.     

A member of the mitogen-activated protein 3-kinase family is involved in the regulation of plant vacuolar glucose uptake

Vacuolar solute accumulation is an important process during plant development, growth and stress responses. Although several vacuolar carriers have been identified recently, knowledge regarding the regulation of transport is still limited. Solute accumulation may be controlled by various factors, such as alterations in carrier abundance or activity. Phosphorylation via kinases is a well-known principle for activation or deactivation of proteins. Several phosphorylated proteins have been identified in the tonoplast proteome; however, kinases that catalyse the phosphorylation of tonoplast proteins are currently unknown. The tonoplast monosaccaride transporter from Arabidopsis (AtTMT1) and its homologue from barley have multiple phosphorylation sites in their extremely large loops. Here we demonstrate that the loop of AtTMT1 interacts with a mitogen-activated triple kinase-like protein kinase (VIK), that an aspartate-rich loop domain is required for effective interaction, and that the presence of VIK stimulates glucose import into isolated vacuoles. Furthermore, the phenotype of VIK loss-of-function plants strikingly resembles that of plants lacking AtTMT1/2. These data suggest that VIK-mediated phosphorylation of the AtTMT1 loop enhances carrier activity and consequently vacuolar sugar accumulation. As many phosphorylated proteins have been identified in the tonoplast, differential phosphorylation may be a general mechanism regulating vacuolar solute import.