dctA permease

K. lactis is able to use mono-and dicarboxylic acids as the sole carbon and energy source and presents mediated transport systems for these acids. S. cerevisiae although presents a carrier for monocarboxylates, is not able to transport dicarboxylic acids. The gene encoding lactic acid permease has been cloned in S. cerevisiae (JEN1 gene). Using the Génolevures database, two clones of K. lactis (named KlJEN1 and KlJEN2) containing a partial sequence of two genes homologues to JEN1 were identified. The RACE technique allowed the identification of the whole sequence of these genes. KlJEN1 and KlJEN2 expression has been assayed, and although in S. cerevisiae JEN1 expression is glucose repressed, a transcript of both JEN1/2 genes is present in glucose grown cells of K. lactis, indicating a different regulation in this species. Indeed, the analysis of the KlJEN1/2 promoters sequenced so far did not reveal cis elements for repressors like mig1p, in opposition to what happens with ScJEN1 promoter. On the second part of this work, a K. lactis gene, associated with the regulation of malate utilization (MUP1) is presented. This gene was isolated by heterologous gene expression in S. cerevisiae. A second copy of this gene is present in the K. lactis genome (MUP2) and functional analysis of MUP1 and MUP2 was carried out by the construction of a K. lactisΔ mupΔ1mup2 deleted strain. The growth in YNMal is very similar in the three strains; however, concerning dicarboxylic acid transport, the double mutant presented half of the activity for this transport system.

Carboxylic acids are widely used in pharmaceutical and food industries and new industrial uses are continually emerging. The first step of carboxylic acids metabolism is their entrance to the cell and understanding in detail this process has significant biotechnological ...

By using a lactose permease mutant containing a single Cys residue in place of Val 331 (helix X), conformational changes induced by ligand binding were studied. With right-side-out membrane vesicles containing Val 331 + Cys permease, lactose transport is inactivated by either N-ethylmaleimide (NEM) or 7-diethylamino-3-(4'maleimidylphenyl)-4-methylcoumarin (CPM). Remarkably, 0,D-galactopyranosyl I-thio-0,D-galactopyranoside (TDG) enhances the rate of inactivation by CPM, a hydrophobic sulfhydryl reagent, whereas NEM inactivation is attenuated by the ligand. Val 33 1 + Cys permease was then purified and studied in dodecyl-0,D-maltoside by site-directed fluorescence spectroscopy. The reactivity of Val 331 "+ Cys permease with 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) is not changed over a low range of TDG concentrations (<0.8 mM), but the fluorescence of the MIANS-labeled protein is quenched in a saturable manner (apparent Kd 0.12 mM) without a change in emission maximum. In contrast, over a higher range of TDG concentrations (1-10 mM), the reactivity of Val 331 + Cys permease with MIANS is enhanced and the emission maximum of MIANS-labeled permease is blue shifted by 3-7 nm. Furthermore, the fluorescence of MIANS-labeled Val 331 --t Cys permease is quenched by both acrylamide and iodide, but the former is considerably more effective. A low concentration of TDG (0.2 mM) does not alter quenching by either compound, whereas a higher concentration of ligand (10mM) decreases the quenching constant for iodide by about 50% and for acrylamide by about 20%. Finally, the EPR spectrum of nitroxide spin-labeled Val 331 + Cys permease exhibits 2 components with different mobilities, and TDG causes the immobilized component to increase. The results provide evidence for the argument that lac permease has more than a single binding site. TDG binding to a higher affinity site quenches the fluorescence of MIANS-labeled Val 33 1 + Cys permease, and occupation of a second lower affinity site causes position 33 1 to become more accessible from a hydrophobic environment.

We present the first account of the structure-function relationships of a protein of the subfamily of urea/H(+) membrane transporters of fungi and plants, using Aspergillus nidulans UreA as a study model. Based on the crystal structures of the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT) and of the Nucleobase-Cation-Symport-1 benzylhydantoin transporter from Microbacterium liquefaciens (Mhp1), we constructed a three-dimensional model of UreA which, combined with site-directed and classical random mutagenesis, led to the identification of amino acids important for UreA function. Our approach allowed us to suggest roles for these residues in the binding, recognition and translocation of urea, and in the sorting of UreA to the membrane. Residues W82, Y106, A110, T133, N275, D286, Y388, Y437 and S446, located in transmembrane helixes 2, 3, 7 and 11, were found to be involved in the binding, recognition and/or translocation of urea and the sorting of UreA to the membrane. Y...

The high affinity iron uptake complex in the yeast plasma membrane (PM) consists of the ferroxidase, Fet3p, and the ferric iron permease, Ftr1p. We used a combination of yeast two-hybrid analysis, confocal fluorescence microscopy, and fluorescence resonance energy transfer (FRET) quantification to delineate the motifs in the two proteins required for assembly and maturation into an uptakecompetent complex. The cytoplasmic, carboxyl-terminal domain of each protein contains a four-residue motif adjacent to the cytoplasm-PM interface that supports an interaction between the proteins. This interaction has been quantified by two-hybrid analysis and is required for assembly and trafficking of the complex to the PM and for the ϳ13% maximum FRET efficiency determined. In contrast, the Fet3p transmembrane domain (TM) can be exchanged with the TM domain from the vacuolar ferroxidase, Fet5p, with no loss of assembly and trafficking. A carboxyl-terminal interaction between the vacuolar proteins, Fet5p and Fth1p, also was quantified. As a measure of the specificity of interaction, no interaction between heterologous ferroxidase permease pairs was observed. Also, whereas FRET was quantified between fluorescent fusions of the copper permease (monomers), Ctr1p, none was observed between Fet3p and Ctr1p. The results are consistent with a (minimal) heterodimer model of the Fet3p⅐Ftr1p complex that supports the trafficking of iron from Fet3p to Ftr1p for iron permeation across the yeast PM.

A transposon library of 5700 mutants was constructed in Caulobacter crescentus strain NA1000. Thirteen mutant strains were isolated that presented growth deficiency in the presence of 20 microM cadmium chloride, and the disrupted genes were identified by DNA sequencing. Among the predicted products were found two putative transcriptional regulators, one histidine kinase/response regulator hybrid protein, two 2Fe-2S family proteins, and also members of a putative metal efflux system. Certain of these mutants also showed an increased sensitivity to zinc and copper, and the genes encoding the efflux system were induced by cadmium and zinc, indicating that this system may be important for homeostasis of both metal ions.

In this review, we present several extracellular proteases, enzymes, membrane permeases, and transporters as essential accessories proteins for nutrient assimilation, conservation, and transportation as determined by nutrient repletion or depletion. As an obligate aerobic pathogen, it is crucial for invading Cryptococcus (C.) neoformans to negotiate its adaptation to human internal organs like the brain and spinal cord, where the oxygen level is low compared to peripheral organs. Besides, essential metals like copper and iron are important cofactors to functional proteins; however, these metals are not usually freely available to invading human pathogens. Again, the phagolysosome low pH with glucose paucity, internal temperature, immune response, and complex extracellular matrixes are challenging environments that must be circumvented by C. neoformans in the systemic tissues for survival, adaptation, and infection in humans. We review extensive works on several extracellular proteas...

Brefeldin A is a commonly used antifungal agent that reversibly blocks protein transport from the endoplasmic reticulum to the Golgi complex. In this study, we aimed to characterize L-leucine uptake in Saccharomyces cerevisiae in the presence of brefeldin A. For this purpose, we used a synthetic medium, containing L-proline and the detergent SDS, which allows the agent to permeate into the yeast cell. The results obtained with a wild type strain and a gap1 mutant indicate that BFA causes either direct or indirect modification of the transport and/or processing of L-leucine permeases. The presence of BFA affects the kinetic parameter values for L-leucine uptake and decreases not only the uptake mediated by the general system (GAP1), but also that through the specific BAP2 (S1) and/or S2 systems.

Candida auris is a serious health concern of current world that possess serious global health threat and is emerging at a high rate. Available antifungal drugs are failing to combat this pathogen as they are growing resistance toward those drugs and some strains have already showed resistant to all three available antifungal drugs in the market. Finding alternative treatments is a must, therefore, to save lives from this foe. To make the way easier for developing new treatments, we have made some insilico analysis of this pathogen to identify suitable targets for designing drugs and also suggested some potential metabolites to test in vivo condition after some computational analysis. After the subtraction of duplicate, non-essential, human homologs, non-metabolic, human microbiome similar and druggable proteins we ended up with three proteins (XP_028890156.1, XP_028891672.1 and XP_028891858.1) from a total of 5441 C. auris proteins. Blocking those proteins will result in the destruc...

Maltodextrin is a mixture of maltooligosaccharides, which are produced by the degradation of starch or glycogen. They are mostly composed of α-1,4- and some α-1,6- linked glucose residues. Genes presumed to code for the Enterococcus faecalis maltodextrin transporter were induced during enterococcal infection. We therefore carried out a detailed study of maltodextrin transport in this organism. Depending on their length (3 to 7 glucose residues), E. faecalis takes up maltodextrins either via MalT, a maltose-specific permease of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), or the ATP binding cassette (ABC) transporter MdxEFG/MsmX. Maltotriose, the smallest maltodextrin, is primarily transported by the PTS permease. A malT mutant therefore exhibits significantly reduced growth on maltose and maltotriose. The residual uptake of the trisaccharide is catalyzed by the ABC transporter, because a malT/mdxF double mutant no longer grows on maltotriose. The trisa...

Trypanosoma cruzi is the etiological agent of Chagas disease, a disease endemic not only in Argentina but also in all of Latin America. T. cruzi presents several metabolic characteristics which are completely absent in its insect vectors and in mammalian hosts. Some of these differences were acquired after millions of years of adaptation to parasitism, during which this protozoan replaced many biosynthetic routes for transport systems. In the present review, we describe the advances in the knowledge of T. cruzi transport processes and the molecules involved. In particular, we focus on amino acid and polyamine transporters from the AAAP family (Amino Acid/Auxin Permeases), because they seem to be exclusive transporters from trypanosomatids. Taking into account that these permeases are completely absent in mammals, they could be considered as a potential target against Trypanosoma cruzi.