Major Facilitator Superfamily Protein Research Articles

Molecular mechanism of choline and ethanolamine transport in humans.

Human feline leukaemia virus subgroup C receptor-related proteins 1 and 2 (FLVCR1 and FLVCR2) are members of the major facilitator superfamily1. Their dysfunction is linked to several clinical disorders, including PCARP, HSAN and Fowler syndrome2-7. Earlier studies concluded that FLVCR1 may function as a haem exporter8-12, whereas FLVCR2 was suggested to act as a haem importer13, yet conclusive biochemical and detailed molecular evidence remained elusive for the function of both transporters14-16. Here, we show that FLVCR1 and FLVCR2 facilitate the transport of choline and ethanolamine across the plasma membrane, using a concentration-driven substrate translocation process. Through structural and computational analyses, we have identified distinct conformational states of FLVCRs and unravelled the coordination chemistry underlying their substrate interactions. Fully conserved tryptophan and tyrosine residues form the binding pocket of both transporters and confer selectivity for choline and ethanolamine through cation-π interactions. Our findings clarify the mechanisms of choline and ethanolamine transport by FLVCR1 and FLVCR2, enhance our comprehension of disease-associated mutations that interfere with these vital processes and shed light on the conformational dynamics of these major facilitator superfamily proteins during the transport cycle.

Characterization and Mechanism Analysis of Sugar Transporter Protein Obtained from Monascus pilosus

Sugar transporter protein family is very common among different organisms in nature and plays a key role in the transportation, absorption, and synthesis of secondary metabolites. To investigate the functional characteristics of sugar transporter proteins in Monascus pilosus, cDNA sequence of sugar transporter protein gene MpST collected from M. pilosus CICC 5045 was cloned in this research. Furthermore, bioinformatics methods and molecular docking techniques were applied to analyze MpST protein. The obtained results revealed that the coding region of MpST was 1677 bp in length, which coded 558 amino acids, and it was a stable protein. Phylogenetic tree analysis found a close relationship between MpST and two other proteins from M. purpureus (BDD61243.1 and TQB74099.1). Bioinformatics analyses indicated that this protein is a hydrophilic protein with 11 transmembrane domains and its functional domains were mainly consisted of the conserved domains of major facilitator superfamily (MFS) and sugar transporter proteins, suggesting that this protein belonged to MFS transporter protein family. Molecular docking results suggested that the receptor protein could spontaneously bind with high affinity to small molecular ligands such as galactose, raffinose, glucose, xylose, and maltose. From the above analyses, it was predicted that this protein might have good transport function for the above five sugars. This study lays the foundation for further in-depth research on the functions and applications of this protein.

Combining Genetic and Transcriptomic Approaches to Identify Transporter-Coding Genes as Likely Responsible for a Repeatable Salt Tolerance QTL in Citrus

The excessive accumulation of chloride (Cl−) in leaves due to salinity is frequently related to decreased yield in citrus. Two salt tolerance experiments to detect quantitative trait loci (QTLs) for leaf concentrations of Cl−, Na+, and other traits using the same reference progeny derived from the salt-tolerant Cleopatra mandarin (Citrus reshni) and the disease-resistant donor Poncirus trifoliata were performed with the aim to identify repeatable QTLs that regulate leaf Cl− (and/or Na+) exclusion across independent experiments in citrus, as well as potential candidate genes involved. A repeatable QTL controlling leaf Cl− was detected in chromosome 6 (LCl-6), where 23 potential candidate genes coding for transporters were identified using the C. clementina genome as reference. Transcriptomic analysis revealed two important candidate genes coding for a member of the nitrate transporter 1/peptide transporter family (NPF5.9) and a major facilitator superfamily (MFS) protein. Cell wall biosynthesis- and secondary metabolism-related processes appeared to play a significant role in differential gene expression in LCl-6. Six likely gene candidates were mapped in LCl-6, showing conserved synteny in C. reshni. In conclusion, markers to select beneficial Cleopatra mandarin alleles of likely candidate genes in LCl-6 to improve salt tolerance in citrus rootstock breeding programs are provided.

Structure Elucidation and Interaction Dynamics of MefA-MsrD Efflux Proteins in Streptococcus pneumoniae: Impact on Macrolide Susceptibility.

Macrolides are empirically used to treat bacterial community-acquired pneumonia (CAP). Streptococcus pneumoniae, being the major pathogen responsible for bacterial CAP with high mortality rates, express MefA-MsrD efflux pumps to hinder macrolide susceptibility. Despite its importance, the structural features of the efflux-protein complex and its impact on macrolide susceptibility have not yet been elucidated explicitly. Therefore, in the present study, combining homology, threading, and dynamics approaches, MefA and MsrD proteins in pathogenic S. pneumoniae were modeled. Both membrane (lipid-bilayer) and cytoplasmic (aqueous) environments were considered to simulate the MefA and MsrD proteins in their ideal cellular conditions followed by dynamics analyses. The simulated MefA structure represented a typical major facilitator superfamily protein structure with 13 transmembrane helices. MefA-MsrD interaction via clustering-based docking revealed low-energy conformers with stable intermolecular interactions. The higher clinical MIC value of azithromycin over erythromycin was reflected upon erythromycin eliciting stronger interactions (dissociation constant or ki = ∼52 μM) with the cytoplasmic ATP-binding MsrD than azithromycin (ki = ∼112 μM). The strong (binding energy = -132.1 ± 9.5 kcal/mol) and highly stable (root-mean-square fluctuation < 1.0 Å) physical association between MefA with MsrD was validated and was found to be unaffected by the antibiotic binding. Higher propensity of the macrolides to interact with MsrD than MefA established the importance of the former in macrolide susceptibility. Ours is probably the first report on the structural arrangements in the MefA-MsrD efflux complex and the macrolide susceptibility in S. pneumoniae. This study provides a novel lead for experimental explorations and efflux-pump inhibitor designs.

CWHM-974 is a fluphenazine derivative with improved antifungal activity against Candida albicans due to reduced susceptibility to multidrug transporter-mediated resistance mechanisms.

Multidrug resistance (MDR) transporters such as ATP-Binding Cassette (ABC) and Major Facilitator Superfamily proteins are important mediators of antifungal drug resistance, particularly with respect to azole class drugs. Consequently, identifying molecules that are not susceptible to this mechanism of resistance is an important goal for new antifungal drug discovery. As part of a project to optimize the antifungal activity of clinically used phenothiazines, we synthesized a fluphenazine derivative (CWHM-974) with 8-fold higher activity against Candida spp. compared to the fluphenazine and with activity against Candida spp. with reduced fluconazole susceptibility due to increased MDR transporters. Here, we show that the improved C. albicans activity is because fluphenazine induces its own resistance by triggering expression of Candida drug resistance (CDR) transporters while CWHM-974 induces expression but does not appear to be a substrate for the transporters or is insensitive to their effects through other mechanisms. We also found that fluphenazine and CWHM-974 are antagonistic with fluconazole in C. albicans but not in C. glabrata, despite inducing CDR1 expression to high levels. Overall, CWHM-974 is one of the few examples of a molecule in which relatively small structural modifications significantly reduced susceptibility to multidrug transporter-mediated resistance.

Evolution and functional diversification of yeast sugar transporters.

While simple sugars such as monosaccharides and disaccharide are the typical carbon source for most yeasts, whether a species can grow on a particular sugar is generally a consequence of presence or absence of a suitable transporter to enable its uptake. The most common transporters that mediate sugar import in yeasts belong to the major facilitator superfamily (MFS). Some of these, for example the Saccharomyces cerevisiae Hxt proteins have been extensively studied, but detailed information on many others is sparce. In part, this is because there are many lineages of MFS transporters that are either absent from, or poorly represented in, the model S. cerevisiae, which actually has quite a restricted substrate range. It is important to address this knowledge gap to gain better understanding of the evolution of yeasts and to take advantage of sugar transporters to exploit or engineer yeasts for biotechnological applications. This article examines the full repertoire of MFS proteins in representative budding yeasts (Saccharomycotina). A comprehensive analysis of 139 putative sugar transporters retrieved from 10 complete genomes sheds new light on the diversity and evolution of this family. Using the phylogenetic lens, it is apparent that proteins have often been misassigned putative functions and this can now be corrected. It is also often seen that patterns of expansion of particular genes reflects the differential importance of transport of specific sugars (and related molecules) in different yeasts, and this knowledge also provides an improved resource for the selection or design of tailored transporters.

Morphophysiological, proteomic and metabolomic analyses reveal cadmium tolerance mechanism in common wheat (Triticum aestivum L.).

Soil cadmium (Cd) contamination can reduce wheat yield and quality, thus threatening food security and human health. Herein, morphological physiology, Cd accumulation and distribution, proteomic and metabolomic analyses were performed (using wheat cultivars 'Luomai23' (LM, Cd-sensitive) and 'Zhongyu10' (ZY, Cd-tolerant) at the seedling stage with sand culture) to reveal Cd tolerance mechanism. Cd inhibited wheat growth, caused oxidative stress, hindered carbon and nitrogen metabolism, and altered the quantity and composition of root exudates. The root Cd concentration was lower in ZY than in LM by about 35% under 15μMCd treatments. ZY reduced Cd uptake through root exudation of amino acids and alkaloids. ZY also reduced Cd accumulation through specific up-regulation (twice) of major facilitator superfamily (MFS) proteins. Furthermore, ZY enhanced Cd cell wall fixation and vacuolar compartmentalization by increasing pectin contents, hemicellulose1 contents, and adenosine triphosphate binding cassette subfamily C member 1 (ABCC1) transporter expression, thus reducing the Cd organelle fraction of ZY by about 12% and 44% in root and shoot, respectively, compared with LM. Additionally, ZY had enhanced resilience to Cd due to increased antioxidant capacity, plasma membrane stability, nitrogen metabolism, and endoplasmic reticulum homeostasis, indicating that the increased Cd tolerance could be because of multi-level coordination. These findings provide a reference for exploring the molecular mechanism of Cd tolerance and accumulation, providing a basis for safe utilization of Cd-contaminated soil by breeding Cd-tolerant and low Cd-accumulating wheat varieties.

Intrinsic fiducials for small membrane protein structure determination.

Solute carrier proteins (SLC) are small membrane transport proteins that play a critical role in many physiological processes. Despite their critical importance, our understanding of how these proteins function is limited due to the difficulty of structure determination. This is particularly true for major facilitator superfamily (MFS) proteins where there are often limited protein features to drive computational alignment of particles. While tools like cryo-EM have greatly enhanced the ability to determine membrane protein structures, fiducial markers are needed to help promote particle alignment. Fragment antigen-binding antibodies (Fab) or nanobodies (Nb) are the most commonly used examples, but generating these is not a trivial task. Often this is the largest barrier for membrane protein structure determination, taking a significant amount of time and resources. In the end, the resulting fiducial may be too flexible to enable high resolution structure determination, requiring the whole process to be done again. A feature intrinsic to the protein sequence would eliminate this barrier as it would require no additional generation time, and can be purified concurrently with the transporter of interest. Here we investigate the use of an intrinsic fiducial consisting of GFP and anti-GFP Nb labeled at the each of the protein's termini to solve a novel structure of an MFS transporter. We have generated three fully functional constructs, each with varying termini lengths, and demonstrated successive improvements in fiducial rigidity and cryo-EM reconstructions. We believe this method will not only enable us to solve a previously unknown structure, but also prove to be an accessible means for membrane protein structure determination.

Identification of a major facilitator superfamily protein that is beneficial to L-lactic acid production by Bacillus coagulans at low pH

BackgroundProduct inhibition is one of the major problems in lactic acid (LA) fermentation. Our previous study revealed that Bacillus coagulans 2–6 was an efficient producer of high-optical-purity L-LA. Its mutant strain B. coagulans Na-2 has better resistance to sodium lactate stress but the resistance mechanism has not been understood.ResultsIn this study, the whole-genome sequencing of B. coagulans Na-2 was performed and one mutant gene mfs coding for the major facilitator superfamily (MFS) protein was revealed by comparative genome analysis. Ten mutation sites were identified between the wild (MFS-2-6) and mutant (MFS-Na-2) proteins, among which T127A and N154T were predicted locating in the center of the transmembrane transport channel. The MFS-2-6 and MFS-Na-2 were expressed separately in a genetically operable strain, B. coagulans DSM1, using the genes’ native promoter. The expression of the two MFS proteins had no effect and a negative effect on L-LA production when the pH was controlled at 6.0 and 7.0 by sodium hydroxide, respectively. However, 4.2 and 4.6-fold of L-LA concentrations were obtained at pH 5.0 by the strains expressing MFS-2-6 and MFS-Na-2 than that by the control strain, respectively. The intracellular pH values of the strains expressing MFS-2-6 and MFS-Na-2 were approximately 0.69 and 0.45 higher than that of the control strain during pH-controlled fermentation at 5.0. Results suggest that the expression of MFS-2-6 and MFS-Na-2 were both conducive to L-LA production at low pH, while the better performance of the latter was probably due to the more appropriate intracellular pH during the whole fermentation process.ConclusionsThe MFS protein identified here can improve the ability of B. coagulans to resist acidic environments and produce more L-LA at low pH. The MFS protein has an application potential in environment-friendly L-LA production.

A genomic approach to analyze the cold adaptation of yeasts isolated from Italian Alps.

Microorganisms including yeasts are responsible for mineralization of organic matter in cold regions, and their characterization is critical to elucidate the ecology of such environments on Earth. Strategies developed by yeasts to survive in cold environments have been increasingly studied in the last years and applied to different biotechnological applications, but their knowledge is still limited. Microbial adaptations to cold include the synthesis of cryoprotective compounds, as well as the presence of a high number of genes encoding the synthesis of proteins/enzymes characterized by a reduced proline content and highly flexible and large catalytic active sites. This study is a comparative genomic study on the adaptations of yeasts isolated from the Italian Alps, considering their growth kinetics. The optimal temperature for growth (OTG), growth rate (Gr), and draft genome sizes considerably varied (OTG, 10°C-20°C; Gr, 0.071-0.0726; genomes, 20.7-21.5 Mpb; %GC, 50.9-61.5). A direct relationship was observed between calculated protein flexibilities and OTG, but not for Gr. Putative genes encoding for cold stress response were found, as well as high numbers of genes encoding for general, oxidative, and osmotic stresses. The cold response genes found in the studied yeasts play roles in cell membrane adaptation, compatible solute accumulation, RNA structure changes, and protein folding, i.e., dihydrolipoamide dehydrogenase, glycogen synthase, omega-6 fatty acid, stearoyl-CoA desaturase, ATP-dependent RNA helicase, and elongation of very-long-chain fatty acids. A redundancy for several putative genes was found, higher for P-loop containing nucleoside triphosphate hydrolase, alpha/beta hydrolase, armadillo repeat-containing proteins, and the major facilitator superfamily protein. Hundreds of thousands of small open reading frames (SmORFs) were found in all studied yeasts, especially in Phenoliferia glacialis. Gene clusters encoding for the synthesis of secondary metabolites such as terpene, non-ribosomal peptide, and type III polyketide were predicted in four, three, and two studied yeasts, respectively.

Spns1 is a lysophospholipid transporter mediating lysosomal phospholipid salvage

The lysosome is central to the degradation of proteins, carbohydrates, and lipids and their salvage back to the cytosol for reutilization. Lysosomal transporters for amino acids, sugars, and cholesterol have been identified, and the metabolic fates of these molecules in the cytoplasm have been elucidated. Remarkably, it is not known whether lysosomal salvage exists for glycerophospholipids, the major constituents of cellular membranes. By using a transport assay screen against orphan lysosomal transporters, we identified the major facilitator superfamily protein Spns1 that is ubiquitously expressed in all tissues as a proton-dependent lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) transporter, with LPC and LPE being the lysosomal breakdown products of the most abundant eukaryotic phospholipids, phosphatidylcholine and phosphatidylethanolamine, respectively. Spns1 deficiency in cells, zebrafish embryos, and mouse liver resulted in lysosomal accumulation of LPC and LPE species with pathological consequences on lysosomal function. Flux analysis using stable isotope-labeled phospholipid apolipoprotein E nanodiscs targeted to lysosomes showed that LPC was transported out of lysosomes in an Spns1-dependent manner and re-esterified back into the cytoplasmic pools of phosphatidylcholine. Our findings identify a phospholipid salvage pathway from lysosomes to the cytosol that is dependent on Spns1 and critical for maintaining normal lysosomal function.

The Putative Major Facilitator Superfamily (MFS) Protein Named Rv1877 in Mycobacterium tuberculosis Behaves as a Multidrug Efflux Pump.

Efflux pumps are one of the major contributors in the intrinsic multidrug resistance of Mycobacterium tuberculosis. These active transporters, localized in the cytoplasmic membrane, often carry an array of unrelated substances, from toxic substances to metabolites and maintain cellular homeostasis. Rv1877, a putative Major Facilitator Superfamily efflux pump from M. tuberculosis, was investigated in this study. Expression of Rv1877 in Escherichia coli resulted in elevated resistance towards antibiotics of various families. A reversal of this resistance was observed in the presence of sub-inhibitory concentration of the uncoupler carbonyl cyanide-m-chlorophenylhydrazone, indicating its dependence on proton motive force (pmf). Lower intracellular accumulation of the fluoroquinolones ofloxacin and levofloxacin in E. coli cells harbouring Rv1877 implied an active efflux of the drugs. Interestingly, real time, energy-dependent efflux was demonstrated by cells expressing Rv1877 with a lipophilic dye Nile Red. In addition, expression of Rv1877 in trans increased the biofilm formation by the host E. coli cells. Moreover, in silico docking analysis of the molecular interactions between Rv1877 and antibiotics corroborated the experimental observations. Based on the in vivo analyses of Rv1877 in E. coli, it could be designated as a pmf-dependent multidrug transporter with the ability of extruding structurally unrelated antibiotics, preferably some of the fluoroquinolones, and a facilitator of biofilm formation.

Utilization of AlphaFold2 to Predict MFS Protein Conformations after Selective Mutation.

The major facilitator superfamily (MFS) is the largest secondary transporter family and is responsible for transporting a broad range of substrates across the biomembrane. These proteins are involved in a series of conformational changes during substrate transport. To decipher the transport mechanism, it is necessary to obtain structures of these different conformations. At present, great progress has been made in predicting protein structure based on coevolutionary information. In this study, AlphaFold2 was used to predict different conformational structures for 69 MFS transporters of E. coli after the selective mutation of residues at the interface between the N- and C-terminal domains. The predicted structures for these mutants had small RMSD values when compared to structures obtained using X-ray crystallography, which indicates that AlphaFold2 predicts the structure of MSF transporters with high accuracy. In addition, different conformations of other transporter family proteins have been successfully predicted based on mutation methods. This study provides a structural basis to study the transporting mechanism of the MFS transporters and a method to probe dynamic conformation changes of transporter family proteins when performing their function.

Spontaneous Suppressors against Debilitating Transmembrane Mutants of CaMdr1 Disclose Novel Interdomain Communication via Signature Motifs of the Major Facilitator Superfamily.

The Major Facilitator Superfamily (MFS) drug:H+ antiporter CaMdr1, from Candida albicans, is responsible for the efflux of structurally diverse antifungals. MFS members share a common fold of 12–14 transmembrane helices (TMHs) forming two N- and C-domains. Each domain is arranged in a pseudo-symmetric fold of two tandems of 3-TMHs that alternatively expose the drug-binding site towards the inside or the outside of the yeast to promote drug binding and release. MFS proteins show great diversity in primary structure and few conserved signature motifs, each thought to have a common function in the superfamily, although not yet clearly established. Here, we provide new information on these motifs by having screened a library of 64 drug transport-deficient mutants and their corresponding suppressors spontaneously addressing the deficiency. We found that five strains recovered the drug-resistance capacity by expressing CaMdr1 with a secondary mutation. The pairs of debilitating/rescuing residues are distributed either in the same TMH (T127ATMH1- > G140DTMH1) or 3-TMHs repeat (F216ATMH4- > G260ATMH5), at the hinge of 3-TMHs repeats tandems (R184ATMH3- > D235HTMH4, L480ATMH10- > A435TTMH9), and finally between the N- and C-domains (G230ATMH4- > P528HTMH12). Remarkably, most of these mutants belong to the different signature motifs, highlighting a mechanistic role and interplay thought to be conserved among MFS proteins. Results also point to the specific role of TMH11 in the interplay between the N- and C-domains in the inward- to outward-open conformational transition.

An updated subtribal classification of Compositae tribe Anthemideae based on extended phylogenetic reconstructions

An updated subtribal classification of the Compositae–Anthemideae is presented based on gene- and species-tree reconstructions considering sequence variation from the plastid (cpDNA ndhF, trnL-trnF) and nuclear genome (nrDNA ITS1-5.8S-ITS2, ETS; nDNA dual specificity phosphatase DSP, plus-3 domain-containing protein of the vernalization independence 5 complex VIP5, major facilitator superfamily protein of the Nitrate Transporter1/Peptide Transporter NPF3.1) for 113 of the 115 hitherto accepted genera of the tribe. As a result, six subtribes are newly circumscribed, i.e. Anthemidinae (including also Xylanthemum), Artemisiinae (including also Hulteniella, Lepidolopha, Opisthopappus and Tridactylina), Glebionidinae (including also Otoglyphis, Endopappus and Nivellea), Handeliinae (including Cancrinia, Polychrysum and excluding Xylanthemum), Leucantheminae (including also Daveaua, Heteromera and Otospermum) and Leucanthemopsidinae (including Phalacrocarpum). Additionally, five unigeneric subtribes (i.e. Brocchiinae, Inulantherinae, Lepidophorinae, Lonadinae and Vogtiinae) are described as new to science due to their phylogenetically isolated positions, resulting in 19 subtribes accepted for Compositae–Anthemideae. Three new combinations are proposed to accommodate the former Aaronsohnia factorovskyi Warb. & Eig and A. pubescens subsp. maroccana (Ball) Fennane & Ibn Tattou in the genus Otoglyphis Pomel (1874), which was found to have priority over Aaronsohnia Warb. & Eig (1927), and to accommodate the former Tanacetum paleaceum Podlech in the genus Xylanthemum Tzvelev. Citation: Oberprieler C., Töpfer A., Dorfner M., Stock M. & Vogt R. 2022: An updated subtribal classification of Compositae tribe Anthemideae based on extended phylogenetic reconstructions. – Willdenowia 52: 117–149. Version of record first published online on 18 May 2022 ahead of inclusion in April 2022 issue.

AaTAS1 and AaMFS1 Genes for Biosynthesis or Efflux Transport of Tenuazonic Acid and Pathogenicity of Alternaria alternata.

Taking tenuazonic acid (TeA) synthetase 1 (TAS1) in Pyricularia oryzae as a reference, the homolog AaTAS1 was first anchored in Alternaria alternata via de novo sequencing. Subsequently, AaMFS1, as a major facilitator superfamily (MFS) protein-encoding gene in the adjacent upstream region, was followed with interest. As hypothesized, AaTAS1 is required for TeA biosynthesis, while AaMFS1 is an efflux pump for the transmembrane transport of TeA. Comparatively, the TeA yield of ΔAaTAS1 and ΔAaMFS1 dropped significantly compared with that of the wild-type strain. Specifically, the A domain of AaTAS1 catalyzed the start of TeA biosynthesis invitro. Simultaneously, the pathogenicity of ΔAaTAS1 was also significantly decreased. Transcriptome analysis confirmed the abovementioned consistency between the TeA-producing phenotypes and related gene expression. Moreover, the proteins AaTAS1 and AaMFS1 were found present in the cytoplasm, plasma membrane, and intracellular membrane system, respectively, by fluorescence localization. Namely, AaTAS1 was responsible for the biosynthesis of TeA, and AaMFS1 was responsible for the efflux transport of TeA. Certainly, AaTAS1 indirectly regulated the expression of AaMFS1 through the level of synthetic TeA. Overall, data on the novel AaTAS1 and AaMFS1 genes mainly contribute to theoretical advances in mycotoxin biosynthesis and the pathogenicity of phytopathogens to agricultural foods.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Comparative analysis of various root active promoters by evaluation of GUS expression in transgenic Arabidopsis.

To prepare various root active promoters for expressing transgenes and prevent gene silencing caused by the repeated use of the same promoter, the expression characteristics of various root active promoters were comparatively evaluated using GUS as a reporter gene. The high-affinity potassium transporter (HKT1;1), the Shaker family potassium ion channel (SKOR), the Shaker family inward rectifying potassium channel (AKT1), the major facilitator superfamily protein (MFS1), and the senescence associated gene 14 (SAG14) promoter from Arabidopsis (Arabidopsis thaliana) were used, and for comparison, four additional constitutive or green tissue specific promoters in the expression vectors were also employed. As the Gateway cloning technology provided by Invitrogen can offer high efficiency and cloning reliability, and easy manipulation of fusion constructs in vitro, our expression vectors are based on binary (destination) vectors compatible with this cloning technique. These destination vectors are also advantageous for stable expression of the transgene, as the heat shock protein terminator is utilized. The AtHKT1;1, SKOR, AKT1, MFS1 and SAG14 promoters were all active in roots but showed slightly different tissue specificities: AtHKT1;1, SKOR, and MFS1 were dominantly active in vascular bundle tissue, while AtHKT1;1 and MFS1- but not SKOR, AKT1, and SAG14-were active in root tips. SKOR showed the strongest root-specificity, and SAG14 showed the highest activity among the five root active promoters. The activity of MFS was developmentally regulated. These destination vectors are now available to express multiple transgenes in transgenic plants, especially in roots.

Evolutionary conservative analysis revealed novel functional sites in the efflux pump NorA of Staphylococcus aureus.

The NorA antiporter of Staphylococcus aureus belongs to the major facilitator superfamily (MFS) and extrudes various kinds of drugs. With no structure available for this drug efflux pump, the aim of this study was to explore its important structural elements that contribute to substrate binding and drug transport. Evolutionary conservative analyses were conducted on different compilations of NorA homologues to identify conservative motifs and residues. Site-directed mutations were constructed to verify the functional changes in NorA efflux capacities and the conformational changes were further measured by fluorescence resonance energy transfer (FRET) and microscale thermophoresis (MST) analysis. Besides Motif-A, Motif-B and Motif-C that were reported previously in MFS proteins, two other motifs, Motif-1 and Motif-2, were identified in NorA. Site-directed mutations of Motif-1 and Motif-2 as well as 11 predicted binding sites all caused remarkable reductions in drug resistance and efflux activity. Among these, mutant F16A/E222A/F303A/D307A showed an altered binding affinity for tetraphenylphosphonium chloride when measured by MST and Motif-1 mutant G114D/A117E/D118G/V119I and Motif-2 mutant Q325E/G326E/A328E/G330E displayed obvious conformational alterations when compared with the wild-type NorA in the FRET signal spectra. The NorA structure agrees well with the typical structures of MFS proteins, with two newly identified motifs (Motif-1 and Motif-2) that are critical to the structural stability of NorA, and sites F16, E222, F303 and D307 are involved in substrate binding.

Molecular insights into the differential efflux mechanism of Rv1634 protein, a multidrug transporter of major facilitator superfamily in Mycobacterium tuberculosis

Currently, multidrug-resistant tuberculosis (MDR-TB) is a public health crisis and a major health security threat globally. In Mycobacterium tuberculosis (Mtb), major facilitator superfamily (MFS) is the largest group of secondary active transporters. Along with the transport of their natural substrates, MFS proteins were involved in a drug efflux mechanism that ultimately lead to resistance against available anti-TB drugs in Mtb. In the present study, the three-dimensional structure model of an MFS protein, Rv1634, a probable multidrug transporter from Mtb, was generated using homology modeling. The protein structure model was found in inward-open conformation having 14 transmembrane helices. In addition, a central transport channel was deduced across the protein, and a single binding pocket was identified halfway through the central cavity by structural alignment with the homologous protein structures. Further, Rv1634 protein was studied based on the differential structural behavior of apo and ligand-bound forms. All the protein systems were inserted into a phospholipid bilayer to characterize the conformational dynamics of the protein using molecular dynamics (MD) simulations. Detailed analysis of the MD trajectories showed the diverse substrate specificity of the binding pocket for the antibiotics that caused differential movement in the ciprofloxacin and norfloxacin, to which Mtb strains have now become resistant. The expulsion of the drugs outside the bacterial cell occurs through the alternating-access mechanism of N and C-terminal domains, which is intriguing and essential to the understanding the drug resistance mechanism in pathogenic bacteria.

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.