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.     

A novel MFS transporter encoding gene in Fusarium verticillioides probably involved in iron-siderophore transport

The major facilitator superfamily (MFS) is a ubiquitous group of proteins involved in the transport of a wide range of compounds, including toxins produced by fungal species. In this paper, a novel MFS encoding gene (Fusarium iron related gene or FIR1), which had shown an up-regulation in fumonisin-inducing conditions, has been identified and characterized. The deduced protein sequence, which predicted 14 transmembrane domains typical of MFS transporters and its phylogenetic relationships with representative members of MFS transporters suggested a possible function of FIR1 as a siderophore transporter. A real-time RT-PCR protocol has been developed to analyse the expression pattern of the FIR1 gene in relation to siderophore production. The results indicated that the synthesis of extracellular siderophores by F. verticillioides observed in absence of extracellular iron was repressed in iron-supplemented cultures and showed a good correspondence with FIR1 gene expression. However, the pattern of FIR1 gene expression observed suggested that this gene did not seem to be functionally related to fumonisin production.     

Identification of Nile red as a fluorescent substrate of the Candida albicans ATP-binding cassette transporters Cdr1p and Cdr2p and the major facilitator superfamily transporter Mdr1p

Clinically relevant azole resistance in the fungal pathogen Candida albicans is most often associated with the increased expression of plasma membrane efflux pumps, specifically the ATP-binding cassette (ABC) transporters CaCdr1p and CaCdr2p and the major facilitator superfamily (MFS) transporter CaMdr1p. Development of potent pump inhibitors that chemosensitize cells to azoles is a promising approach to overcome antifungal resistance. Here we identify Nile red as a new fluorescent substrate for CaCdr1p, CaCdr2p, and CaMdr1p. Nile red was effluxed efficiently from Saccharomyces cerevisiae cells heterologously expressing these transporters. Enniatin selectively inhibited the efflux of Nile red from S. cerevisiae cells expressing CaCdr1p or CaMdr1p but not from cells expressing CaCdr2p. This indicates that Nile red can be used for the identification of inhibitors specific for particular transporters mediating antifungal resistance in pathogenic yeast.     

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.     

Specificity of drug transport mediated by CaMDR1: a major facilitator of Candida albicans

CaMDR1 encodes a major facilitator superfamily (MFS) protein inCandida albicans whose expression has been linked to azole resistance and which is frequently encountered in this human pathogenic yeast. In this report we have overexpressed CaMdr1p inSf9 insect cells and demonstrated for the first time that it can mediate methotrexate (MTX) and fluconazole (FLC) transport. MTX appeared to be a better substrate for CaMdr1p among these two tested drugs. Due to severe toxicity of these drugs to insect cells, further characterization of CaMdr1p as a drug transporter could not be done with this system. Therefore, as an alternative, CaMdr1p and Cdr1p, which is an ABC protein (ATP binding cassette) also involved in azole resistance inC. albicans, were independently expressed in a common hypersensitive host JG436 ofSaccharomyces cerevisiae. This allowed a better comparison between the functionality of the two export pumps. We observed that while both FLC and MTX are effluxed by CaMdr1p, MTX appeared to be a poor substrate for Cdr1p. JG436 cells expressing Cdr1p thus conferred resistance to other antifungal drugs but remained hypersensitive to MTX. Since MTX is preferentially transported by CaMdr1p, it can be used for studying the function of this MFS protein.     

Structural Requirements for the Activity of the MirB Ferrisiderophore Transporter of Aspergillus fumigatus

Siderophores have been identified as virulence factors in the opportunistic fungal pathogen Aspergillus fumigatus. The 14-pass transmembrane protein MirB is postulated to function as a siderophore transporter, responsible for uptake of the hydroxamate siderophore N,N′,N″-triacetylfusarinine C (TAFC). Our aim was to identify amino acids of A. fumigatus MirB that are crucial for uptake of TAFC. Site-directed mutagenesis was used to create MirB mutants. Expression of wild-type and mutant proteins in the Saccharomyces cerevisiae strain PHY14, which lacks endogenous siderophore transporters, was confirmed by Western blotting. TAFC transport assays using ⁵⁵Fe-labeled TAFC and growth assays with Fe-TAFC as the sole iron source identified alanine 125, tyrosine 577, loop 3, and the second half of loop 7 (Loop7Del2) as crucial for function, since their substitution or deletion abrogated uptake completely. Wild-type MirB transported ferricrocin and coprogen as well as TAFC but not ferrichrysin. MirB was localized by fluorescence microscopy using antisera raised against a MirB extracellular loop peptide. Immunofluorescence microscopy showed that in yeast, wild-type MirB had a punctate distribution under the plasma membrane, as did the A125D and Y577A strains, indicating that the defect in transport of these mutants was unlikely to be due to mislocalization or degradation. MirB immunolocalization in A. fumigatus showed that the transporter was found in vesicles which cycled between the cytoplasm and the plasma membrane and was concentrated at the hyphal tips. The location of MirB was not influenced by the presence of the siderophore TAFC but was sensitive to internal iron stores.     

Fungal transporters involved in efflux of natural toxic compounds and fungicides

Survival of microorganisms in natural environments is favored by the capacity to produce compounds toxic to competing organisms and the ability to resist the effects of such toxic compounds. Both factors contribute to a competitive advantage of organisms in ecosystems. All organisms have evolved active transport mechanisms by which endogenous and exogenous toxicants can be secreted. Two major classes of transporter proteins are the ATP-binding cassette (ABC) and the major facilitator superfamily (MFS) transporters. Members of both classes can have broad and overlapping substrate specificities for natural toxic compounds and can be regarded as a "first-line defense barrier" in survival mechanisms. In plant pathogens, these transporters can play an essential role in protection against plant defense compounds during pathogenesis. Also, some transporters actively secrete host-specific and non-host-specific toxins. Remarkably, ABC and MFS transporters can also play a major role in fungicide sensitivity and resistance. Their role in multidrug resistance of Aspergillus nidulans, Candida albicans, and Saccharomyces cerevisiae to azoles and other fungitoxic compounds is well established. Knowledge of ABC and MFS transporters opens possibilities of developing novel strategies for controlling plant diseases, either by modulation of transporter activity or by transgenic expression of transporter genes in plants.     

Expression of a tomato sugar transporter is increased in leaves of mycorrhizal or Phytophthora parasitica-infected plants

A full-length cDNA clone (LeST3), encoding a putative tomato sugar transporter, was isolated from mycorrhizal roots by using a PCR-based approach. Based on sequence similarity, conserved motifs and predicted membrane topology, LeST3 was classified as a putative monosaccharide transporter of the sugar transporter subgroup of the major facilitator superfamily. Southern blot analysis showed that LeST3 represents a single-copy gene in tomato. To investigate its function, LeST3 was expressed in a hexose transport-deficient mutant of Saccharomyces cerevisiae. Although LeST3 was correctly transcribed in yeast, it did not restore growth on hexoses of the S. cerevisiae mutant. LeST3 gene expression was increased in the leaves of plants colonised by the arbuscular mycorrhizal (AM) fungi Glomus mosseae or Glomus intraradices and in those of plants infected with the root pathogen Phytophthora parasitica. These data suggest that LeST3 plays a role in the transport of sugars into the sink tissues and responds to the increased demand for carbohydrates exerted by two AM fungi and by a root pathogen to cope with the increased metabolic activity of the colonised/infected tissues or to supply carbohydrates to the AM fungus.     

Functional production of mammalian concentrative nucleoside transporters in Saccharomyces cerevisiae

The transport of nucleosides and nucleobases in the yeast Saccharomyces cerevisiae is reviewed and the use of this organism to study recombinant mammalian concentrative nucleoside transport (CNT) proteins is described. A selection strategy based on the ability of an expressed nucleoside transporter cDNA to mediate thymidine uptake by yeast under a selective condition that depletes endogenous thymidylate was used to assess the transport capacity of heterologous transporter proteins. The pyrimidine-nucleoside selective concentrative transporters from human (hCNT1) and rat (rCNT1) complemented the imposed thymidylate depletion in S. cerevisiae, as did N-terminally truncated versions of hCNT1 and rCNT1 lacking up to 31 amino acids. Transporter-mediated rescue of S. cerevisiae by both nucleoside transporters was inhibited by cytidine, uridine and adenosine, but not by guanosine or inosine. This work represents the development of a new model system for the functional production of recombinant nucleoside transporters of the CNT family of membrane proteins.     

马克斯克鲁维酵母GX-UN120糖转运蛋白基因的克隆及表征

【背景】马克斯克鲁维酵母(Kluyveromyces marxianus)具有完整的木糖代谢途径,可以高效利用木质纤维素中的木糖,因此对其糖转运蛋白基因的研究或可有效解决酵母木糖转运的相关问题。【目的】根据马克斯克鲁维酵母DMKU3-1042中KLMA＿70145和KLMA＿80101基因位点的功能预测,获得马克斯克鲁维酵母GX-UN120相应的糖转运蛋白基因序列并探究其功能。【方法】将转运蛋白基因分别克隆表达至酿酒酵母EBY.VW4000中考察重组菌株生长特性,以此间接评价对应转运蛋白的转运能力。【结果】Km＿SUT2基因编码的糖转运蛋白可有效提高宿主细胞转运木糖、阿拉伯糖、山梨糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖、果糖、蔗糖和半乳糖。类似地,Km＿SUT3基因编码的糖转运蛋白可提高细胞转运木糖、阿拉伯糖、山梨糖、半乳糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖和果糖。然而在葡萄糖存在的条件下,重组菌株对各种碳源的利用均受抑制,但Km＿SUT3转运木糖和核糖过程中受葡萄糖的抑制作用较小。【结论】马克斯克鲁维酵母GX-UN120中转运蛋白Km＿SUT2和Km＿SUT3可...     

Modeling of glycerol-3-phosphate transporter suggests a potential 'tilt' mechanism involved in its function

Many major facilitator superfamily (MFS) transporters have similar 12-transmembrane alpha-helical topologies with two six-helix halves connected by a long loop. In humans, these transporters participate in key physiological processes and are also, as in the case of members of the organic anion transporter (OAT) family, of pharmaceutical interest. Recently, crystal structures of two bacterial representatives of the MFS family--the glycerol-3-phosphate transporter (GlpT) and lac-permease (LacY)--have been solved and, because of assumptions regarding the high structural conservation of this family, there is hope that the results can be applied to mammalian transporters as well. Based on crystallography, it has been suggested that a major conformational "switching" mechanism accounts for ligand transport by MFS proteins. This conformational switch would then allow periodic changes in the overall transporter configuration, resulting in its cyclic opening to the periplasm or cytoplasm. Following this lead, we have modeled a possible "switch" mechanism in GlpT, using the concept of rotation of protein domains as in the DynDom program17 and membranephilic constraints predicted by the MAPAS program.(23) We found that the minima of energies of intersubunit interactions support two alternate positions consistent with their transport properties. Thus, for GlpT, a "tilt" of 9 degrees -10 degrees rotation had the most favorable energetics of electrostatic interaction between the two halves of the transporter; moreover, this confirmation was sufficient to suggest transport of the ligand across the membrane. We conducted steered molecular dynamics simulations of the GlpT-ligand system to explore how glycerol-3-phosphate would be handled by the "tilted" structure, and obtained results generally consistent with experimental mutagenesis data. While biochemical data remain most consistent with a single-site alternating access model, our results raise the possibility that, while the "rocker switch" may apply to certain MFS transporters, intermediate "tilted" states may exist under certain circumstances or as transitional structures. Although wet lab experimental confirmation is required, our results suggest that transport mechanisms in this transporter family should probably not be assumed to be conserved simply based on standard structural homology considerations. Furthermore, steered molecular dynamics elucidating energetic interactions of ligands with amino acid residues in an appropriately modeled transporter may have predictive value in understanding the impact of mutations and/or polymorphisms on transporter function.     

Fungal siderophore biosynthesis is partially localized in peroxisomes

Siderophores play a central role in iron metabolism and virulence of most fungi. Both Aspergillus fumigatus and Aspergillus nidulans excrete the siderophore triacetylfusarinine C (TAFC) for iron acquisition. In A. fumigatus, green fluorescence protein‐tagging revealed peroxisomal localization of the TAFC biosynthetic enzymes SidI (mevalonyl‐CoA ligase), SidH (mevalonyl‐CoA hydratase) and SidF (anhydromevalonyl‐CoA transferase), while elimination of the peroxisomal targeting signal (PTS) impaired both, peroxisomal SidH‐targeting and TAFC biosynthesis. The analysis of A. nidulans mutants deficient in peroxisomal biogenesis, ATP import or protein import revealed that cytosolic mislocalization of one or two but, interestingly, not all three enzymes impairs TAFC production during iron starvation. The PTS motifs are conserved in fungal orthologues of SidF, SidH and SidI. In agreement with the evolutionary conservation of the partial peroxisomal compartmentalization of fungal siderophore biosynthesis, the SidI orthologue of coprogen‐type siderophore‐producing Neurospora crassa was confirmed to be peroxisomal. Taken together, this study identified and characterized a novel, evolutionary conserved metabolic function of peroxisomes.     

Substrate-bound structure of the E. coli multidrug resistance transporter MdfA

Multidrug resistance is a serious threat to public health. Proton motive force-driven antiporters from the major facilitator superfamily (MFS) constitute a major group of multidrug-resistance transporters. Currently, no reports on crystal structures of MFS antiporters in complex with their substrates exist. The E. coli MdfA transporter is a well-studied model system for biochemical analyses of multidrug-resistance MFS antiporters. Here, we report three crystal structures of MdfA-ligand complexes at resolutions up to 2.0 Å, all in the inward-facing conformation. The substrate-binding site sits proximal to the conserved acidic residue, D34. Our mutagenesis studies support the structural observations of the substrate-binding mode and the notion that D34 responds to substrate binding by adjusting its protonation status. Taken together, our data unveil the substrate-binding mode of MFS antiporters and suggest a mechanism of transport via this group of transporters.     

The hexose transporter family of Saccharomyces cerevisiae

Saccharomyces cerevisiae accomplishes high rates of hexose transport. The kinetics of hexose transport are complex. The capacity and kinetic complexity of hexose transport in yeast are reflected in the large number of sugar transporter genes in the genome. Twenty hexose transporter genes exist in S. cerevisiae. Some of these have been found by genetic means; many have been discovered by the comprehensive sequencing of the yeast genome. This review codifies the nomenclature of the hexose transporter genes and describes the sequence homology and structural similarity of the proteins they encode. Information about the expression and function of the transporters is presented. Access to the sequences of the genes and proteins at three sequence databases is provided via the World Wide Web. Received: 24 June 1996 / Accepted: 29 July 1996     

Cloning and expression study of a putative high-affinity nitrate transporter gene from Dunaliella salina

A nitrate transporter gene, named DsNRT2.1 (GeneBank accession number AY621079), from Dunaliella salina has been cloned by screening a cDNA library, which was constructed with mRNAs from D. salina after 60 min treatment with 5 mM nitrate, with a 342 bp NRT2 cDNA fragment from D. salina as a probe. DsNRT2.1 exhibits sequence similarity to those known nitrate transporters of the NRT2 family. A hydrophobicity blot indicated that DsNRT2.1 belongs to the major facilitator superfamily (MFS). Northern analysis showed that an mRNA species of 1.9 kb can be rapidly induced by NO^– ₃, but not by NH⁺ ₄. Northern analysis also showed that NaCl could significantly increase the expression of DsNRT2.1.