Modelling,substrate docking and mutational analysis identify residues essential for function and specificity of the major fungal purine transporter AzgA

The AzgA purine/H⁺ symporter of Aspergillus nidulans is the founding member of a functionally and phylogenetically distinct transporter family present in fungi, bacteria and plants. Here a valid AzgA topological model is built based on the crystal structure of the Escherichia coli uracil transporter UraA, a member of the nucleobase‐ascorbate transporter (NAT/NCS2) family. The model consists of 14 transmembrane, mostly α‐helical, segments (TMSs) and cytoplasmic N‐ and C‐tails. A distinct compact core of 8 TMSs, made of two intertwined inverted repeats (TMSs 1–4 and 8–11), is topologically distinct from a flexible domain (TMSs 5–7 and 12–14). A putative substrate binding cavity is visible between the core and the gate domains. Substrate docking, molecular dynamics and mutational analysis identified several residues critical for purine binding and/or transport in TMS3, TMS8 and TMS10. Among these, Asn131 (TMS3), Asp339 (TMS8) and Glu394 (TMS10) are proposed to directly interact with substrates, while Asp342 (TMS8) might be involved in subsequent substrate translocation, through H⁺ binding and symport. Thus, AzgA and other NAT transporters use topologically similar TMSs and amino acid residues for substrate binding and transport, which in turn implies that AzgA‐like proteins constitute a distant subgroup of the ubiquitous NAT family.     

Two functionally distinct members of the MATE (multi‐drug and toxic compound extrusion) family of transporters potentially underlie two major aluminum tolerance QTLs in maize

Crop yields are significantly reduced by aluminum (Al) toxicity on acidic soils, which comprise up to 50% of the world’s arable land. Al‐activated release of ligands (such as organic acids) from the roots is a major Al tolerance mechanism in plants. In maize, Al‐activated root citrate exudation plays an important role in tolerance. However, maize Al tolerance is a complex trait involving multiple genes and physiological mechanisms. Recently, transporters from the MATE family have been shown to mediate Al‐activated citrate exudation in a number of plant species. Here we describe the cloning and characterization of two MATE family members in maize, ZmMATE1 and ZmMATE2, which co‐localize to major Al tolerance QTL. Both genes encode plasma membrane proteins that mediate significant anion efflux when expressed in Xenopus oocytes. ZmMATE1 expression is mostly concentrated in root tissues, is up‐regulated by Al and is significantly higher in Al‐tolerant maize genotypes. In contrast, ZmMATE2 expression is not specifically localized to any particular tissue and does not respond to Al. [¹⁴C]‐citrate efflux experiments in oocytes demonstrate that ZmMATE1 is a citrate transporter. In addition, ZmMATE1 expression confers a significant increase in Al tolerance in transgenic Arabidopsis. Our data suggests that ZmMATE1 is a functional homolog of the Al tolerance genes recently characterized in sorghum, barley and Arabidopsis, and is likely to underlie the largest maize Al tolerance QTL found on chromosome 6. However, ZmMATE2 most likely does not encode a citrate transporter, and could be involved in a novel Al tolerance mechanism.     

Handedness preference and switching of peptide helices. Part I: Helices based on protein amino acids

In this article, we review the relevant results obtained during almost 60 years of research on a specific aspect of stereochemistry, namely handedness preference and switches between right‐handed and left‐handed helical peptide structures generated by protein amino acids or appropriately designed, side‐chain modified analogs. In particular, we present and discuss here experimental and theoretical data on three categories of those screw‐sense issues: (i) right‐handed/left‐handed α‐helix transitions underwent by peptides rich in Asp, specific Asp β‐esters, and Asn; (ii) comparison of the preferred conformations adopted by helical host–guest peptide series, each characterized by an amino acid residue (e.g. Ile or its diastereomer aIle) endowed with two chiral centers in its chemical structure; and (iii) right‐handed (type I)/left‐handed (type II) poly‐(Pro)_n helix transitions monitored for peptides rich in Pro itself or its analogs with a pyrrolidine ring substitution, particularly at the biologically important position 4. The unique modular and chiral properties of peptides, combined with their relatively easy synthesis, the chance to shape them into the desired conformation, and the enormous chemical diversity of their coded and non‐coded α‐amino acid building blocks, offer a huge opportunity to structural chemists for applications to bioscience and nanoscience problems. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

BAT1, a bidirectional amino acid transporter in <Emphasis Type="Italic">Arabidopsis</Emphasis>

The Arabidopsis thaliana At2g01170 gene is annotated as a putative gamma amino butyric acid (GABA) permease based on its sequence similarity to a yeast GABA transporting gene (UGA4). A cDNA of At2g01170 was expressed in yeast and analyzed for amino acid transport activity. Both direct measurement of amino acid transport and yeast growth experiments demonstrated that the At2g01170 encoded-protein exhibits transport activity for alanine, arginine, glutamate and lysine, but not for GABA or proline. Significantly, unlike other amino acid transporters described in plants to date, At2g01170 displayed both export and import activity. Based on that observation, it was named bidirectional amino acid transporter 1 (BAT1). Sequence comparisons show BAT1 is not a member of any previously defined amino acid transporter family. It does share, however, several conserved protein domains found in a variety of prokaryotic and eukaryotic amino acid transporters, suggesting membership in an ancient family of transporters. BAT1 is a single copy gene in the Arabidopsis genome, and its mRNA is ubiquitously expressed in all organs. A transposon—GUS gene-trap insert in the BAT1 gene displays GUS localization in the vascular tissues (Dundar in Ann Appl Biol, 2009) suggesting BAT1 may function in amino acid export from the phloem into sink tissues.     

Disruption of the OLE ribonucleoprotein complex causes magnesium toxicity in Bacillus halodurans

OLE RNAs represent an unusual class of bacterial noncoding RNAs common in Gram‐positive anaerobes. The OLE RNA of the alkaliphile Bacillus halodurans is highly expressed and naturally interacts with at least two RNA‐binding proteins called OapA and OapB. The phenotypes of the corresponding knockouts include growth inhibition when exposed to ethanol or other short‐chain alcohols or when incubated at modestly reduced temperatures (e.g. 20°C). Intriguingly, the OapA ‘PM1’ mutant, which carries two amino acid changes to a highly conserved region, yields a dominant‐negative phenotype that causes more severe growth defects under these same stress conditions. Herein, we report that the PM1 strain also exhibits extreme sensitivity to elevated Mg²⁺ concentrations, beginning as low as 2 mM. Suppressor mutants predominantly map to genes for aconitate hydratase and isocitrate dehydrogenase, which are expected to alter cellular citrate concentrations. Citrate reduces the severity of the Mg²⁺ toxicity phenotype, but neither the genomic mutations nor the addition of citrate to the medium overcomes ethanol toxicity or temperature sensitivity. These findings reveal that OLE RNA and its protein partners are involved in biochemical responses under several stress conditions, wherein the unusual sensitivity to Mg²⁺ can be independently suppressed by specific genomic mutations.     

New Glycoprotein-Associated Amino Acid Transporters

The L-type amino acid transporter LAT1 has recently been identified as being a disulfide-linked ``light chain' of the ubiquitously expressed glycoprotein 4F2hc/CD98. Several LAT1-related transporters have been identified, which share the same putative 12-transmembrane segment topology and also associate with the single transmembrane domain 4F2hc protein. They display differing amino acid substrate specificities, transport kinetics and localizations such as, for instance, y⁺LAT1 which is localized at the basolateral membrane of transporting epithelia, and the defect of which causes lysinuric protein intolerance. The b^0,+AT transporter which associates with the 4F2hc-related rBAT protein to form the luminal high-affinity diamino acid transporter defective in cystinuria, belongs to the same family of glycoprotein-associated amino acid transporters (gpaATs). These glycoprotein-associated transporters function as amino acid exchangers. They extend the specificity range of vectorial amino acid transport when located in the same membrane as carriers that unidirectionally transport one of the exchanged substrates. gpaATs belong to a phylogenetic cluster within the amino acid/polyamine/choline (APC) superfamily of transporters. This cluster, which we designate the LAT family (named after its first vertebrate member), includes some members from nematodes, yeast and bacteria. The latter of these proteins presumably lack association with a second subunit. In this review, we focus on the animal members of the LAT cluster that form, together with some of the nematode members, the family of glycoprotein-associated amino acid transporters (gpaAT family). Received: 20 July 1999/Revised: 7 September 1999     

Structural studies of bovine,equine, and leporine serum albumin complexes with naproxen

Serum albumin, a protein naturally abundant in blood plasma, shows remarkable ligand binding properties of numerous endogenous and exogenous compounds. Most of serum albumin binding sites are able to interact with more than one class of ligands. Determining the protein‐ligand interactions among mammalian serum albumins is essential for understanding the complexity of this transporter. We present three crystal structures of serum albumins in complexes with naproxen (NPS): bovine (BSA‐NPS), equine (ESA‐NPS), and leporine (LSA‐NPS) determined to 2.58 Å (C2), 2.42 Å (P6₁), and 2.73 Å (P2₁2₁2₁) resolutions, respectively. A comparison of the structurally investigated complexes with the analogous complex of human serum albumin (HSA‐NPS) revealed surprising differences in the number and distribution of naproxen binding sites. Bovine and leporine serum albumins possess three NPS binding sites, but ESA has only two. All three complexes of albumins studied here have two common naproxen locations, but BSA and LSA differ in the third NPS binding site. None of these binding sites coincides with the naproxen location in the HSA‐NPS complex, which was obtained in the presence of other ligands besides naproxen. Even small differences in sequences of serum albumins from various species, especially in the area of the binding pockets, influence the affinity and the binding mode of naproxen to this transport protein. Proteins 2014; 82:2199–2208. © 2014 Wiley Periodicals, Inc.     

The Escherichia coli SLC26 homologue YchM (DauA) is a C4‐dicarboxylic acid transporter

The SLC26/SulP (solute carrier/sulphate transporter) proteins are a ubiquitous superfamily of secondary anion transporters. Prior studies have focused almost exclusively on eukaryotic members and bacterial members are frequently classified as sulphate transporters based on their homology with SulP proteins from plants and fungi. In this study we have examined the function and physiological role of the Escherichia coli Slc26 homologue, YchM. We show that there is a clear YchM‐dependent growth defect when succinate is used as the sole carbon source. Using an in vivo succinate transport assay, we show that YchM is the sole aerobic succinate transporter active at acidic pH. We demonstrate that YchM can also transport other C₄‐dicarboxylic acids and that its substrate specificity differs from the well‐characterized succinate transporter, DctA. Accordingly ychM was re‐designated dauA (dicarboxylic acid uptake system A). Finally, our data suggest that DauA is a protein with transport and regulation activities. This is the first report that a SLC26/SulP protein acts as a C₄‐dicarboxylic acid transporter and an unexpected new function for a prokaryotic member of this transporter family.     

Structure,function, and expression pattern of a novel sodium-coupled citrate transporter (NaCT) cloned from mammalian brain

Citrate plays a pivotal role not only in the generation of metabolic energy but also in the synthesis of fatty acids, isoprenoids, and cholesterol in mammalian cells. Plasma levels of citrate are the highest ( approximately 135 microm) among the intermediates of the tricarboxylic acid cycle. Here we report on the cloning and functional characterization of a plasma membrane transporter (NaCT for Na+ -coupled citrate transporter) from rat brain that mediates uphill cellular uptake of citrate coupled to an electrochemical Na+ gradient. NaCT consists of 572 amino acids and exhibits structural similarity to the members of the Na+-dicarboxylate cotransporter/Na+ -sulfate cotransporter (NaDC/NaSi) gene family including the recently identified Drosophila Indy. In rat, the expression of NaCT is restricted to liver, testis, and brain. When expressed heterologously in mammalian cells, rat NaCT mediates the transport of citrate with high affinity (Michaelis-Menten constant, approximately 20 microm) and with a Na+:citrate stoichiometry of 4:1. The transporter does interact with other dicarboxylates and tricarboxylates but with considerably lower affinity. In mouse brain, the expression of NaCT mRNA is evident in the cerebral cortex, cerebellum, hippocampus, and olfactory bulb. NaCT represents the first transporter to be identified in mammalian cells that shows preference for citrate over dicarboxylates. This transporter is likely to play an important role in the cellular utilization of citrate in blood for the synthesis of fatty acids and cholesterol (liver) and for the generation of energy (liver and brain). NaCT thus constitutes a potential therapeutic target for the control of body weight, cholesterol levels, and energy homeostasis.     

Modelling and mutational evidence identify the substrate binding site and functional elements in APC amino acid transporters

The Amino acid-Polyamine-Organocation (APC) superfamily is the main family of amino acid transporters found in all domains of life and one of the largest families of secondary transporters. Here, using a sensitive homology threading approach and modelling we show that the predicted structure of APC members is extremely similar to the crystal structures of several prokaryotic transporters belonging to evolutionary distinct protein families with different substrate specificities. All of these proteins, despite having no primary amino acid sequence similarity, share a similar structural core, consisting of two V-shaped domains of five transmembrane domains each, intertwined in an antiparallel topology. Based on this model, we reviewed available data on functional mutations in bacterial, fungal and mammalian APCs and obtained novel mutational data, which provide compelling evidence that the amino acid binding pocket is located in the vicinity of the unwound part of two broken helices, in a nearly identical position to the structures of similar transporters. Our analysis is fully supported by the evolutionary conservation and specific amino acid substitutions in the proposed substrate binding domains. Furthermore, it allows predictions concerning residues that might be crucial in determining the specificity profile of APC members. Finally, we show that two cytoplasmic loops constitute important functional elements in APCs. Our work along with different kinetic and specificity profiles of APC members in easily manipulated bacterial and fungal model systems could form a unique framework for combining genetic, in-silico and structural studies, for understanding the function of one of the most important transporter families.     

An amino acid code for β‐sheet packing structure

To understand the relationship between protein sequence and structure, this work extends the knob‐socket model in an investigation of β‐sheet packing. Over a comprehensive set of β‐sheet folds, the contacts between residues were used to identify packing cliques: sets of residues that all contact each other. These packing cliques were then classified based on size and contact order. From this analysis, the two types of four‐residue packing cliques necessary to describe β‐sheet packing were characterized. Both occur between two adjacent hydrogen bonded β‐strands. First, defining the secondary structure packing within β‐sheets, the combined socket or XY:HG pocket consists of four residues i, i+2 on one strand and j, j+2 on the other. Second, characterizing the tertiary packing between β‐sheets, the knob‐socket XY:H+B consists of a three‐residue XY:H socket (i, i+2 on one strand and j on the other) packed against a knob B residue (residue k distant in sequence). Depending on the packing depth of the knob B residue, two types of knob‐sockets are found: side‐chain and main‐chain sockets. The amino acid composition of the pockets and knob‐sockets reveal the sequence specificity of β‐sheet packing. For β‐sheet formation, the XY:HG pocket clearly shows sequence specificity of amino acids. For tertiary packing, the XY:H+B side‐chain and main‐chain sockets exhibit distinct amino acid preferences at each position. These relationships define an amino acid code for β‐sheet structure and provide an intuitive topological mapping of β‐sheet packing. Proteins 2014; 82:2128–2140. © 2014 Wiley Periodicals, Inc.     

Evidence for the transport of neutral as well as cationic amino acids by ATA3, a novel and liver-specific subtype of amino acid transport system A

We report here on the cloning and functional characterization of the third subtype of amino acid transport system A, designated ATA3 (amino acid transporter A3), from a human liver cell line. This transporter consists of 547 amino acids and is structurally related to the members of the glutamine transporter family. The human ATA3 (hATA3) exhibits 88% identity in amino acid sequence with rat ATA3. The gene coding for hATA3 contains 16 exons and is located on human chromosome 12q13. It is expressed almost exclusively in the liver. hATA3 mediates the transport of neutral amino acids including α-(methylamino)isobutyric acid (MeAIB), the model substrate for system A, in a Na⁺-coupled manner and the transport of cationic amino acids in a Na⁺-independent manner. The affinity of hATA3 for cationic amino acids is higher than for neutral amino acids. The transport function of hATA3 is thus similar to that of system y⁺L. The ability of hATA3 to transport cationic amino acids with high affinity is unique among the members of the glutamine transporter family. hATA1 and hATA2, the other two known members of the system A subfamily, show little affinity toward cationic amino acids. hATA3 also differs from hATA1 and hATA2 in exhibiting low affinity for MeAIB. Since liver does not express any of the previously known high-affinity cationic amino acid transporters, ATA3 is likely to provide the major route for the uptake of arginine in this tissue.     

Digestive capacities,inbreeding and growth capacities in juvenile Arctic charr Salvelinus alpinus

Genetic variation in growth performance was estimated in 26 families from two commercial strains of Arctic charr Salvelinus alpinus. Physiological determinants of growth and metabolic capacities were also assessed through enzymatic assays. A relatedness coefficient was attributed to each family using parental genotypes at seven microsatellite loci. After 15 months of growth, faster growing families had significantly lower relatedness coefficients than slower growing families, suggesting their value as indicators of growth potential. Individual fish that exhibited higher trypsin activity also displayed higher growth rate, suggesting that superior protein digestion capacities can be highly advantageous at early stages. Capacities to use amino acids as expressed by glutamate dehydrogenase (GDH) activities were lower in the liver of fast‐growing fish (13–20%), whereas white muscle of fast‐growing fish showed higher activities than that of slow‐growing fish for amino acid metabolism and aerobic capacity [22–32% increase for citrate synthase (CS), aspartate aminotransferase (AAT) and GDH]. The generally higher glycolytic capacities (PK and LDH) in white muscle of fast‐growing fish indicated higher burst swimming capacities and hence better access to food.     

Interactions between hydrogen sulphide and nitric oxide regulate two soybean citrate transporters during the alleviation of aluminium toxicity

Hydrogen sulphide (H₂S) is emerging as an important signalling molecule involved in plant resistance to various stresses. However, the underlying mechanism of H₂S in aluminium (Al) resistance and the crosstalk between H₂S and nitric oxide (NO) in Al stress signalling remain elusive. Citrate secretion is a wide‐spread strategy for plants against Al toxicity. Here, two citrate transporter genes, GmMATE13 and GmMATE47, were identified and characterized in soybean. Functional analysis in Xenopus oocytes and transgenic Arabidopsis showed that GmMATE13 and GmMATE47 mediated citrate exudation and enhanced Al resistance. Al treatment triggered H₂S generation and citrate exudation in soybean roots. Pretreatment with an H₂S donor significantly elevated Al‐induced citrate exudation, reduced Al accumulation in root tips, and alleviated Al‐induced inhibition of root elongation, whereas application of an H₂S scavenger elicited the opposite effect. Furthermore, H₂S and NO mediated Al‐induced GmMATE expression and plasma membrane (PM) H⁺‐ATPase activity and expression. Further investigation showed that NO induced H₂S production by regulating the key enzymes involved in biosynthesis and degradation of H₂S. These findings indicate that H₂S acts downstream of NO in mediating Al‐induced citrate secretion through the upregulation of PM H⁺‐ATPase‐coupled citrate transporter cotransport systems, thereby conferring plant resistance to Al toxicity.