Member Of Major Facilitator Superfamily Research Articles

Discovery of a glutathione utilization pathway in Francisella that shows functional divergence between environmental and pathogenic species

Glutathione (GSH) is an abundant metabolite within eukaryotic cells that can act as a signal, a nutrient source, or serve in a redox capacity for intracellular bacterial pathogens. For Francisella, GSH is thought to be a critical invivo source of cysteine; however, the cellular pathways permitting GSH utilization by Francisella differ between strains and have remained poorly understood. Using genetic screening, we discovered a unique pathway for GSH utilization in Francisella. Whereas prior work suggested GSH catabolism initiates in the periplasm, the pathway we define consists of a major facilitator superfamily (MFS) member that transports intact GSH and a previously unrecognized bacterial cytoplasmic enzyme that catalyzes the first step of GSH degradation. Interestingly, we find that the transporter gene for this pathway is pseudogenized in pathogenic Francisella,explaining phenotypic discrepancies in GSH utilization among Francisella spp. and revealing a critical role for GSH in the environmental niche of these bacteria.

Molecular modeling, simulation and docking of Rv1250 protein from Mycobacterium tuberculosis.

Computational prediction and protein structure modeling have come to the aid of various biological problems in determining the structure of proteins. These technologies have revolutionized the biological world of research, allowing scientists and researchers to gain insights into their biological questions and design experimental research much more efficiently. Pathogenic Mycobacterium spp. is known to stay alive within the macrophages of its host. Mycobacterium tuberculosis is an acid-fast bacterium that is the most common cause of tuberculosis and is considered to be the main cause of resistance of tuberculosis as a leading health issue. The genome of Mycobacterium tuberculosis contains more than 4,000 genes, of which the majority are of unknown function. An attempt has been made to computationally model and dock one of its proteins, Rv1250 (MTV006.22), which is considered as an apparent drug-transporter, integral membrane protein, and member of major facilitator superfamily (MFS). The most widely used techniques, i.e., homology modeling, molecular docking, and molecular dynamics (MD) simulation in the field of structural bioinformatics, have been used in the present work to study the behavior of Rv1250 protein from M. tuberculosis. The structure of unknown TB protein, i.e., Rv1250 was retrived using homology modeling with the help of I-TASSER server. Further, one of the sites responsible for infection was identified and docking was done by using the specific Isoniazid ligand which is an inhibitor of this protein. Finally, the stability of protein model and analysis of stable and static interaction between protein and ligand molecular dynamic simulation was performed at 100ns The designing of novel Rv1250 enzyme inhibitors is likely achievable with the use of proposed predicted model, which could be helpful in preventing the pathogenesis caused by M. tuberculosis. Finally, the MD simulation was done to evaluate the stability of the ligand for the specific protein.

Integrating Hydrogen Deuterium Exchange–Mass Spectrometry with Molecular Simulations Enables Quantification of the Conformational Populations of the Sugar Transporter XylE

A yet unresolved challenge in structural biology is to quantify the conformational states of proteins underpinning function. This challenge is particularly acute for membrane proteins owing to the difficulties in stabilizing them for in vitro studies. To address this challenge, we present an integrative strategy that combines hydrogen deuterium exchange-mass spectrometry (HDX-MS) with ensemble modeling. We benchmark our strategy on wild-type and mutant conformers of XylE, a prototypical member of the ubiquitous Major Facilitator Superfamily (MFS) of transporters. Next, we apply our strategy to quantify conformational ensembles of XylE embedded in different lipid environments. Further application of our integrative strategy to substrate-bound and inhibitor-bound ensembles allowed us to unravel protein-ligand interactions contributing to the alternating access mechanism of secondary transport in atomistic detail. Overall, our study highlights the potential of integrative HDX-MS modeling to capture, accurately quantify, and subsequently visualize co-populated states of membrane proteins in association with mutations and diverse substrates and inhibitors.

Golgi-localized putative S-adenosyl methionine transporters required for plant cell wall polysaccharide methylation.

Polysaccharide methylation, especially that of pectin, is a common and important feature of land plant cell walls. Polysaccharide methylation takes place in the Golgi apparatus and therefore relies on the import of S-adenosyl methionine (SAM) from the cytosol into the Golgi. However, so far, no Golgi SAM transporter has been identified in plants. Here we studied major facilitator superfamily members in Arabidopsis that we identified as putative Golgi SAM transporters (GoSAMTs). Knockout of the two most highly expressed GoSAMTs led to a strong reduction in Golgi-synthesized polysaccharide methylation. Furthermore, solid-state NMR experiments revealed that reduced methylation changed cell wall polysaccharide conformations, interactions and mobilities. Notably, NMR revealed the existence of pectin 'egg-box' structures in intact cell walls and showed that their formation is enhanced by reduced methyl esterification. These changes in wall architecture were linked to substantial growth and developmental phenotypes. In particular, anisotropic growth was strongly impaired in the double mutant. The identification of putative transporters involved in import of SAM into the Golgi lumen in plants provides new insights into the paramount importance of polysaccharide methylation for plant cell wall structure and 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.

Interactions of cytosolic tails in the Jen1 carboxylate transporter are critical for trafficking and transport activity.

Plasma membrane (PM) transporters of the major facilitator superfamily (MFS) are essential for cell metabolism, growth and response to stress or drugs. In Saccharomyces cerevisiae, Jen1 is a monocarboxylate/H+ symporter that provides a model to dissect the molecular details underlying cellular expression, transport mechanism and turnover of MFS transporters. Here, we present evidence revealing novel roles of the cytosolic N- and C-termini of Jen1 in its biogenesis, PM stability and transport activity, using functional analyses of Jen1 truncations and chimeric constructs with UapA, an endocytosis-insensitive transporter of Aspergillus nidulans. Our results show that both N- and C-termini are critical for Jen1 trafficking to the PM, transport activity and endocytosis. Importantly, we provide evidence that Jen1 N- and C-termini undergo transport-dependent dynamic intramolecular interactions, which affect the transport activity and turnover of Jen1. Our results support an emerging concept where the cytoplasmic termini of PM transporters control transporter cell surface stability and function through flexible intramolecular interactions with each other. These findings might be extended to other MFS members to understand conserved and evolving mechanisms underlying transporter structure-function relationships. This article has an associated First Person interview with the first authors of the paper.

Insights into the structure and function of the human organic anion transporter 1 in lipid bilayer membranes

The human SLC22A6/OAT1 plays an important role in the elimination of a broad range of endogenous substances and xenobiotics thus attracting attention from the pharmacological community. Furthermore, OAT1 is also involved in key physiological events such as the remote inter-organ communication. Despite its significance, the knowledge about hOAT1 structure and the transport mechanism at the atomic level remains fragmented owing to the lack of resolved structures. By means of protein-threading modeling refined by μs-scaled Molecular Dynamics simulations, the present study provides the first robust model of hOAT1 in outward-facing conformation. Taking advantage of the AlphaFold 2 predicted structure of hOAT1 in inward-facing conformation, we here provide the essential structural and functional features comparing both states. The intracellular motifs conserved among Major Facilitator Superfamily members create a so-called “charge-relay system” that works as molecular switches modulating the conformation. The principal element of the event points at interactions of charged residues that appear crucial for the transporter dynamics and function. Moreover, hOAT1 model was embedded in different lipid bilayer membranes highlighting the crucial structural dependence on lipid-protein interactions. MD simulations supported the pivotal role of phosphatidylethanolamine components to the protein conformation stability. The present model is made available to decipher the impact of any observed polymorphism and mutation on drug transport as well as to understand substrate binding modes.

Strategies for single-particle cryo-electron microscopy studies of small integral membrane proteins

Membrane proteins comprise nearly half of the drug targets and are the preferred targets for majority of the FDA approved small-molecule drugs. GPCRs, ion channels, and kinases are leading drug targets because of their druggability, and because of availability of high-resolution three-dimensional structures. Solute carriers (SLC) and major facilitator superfamily (MFS) members are linked to more than 100 human diseases and have been pursued as lucrative drug targets. Functional mapping and attempts at structural characterization of SLCs and MFSs belonging to unique transporter classes have enabled structure-based drug design and pharmacological targeting of those families.

Molecular modeling investigation of proton coupling in XylE

Sugar transporters are major facilitator superfamily (MFS) members that play a fundamental role in cell biology: they take up energy into cells in the form of glucose and other hexoses using a rocker-switch alternating access mechanism of transport. XylE is an Escherichia coli homolog that has been often used as a model system to study the molecular basis for sugar transport. In contrast to most known GLUTs, sugar-transport by XylE is proton-coupled. Previous studies have shown that substitution of a single aspartate residue (D27) in TM1 with an asparagine converts XylE into a passive transporter.

The Role of Mfsd2a in Nervous System Diseases.

Major facilitator superfamily (MFS) is the maximum and most diversified membrane transporter, acting as uniporters, symporters and antiporters. MFS is considered to have a good development potential in the transport of drugs for the treatment of brain diseases. The major facilitator superfamily domain containing protein 2a (Mfsd2a) is a member of MFS. Mfsd2a-knockout mice have shown a marked decrease of docosahexaenoic acid (DHA) level in brain, exhibiting neuron loss, microcephaly and cognitive deficits, as DHA acts essentially in brain growth and integrity. Mfsd2a has attracted more and more attention in the study of nervous system diseases because of its critical role in maintaining the integrity of the blood-brain barrier (BBB) and transporting DHA, including inhibiting cell transport in central nervous system endothelial cells, alleviating BBB injury, avoiding BBB injury in cerebral hemorrhage model, acting as a carrier etc. Up to now, the clinical research of Mfsd2a in nervous system diseases is rare. This article reviewed the current research progress of Mfsd2a in nervous system diseases. It summarized the physiological functions of Mfsd2a in the occurrence and development of intracranial hemorrhage (ICH), Alzheimer’s disease (AD), sepsis-associated encephalopathy (SAE), autosomal recessive primary microcephaly (MCPH) and intracranial tumor, aiming to provide ideas for the basic research and clinical application of Mfsd2a.

The membrane transporter lactose permease increases lipid bilayer bending rigidity

Cellular life relies on membranes, which provide a resilient and adaptive cell boundary. Many essential processes depend upon the ease with which the membrane is able to deform and bend, features that can be characterized by the bending rigidity. Quantitative investigations of such mechanical properties of biological membranes have primarily been undertaken in solely lipid bilayers and frequently in the absence of buffers. In contrast, much less is known about the influence of integral membrane proteins on bending rigidity under physiological conditions. We focus on an exemplar member of the ubiquitous major facilitator superfamily of transporters and assess the influence of lactose permease on the bending rigidity of lipid bilayers. Fluctuation analysis of giant unilamellar vesicles (GUVs) is a useful means to measure bending rigidity. We find that using a hydrogel substrate produces GUVs that are well suited to fluctuation analysis. Moreover, the hydrogel method is amenable to both physiological salt concentrations and anionic lipids, which are important to mimic key aspects of the native lactose permease membrane. Varying the fraction of the anionic lipid in the lipid mixture DOPC/DOPE/DOPG allows us to assess the dependence of membrane bending rigidity on the topology and concentration of an integral membrane protein in the lipid bilayer of GUVs. The bending rigidity gradually increases with the incorporation of lactose permease, but there is no further increase with greater amounts of the protein in the membrane.

Structural basis of inhibition of a transporter from Staphylococcus aureus, NorC, through a single-domain camelid antibody

Transporters play vital roles in acquiring antimicrobial resistance among pathogenic bacteria. In this study, we report the X-ray structure of NorC, a 14-transmembrane major facilitator superfamily member that is implicated in fluoroquinolone resistance in drug-resistant Staphylococcus aureus strains, at a resolution of 3.6 Å. The NorC structure was determined in complex with a single-domain camelid antibody that interacts at the extracellular face of the transporter and stabilizes it in an outward-open conformation. The complementarity determining regions of the antibody enter and block solvent access to the interior of the vestibule, thereby inhibiting alternating-access. NorC specifically interacts with an organic cation, tetraphenylphosphonium, although it does not demonstrate an ability to transport it. The interaction is compromised in the presence of NorC-antibody complex, consequently establishing a strategy to detect and block NorC and related transporters through the use of single-domain camelid antibodies.

Structures and General Transport Mechanisms by the Major Facilitator Superfamily (MFS).

The major facilitator superfamily (MFS) is the largest known superfamily of secondary active transporters. MFS transporters are responsible for transporting a broad spectrum of substrates, either down their concentration gradient or uphill using the energy stored in the electrochemical gradients. Over the last 10 years, more than a hundred different MFS transporter structures covering close to 40 members have provided an atomic framework for piecing together the molecular basis of their transport cycles. Here, we summarize the remarkable promiscuity of MFS members in terms of substrate recognition and proton coupling as well as the intricate gating mechanisms undergone in achieving substrate translocation. We outline studies that show how residues far from the substrate binding site can be just as important for fine-tuning substrate recognition and specificity as those residues directly coordinating the substrate, and how a number of MFS transporters have evolved to form unique complexes with chaperone and signaling functions. Through a deeper mechanistic description of glucose (GLUT) transporters and multidrug resistance (MDR) antiporters, we outline novel refinements to the rocker-switch alternating-access model, such as a latch mechanism for proton-coupled monosaccharide transport. We emphasize that a full understanding of transport requires an elucidation of MFS transporter dynamics, energy landscapes, and the determination of how rate transitions are modulated by lipids.

Transcriptome analysis reveals Nitrogen deficiency induced alterations in leaf and root of three cultivars of potato (Solanum tuberosum L.).

Nitrogen (N) is a key element for the production of potato. The N uptake efficiency, N use efficiency and increased N utilization efficiency can be decreased by N deficiency treatment. We performed this study to investigate the association between transcriptomic profiles and the efficiencies of N in potato. Potato cultivars "Yanshu 4" (short for Y), "Xiabodi" (cv. Shepody, short for X) and "Chunshu 4" (short for C) were treated with sufficient N fertilizer and deficient N fertilizer. Then, the growth parameters and tuber yield were recorded; the contents of soluble sugar and protein were measured; and the activities of enzymes were detected. Leaf and root transcriptomes were analyzed and differentially expressed genes (DEGs) in response to N deficiency were identified. The results showed that N deficiency decreased the nitrate reductase (NR), glutamine synthetase (GS) and root activity. Most of the DEGs between N-treated and N-deficiency participate the processes of transport, nitrate transport, nitrogen compound transport and N metabolism in C and Y, not in X, indicating the cultivar-dependent response to N deficiency. DEGs like glutamate dehydrogenase (StGDH), glutamine synthetase (StGS) and carbonic anhydrase (StCA) play key roles in these processes mentioned above. DEGs related to N metabolism showed a close relationship with the N utilization efficiency (UTE), but not with N use efficiency (NUE). The Major Facilitator Superfamily (MFS) members, like nitrate transporter 2.4 (StNRT2.4), 2.5 (StNRT2.5) and 2.7 (StNRT2.7), were mainly enriched in the processes associated with response to stresses and defense, indicating that N deficiency induced stresses in all cultivars.

Transcriptional responses in Parascaris univalens after in vitro exposure to ivermectin, pyrantel citrate and thiabendazole

BackgroundParascaris univalens is a pathogenic parasite of foals and yearlings worldwide. In recent years, Parascaris spp. worms have developed resistance to several of the commonly used anthelmintics, though currently the mechanisms behind this development are unknown. The aim of this study was to investigate the transcriptional responses in adult P. univalens worms after in vitro exposure to different concentrations of three anthelmintic drugs, focusing on drug targets and drug metabolising pathways.MethodsAdult worms were collected from the intestines of two foals at slaughter. The foals were naturally infected and had never been treated with anthelmintics. Worms were incubated in cell culture media containing different concentrations of either ivermectin (10−9 M, 10−11 M, 10−13 M), pyrantel citrate (10−6 M, 10−8 M, 10−10 M), thiabendazole (10−5 M, 10−7 M, 10−9 M) or without anthelmintics (control) at 37 °C for 24 h. After incubation, the viability of the worms was assessed and RNA extracted from the anterior region of 36 worms and sequenced on an Illumina NovaSeq 6000 system.ResultsAll worms were alive at the end of the incubation but showed varying degrees of viability depending on the drug and concentration used. Differential expression (Padj < 0.05 and log2 fold change ≥ 1 or ≤ − 1) analysis showed similarities and differences in the transcriptional response after exposure to the different drug classes. Candidate genes upregulated or downregulated in drug exposed worms include members of the phase I metabolic pathway short-chain dehydrogenase/reductase superfamily (SDR), flavin containing monooxygenase superfamily (FMO) and cytochrome P450-family (CYP), as well as members of the membrane transporters major facilitator superfamily (MFS) and solute carrier superfamily (SLC). Generally, different targets of the anthelmintics used were found to be upregulated and downregulated in an unspecific pattern after drug exposure, apart from the GABA receptor subunit lgc-37, which was upregulated only in worms exposed to 10−9 M of ivermectin.ConclusionsTo our knowledge, this is the first time the expression of lgc-37 and members of the FMO, SDR, MFS and SLC superfamilies have been described in P. univalens and future work should be focused on characterising these candidate genes to further explore their potential involvement in drug metabolism and anthelmintic resistance.

A Novel MFS-MDR Transporter, MdrP, Employs D223 as a Key Determinant in the Na+ Translocation Coupled to Norfloxacin Efflux.

Multidrug resistance (MDR) transporters of the major facilitator superfamily (MFS) were previously believed to drive the extrusion of multiple antimicrobial drugs through the coupling to proton translocation. Here, we present the identification of the first Na+-coupled MFS-MDR transporter, MdrP, which also can achieve H+-coupled drug efflux independently of Na+. Importantly, we propose that MdrP can extrude norfloxacin in a mode of drug/Na+ antiport, which has not yet been reported in any MFS member. On this basis, we further provide the insights into a novel Na+ and H+ coupling mechanism of MFS-MDR transporters, even for all secondary transporters. The most important finding lies in that D223 should mainly act as a key determinant in the Na+ translocation coupled to norfloxacin efflux. Furthermore, our results partially modify the knowledge of the conformational stability-related residues in the motif A of MFS transporters and imply the importance of a new positively charged residue, R361, for the stabilization of outward-facing conformation of MFS transporters. These novel findings positively contribute to the knowledge of MFS-MDR transporters, especially about Na+ and H+ coupling mechanism. This study is based mainly on measurements in intact cells or everted membranes, and a biochemical assay with a reconstituted MdrP protein should be necessary to come to conclusion to be assured.

Arabidopsis PIC30 encodes a major facilitator superfamily transporter responsible for the uptake of picolinate herbicides.

Picloram is an auxinic herbicide that is widely used for controlling broad leaf weeds. However, its mechanism of transport into plants is poorly understood. In a genetic screen for picloram resistance, we identified three Arabidopsis mutant alleles of PIC30 (PICLORAM RESISTANT30) that are specifically resistant to picolinates, but not to other auxins. PIC30 is a previously uncharacterized gene that encodes a major facilitator superfamily (MFS) transporter. Similar to most members of MFS, PIC30 contains 12 putative transmembrane domains, and PIC30-GFP fusion protein selectively localizes to the plasma membrane. In planta transport assays demonstrate that PIC30 specifically transports picloram, but not indole-3-acetic acid (IAA). Functional analysis of Xenopus laevis oocytes injected with PIC30 cRNA demonstrated PIC30 mediated transport of picloram and several anions, including nitrate and chloride. Consistent with these roles of PIC30, three allelic pic30 mutants are selectively insensitive to picolinate herbicides, while pic30-3 is also defective in chlorate (analogue of nitrate) transport and also shows reduced uptake of . Overexpression of PIC30 fully complements both picloram and chlorate insensitive phenotypes of pic30-3. Despite the continued use of picloram as an herbicide, a transporter for picloram was not known until now. This work provides insight into the mechanisms of plant resistance to picolinate herbicides and also shed light on the possible endogenous function of PIC30 protein.

Cultivation at high osmotic pressure confers ubiquinone 8–independent protection of respiration on Escherichia coli

Cultivation at high osmotic pressure confers ubiquinone 8–independent protection of respiration on Escherichia coli

A conserved major facilitator superfamily member orchestrates a subset of O-glycosylation to aid macrophage tissue invasion.

Aberrant display of the truncated core1 O-glycan T-antigen is a common feature of human cancer cells that correlates with metastasis. Here we show that T-antigen in Drosophila melanogaster macrophages is involved in their developmentally programmed tissue invasion. Higher macrophage T-antigen levels require an atypical major facilitator superfamily (MFS) member that we named Minerva which enables macrophage dissemination and invasion. We characterize for the first time the T and Tn glycoform O-glycoproteome of the Drosophila melanogaster embryo, and determine that Minerva increases the presence of T-antigen on proteins in pathways previously linked to cancer, most strongly on the sulfhydryl oxidase Qsox1 which we show is required for macrophage tissue entry. Minerva's vertebrate ortholog, MFSD1, rescues the minerva mutant's migration and T-antigen glycosylation defects. We thus identify a key conserved regulator that orchestrates O-glycosylation on a protein subset to activate a program governing migration steps important for both development and cancer metastasis.

Transporter oligomerisation: roles in structure and function.

Oligomerisation is a key feature of integral membrane transporters with roles in structure, function and stability. In this review, we cover some very recent advances in our understanding of how oligomerisation affects these key transporter features, with emphasis on a few groups of transporters, including the nucleobase ascorbate transporters, neurotransmitter sodium symporters and major facilitator superfamily members.