Nomenclature of the GLUT/SLC2A family of sugar/polyol transport facilitators

The recent identification of several additional members of the family of sugar transport facilitators (gene symbol SLC2A, protein symbol GLUT) has created a heterogeneous and, in part, confusing nomenclature. Therefore, this letter provides a summary of the family members and suggests a systematic nomenclature for SLC2A and GLUT symbols.

The extended GLUT-family of sugar/polyol transport facilitators: nomenclature, sequence characteristics, and potential function of its novel members (review).

During the last 2 years, several novel genes that encode glucose transporter-like proteins have been identified and characterized. Because of their sequence similarity with GLUT1, these genes appear to belong to the family of solute carriers 2A (SLC2A, protein symbol GLUT). Sequence comparisons of all 13 family members allow the definition of characteristic sugar/polyol transporter signatures: (1) the presence of 12 membrane-spanning helices, (2) seven conserved glycine residues in the helices, (3) several basic and acidic residues at the intracellular surface of the proteins, (4) two conserved tryptophan residues, and (5) two conserved tyrosine residues. On the basis of sequence similarities and characteristic elements, the extended GLUT family can be divided into three subfamilies, namely class I (the previously known glucose transporters GLUT1-4), class II (the previously known fructose transporter GLUT5, the GLUT7, GLUT9 and GLUT11), and class III (GLUT6, 8, 10, 12, and the myo-inositol transporter HMIT1). Functional characteristics have been reported for some of the novel GLUTs. Like GLUT1-4, they exhibit a tissue/cell-specific expression (GLUT6, leukocytes, brain; GLUT8, testis, blastocysts, brain, muscle, adipocytes; GLUT9, liver, kidney; GLUT10, liver, pancreas; GLUT11, heart, skeletal muscle). GLUT6 and GLUT8 appear to be regulated by sub-cellular redistribution, because they are targeted to intra-cellular compartments by dileucine motifs in a dynamin dependent manner. Sugar transport has been reported for GLUT6, 8, and 11; HMIT1 has been shown to be a H+/myo-inositol co-transporter. Thus, the members of the extended GLUT family exhibit a surprisingly diverse substrate specificity, and the definition of sequence elements determining this substrate specificity will require a full functional characterization of all members.

Nomenclature of the GLUT/SLC2A family of sugar/polyol transport facilitators.

The recent identification of several additional members of the family of sugar transport facilitators (gene symbol SLC2A, protein symbol GLUT) has created a heterogeneous and, in part, confusing nomenclature. Therefore, this letter provides a summary of the family members and suggests a systematic nomenclature for SLC2A and GLUT symbols.

Lipid, sugar and liposaccharide based delivery systems

Although there are formidable barriers to the oral delivery of biologically active drugs, considerable progress in the field has been made, using both physical and chemical strategies of absorption enhancement. A possible method to enhance oral absorption is to exploit the phenomenon of lipophilic modification and mono and oligosaccharide conjugation. Depending on the uptake mechanism targeted, different modifications can be employed. To target passive diffusion, lipid modification has been used, whereas the targeting of sugar transport systems has been achieved through drugs conjugated with sugars. These drug delivery units can be specifically tailored to transport a wide variety of poorly absorbed drugs through the skin, and across the barriers that normally inhibit absorption from the gut or into the brain. The delivery system can be conjugated to the drug in such a way as to release the active compound after it has been absorbed (i.e. the drug becomes a prodrug), or to form a biologically stable and active molecule (i.e. the conjugate becomes a new drug moiety). Examples where lipid, sugar and lipid-sugar conjugates have resulted in enhanced drug delivery will be highlighted in this review.

Conformational changes couple Na+ and glucose transport

The mechanism by which cotransport proteins couple their substrates across cell membranes is not known. A commonly proposed model is that cotransport results from ligand-induced conformational transitions that change the accessibility of ligand-binding sites from one side of the membrane to the other. To test this model, we have measured the accessibility of covalent probes to a cysteine residue (Q457C) placed in the putative sugar-translocation domain of the Na+/glucose cotransporter (SGLT1). The mutant protein Q457C was able to transport sugar, but transport was abolished after alkylation by methanethiosulfonate reagents. Alkylation blocked sugar translocation but not sugar binding. Accessibility of Q457C to alkylating reagents required external Na+ and was blocked by external sugar and phlorizin. The voltage dependence of accessibility was directly correlated with the presteady–state charge movement of SGLT1. Voltage-jump experiments with rhodamine-6-maleimide-labeled Q457C showed that the time course and level of changes in fluorescence closely followed the presteady–state charge movement. We conclude that conformational changes are responsible for the coupling of Na+ and sugar transport and that Q457 plays a critical role in sugar translocation by SGLT1.

Stepwise functional evolution in the Ffz sugar transporter family

Abstract of the poster presented at the Microbiotec’15 Congress, 10-12 December 2015, University of Évora, Portugal.

The expression in saccharomyces cerevisiae of a glucose/xylose symporter from Candida intermedia is affected by the presence of a glucose/xylose facilitator

Microbiology, 154

The influence of kaolin application on key metabolic pathways associated with grape berry quality: a molecular and biochemical analysis

Dissertação de mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em Plantas

Genome sequence of the pea aphid Acyrthosiphon pisum

Aphids are important agricultural pests and also biological models for studies of insect-plant interactions, symbiosis, virus vectoring, and the developmental causes of extreme phenotypic plasticity. Here we present the 464 Mb draft genome assembly of the pea aphid Acyrthosiphon pisum. This first published whole genome sequence of a basal hemimetabolous insect provides an outgroup to the multiple published genomes of holometabolous insects. Pea aphids are host-plant specialists, they can reproduce both sexually and asexually, and they have coevolved with an obligate bacterial symbiont. Here we highlight findings from whole genome analysis that may be related to these unusual biological features. These findings include discovery of extensive gene duplication in more than 2000 gene families as well as loss of evolutionarily conserved genes. Gene family expansions relative to other published genomes include genes involved in chromatin modification, miRNA synthesis, and sugar transport. Gene losses include genes central to the IMD immune pathway, selenoprotein utilization, purine salvage, and the entire urea cycle. The pea aphid genome reveals that only a limited number of genes have been acquired from bacteria; thus the reduced gene count of Buchnera does not reflect gene transfer to the host genome. The inventory of metabolic genes in the pea aphid genome suggests that there is extensive metabolite exchange between the aphid and Buchnera, including sharing of amino acid biosynthesis between the aphid and Buchnera. The pea aphid genome provides a foundation for post-genomic studies of fundamental biological questions and applied agricultural problems.

Glucosamine resistance in the yeast, Saccharomyces cerivisae [sic]

By using glucosamine resistant mutants of Saccharomyces ceriv~sa~ an attempt was made to discover the mechanisms which cause glucose repression and/or the Crabtree effect. The strains used are 4B2, GR6, lOP3r, GR8l and GRI08. 4B2 is a wild type yeast while the others are its mutants. To characterize the biochemical reactions which made these mutants resistant to glucosamine poisoning the following experiments were done~ 1. growth and respiration; 2. transport of sugars; 3. effect of inorganic phosphate (Pi): 4. Hexokinase; 5. In yivo phosphorylation. From the above experiments the following conclusions may be drawn: (i) GR6 and lOP3r have normal respiratory and fermentative pathways. These mutants are resistant to glucosamine poisoning due to a slow rate of sugar transport which is due to change in the cell membrane. (ii) GR8l has a normal respiratory pathway. The slow growth on fermentable carbon sourCEE indicates that in GR8l the lesion is in or associated with the glycolytic pathway. The lower rate of sugar transport may be due to a change in energy metabolism. The invivo phosphorylation rate indicates that in GR81 facilitated diffusion is the dominant transport mechanism. (iii) GR108 msa normal glycolytic pathway but the respiratory pathway is abnormal. The slow rate of sugar transport is due to a change in energy metabolism. The lower percentage of in vivo phosphorylation is probably due to a lowered availability of ATP because of the mitochondrial lesion. In all mutants resistance to glucosamine poisoning is due to a lower rate of utilization of ATP. which is caused by various mechanisms (see above), making less ADP available for phosphorylation via ATP synthase which utilizes inorganic phosphate. Because of the lower utilization of Pi, the concentration of intra-mitochondrial Pi does not go down thus protecting mutants from glucosamine poisoning.

Caractérisation du co-transporteur Na+/myo-inositol SMIT2 dans les membranes en bordure en brosse de rein de lapin et d’intestin de rat

Le myo-inositol (MI) est un soluté organique impliqué dans diverses fonctions physiologiques de la cellule dont la signalisation cellulaire. Il est également un osmolyte compatible reconnu. Trois co-transporteurs de type actif secondaire responsables de son absorption ont été identifiés. Deux d’entre eux sont couplés au transport du sodium (SMIT1 et SMIT2) et le troisième est couplé au transport de protons (HMIT). L’objectif de cette étude a été la caractérisation du transport du MI par SMIT2 dans des membranes en bordure en brosse (BBMv) issues du rein de lapin et de l’intestin de rat ainsi qu’après expression dans les ovocytes de Xenopus laevis. La quantification de l’ARNm de SMIT1 et de SMIT2 dans le rein nous a appris que SMIT1 est majoritairement présent dans la médullaire alors que SMIT2 est principalement localisé dans le cortex. Ces résultats ont été confirmés par immunobuvardage en utilisant un anticorps dirigé contre SMIT2. Grâce à l’inhibition sélective de SMIT1 par le L-Fucose et de SMIT2 par le D-chiro-inositol (DCI), nous avons démontré que SMIT2 semble le seul responsable du transport luminal de MI dans le tubule contourné proximal avec un Km de 57 ± 14 µM. Pour ce qui est de l’intestin, des études de transport de MI radioactif ont démontré une absence de transport de MI chez le lapin alors que l’intestin de rat présente un transport de MI très actif. Une quantification par qRT-PCR nous a permis de constater que l’intestin de lapin ne semble pas posséder les transporteurs de MI nécessaires. Comme pour le rein, SMIT2 semble le seul transporteur de MI présent au niveau du pôle apical des entérocytes intestinaux chez le rat. Il est chargé du prélèvement du MI de l'alimentation avec un Km de 150 ± 40 µM. Les analyses fonctionnelles exécutées sur SMIT2 de rat en électrophysiologie après expression dans les ovocytes de Xenopus laevis donnent sensiblement les mêmes résultats que pour les BBMv de rein de lapin et d’intestin de rat. Dans les ovocytes, SMIT2 présente une grande affinité pour le MI (270 ± 19 µM) et le DCI (310 ± 60 µM) et aucune affinité pour le L-fucose. Il est ii également très sensible à la phlorizine (16 ± 7 µM). Une seule exception persiste : la constante d’affinité pour le glucose dans les BBMv d’intestin de rat est 40 fois plus petite que celle observée sur les ovocytes de Xenopus laevis. Nous avons également testé la capacité de certains transporteurs de sucre présents à la surface des membranes apicales des entérocytes à prélever le MI. Vu que l'inhibition de ces transporteurs (SGLT1 et GLUT5) ne changeait rien au taux de MI radioactif transporté, nous en avons conclu qu'ils ne sont pas impliqués dans son transport. Finalement, l’efflux de MI à partir du pôle basolatéral des entérocytes n’est pas effectué par GLUT2 puisque ce dernier lorsqu'il est exprimé dans des ovocytes, est incapable de transporter le MI.

Molecular cloning and comparative analysis of four beta-galactosidase genes from Bifidobacterium bifidum NCIMB41171

Bifidobacterium bifidum NCIMB41171 carries four genes encoding different beta-galactosidases. One of them, named bbgIII, consisted of an open reading frame of 1,935 amino acid (a.a.) residues encoding a protein with a multidomain structure, commonly identified on cell wall bound enzymes, having a signal peptide, a membrane anchor, FIVAR domains, immunoglobulin Ig-like and discoidin-like domains. The other three genes, termed bbgI, bbgII and bbgIV, encoded proteins of 1,291, 689 and 1,052 a.a. residues, respectively, which were most probably intracellularly located. Two cases of protein evolution between strains of the same species were identified when the a.a. sequences of the BbgI and BbgIII were compared with homologous proteins from B. bifidum DSM20215. The homologous proteins were found to be differentiated at the C-terminal a.a. part either due to a single nucleotide insertion or to a whole DNA sequence insertion, respectively. The bbgIV gene was located in a gene organisation surrounded by divergently transcribed genes putatively for sugar transport (galactoside-symporter) and gene regulation (LacI-transcriptional regulator), a structure that was found to be highly conserved in B. longum, B. adolescentis and B. infantis, suggesting optimal organisation shared amongst those species.

Genomic DNA microarray comparison of gene expression patterns in Paracoccidioides brasiliensis mycelia and yeasts in vitro

Paracoccidioides brasiliensis is a thermally dimorphic fungus, and causes the most prevalent systemic mycosis in Latin America. Infection is initiated by inhalation of conidia or mycelial fragments by the host, followed by further differentiation into the yeast form. Information regarding gene expression by either form has rarely been addressed with respect to multiple time points of growth in culture. Here, we report on the construction of a genomic DNA microarray, covering approximately 25% of the genome of the organism, and its utilization in identifying genes and gene expression patterns during growth in vitro. Cloned, amplified inserts from randomly sheared genomic DNA (gDNA) and known control genes were printed onto glass slides to generate a microarray of over 12 000 elements. To examine gene expression, mRNA was extracted and amplified from mycelial or yeast cultures grown in semi-defined medium for 5, 8 and 14 days. Principal components analysis and hierarchical clustering indicated that yeast gene expression profiles differed greatly from those of mycelia, especially at earlier time points, and that mycelial gene expression changed less than gene expression in yeasts over time. Genes upregulated in yeasts were found to encode proteins shown to be involved in methionine/cysteine metabolism, respiratory and metabolic processes (of sugars, amino acids, proteins and lipids), transporters (small peptides, sugars, ions and toxins), regulatory proteins and transcription factors. Mycelial genes involved in processes such as cell division, protein catabolism, nucleotide biosynthesis and toxin and sugar transport showed differential expression. Sequenced clones were compared with Histoplasma capsulatum and Coccidioides posadasii genome sequences to assess potentially common pathways across species, such as sulfur and lipid metabolism, amino acid transporters, transcription factors and genes possibly related to virulence. We also analysed gene expression with time in culture and found that while transposable elements and components of respiratory pathways tended to increase in expression with time, genes encoding ribosomal structural proteins and protein catabolism tended to sharply decrease in expression over time, particularly in yeast. These findings expand our knowledge of the different morphological forms of P. brasiliensis during growth in culture.

Topology of allosteric regulation of lactose permease

Sugar transport by some permeases in Escherichia coli is allosterically regulated by the phosphorylation state of the intracellular regulatory protein, enzyme IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system. A sensitive radiochemical assay for the interaction of enzyme IIAglc with membrane-associated lactose permease was used to characterize the binding reaction. The binding is stimulated by transportable substrates such as lactose, melibiose, and raffinose, but not by sugars that are not transported (maltose and sucrose). Treatment of lactose permease with N-ethylmaleimide, which blocks ligand binding and transport by alkylating Cys-148, also blocks enzyme IIAglc binding. Preincubation with the substrate analog β-d-galactopyranosyl 1-thio-β-d-galactopyranoside protects both lactose transport and enzyme IIAglc binding against inhibition by N-ethylmaleimide. A collection of lactose permease replacement mutants at Cys-148 showed, with the exception of C148V, a good correlation of relative transport activity and enzyme IIAglc binding. The nature of the interaction of enzyme IIAglc with the cytoplasmic face of lactose permease was explored. The N- and C-termini, as well as five hydrophilic loops in the permease, are exposed on the cytoplasmic surface of the membrane and it has been proposed that the central cytoplasmic loop of lactose permease is the major determinant for interaction with enzyme IIAglc. Lactose permease mutants with polyhistidine insertions in cytoplasmic loops IV/V and VI/VII and periplasmic loop VII/VIII retain transport activity and therefore substrate binding, but do not bind enzyme IIAglc, indicating that these regions of lactose permease may be involved in recognition of enzyme IIAglc. Taken together, these results suggest that interaction of lactose permease with substrate promotes a conformational change that brings several cytoplasmic loops into an arrangement optimal for interaction with the regulatory protein, enzyme IIAglc. A topological map of the proposed interaction is presented.

Transcriptome, carbohydrate, and phytohormone analysis of Petunia hybrida reveals a complex disturbance of plant functional integrity under mild chilling stress

Cultivation of chilling-tolerant ornamental crops at lower temperature could reduce the energy demands of heated greenhouses. To provide a better understanding of how sub-optimal temperatures (12 degrees C vs. 16 degrees C) affect growth of the sensitive Petunia hybrida cultivar 'SweetSunshine Williams', the transcriptome, carbohydrate metabolism, and phytohormone homeostasis were monitored in aerial plant parts over 4 weeks by use of a microarray, enzymatic assays and GC-MS/MS. The data revealed three consecutive phases of chilling response. The first days were marked by a strong accumulation of sugars, particularly in source leaves, preferential up-regulation of genes in the same tissue and down-regulation of several genes in the shoot apex, especially those involved in the abiotic stress response. The midterm phase featured a partial normalization of carbohydrate levels and gene expression. After 3 weeks of chilling exposure, a new stabilized balance was established. Reduced hexose levels in the shoot apex, reduced ratios of sugar levels between the apex and source leaves and a higher apical sucrose/hexose ratio, associated with decreased activity and expression of cell wall invertase, indicate that prolonged chilling induced sugar accumulation in source leaves at the expense of reduced sugar transport to and reduced sucrose utilization in the shoot. This was associated with reduced levels of indole-3-acetic acid and abscisic acid in the apex and high numbers of differentially, particularly up-regulated genes, especially in the source leaves, including those regulating histones, ethylene action, transcription factors, and a jasmonate-ZIM-domain protein. Transcripts of one Jumonji C domain containing protein and one expansin accumulated in source leaves throughout the chilling period. The results reveal a dynamic and complex disturbance of plant function in response to mild chilling, opening new perspectives for the comparative analysis of differently tolerant cultivars.