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Proceedings of the National Academy of... May 1997
Review
Topics: Amino Acid Sequence; Bacteria; Bacteriorhodopsins; Biological Transport, Active; Carrier Proteins; Cell Membrane; Escherichia coli; Escherichia coli Proteins; Membrane Transport Proteins; Monosaccharide Transport Proteins; Symporters
PubMed: 9159101
DOI: 10.1073/pnas.94.11.5508 -
The Journal of Biological Chemistry Sep 1989In the present study, lactose permease mutants were isolated which have an enhanced recognition toward maltose (an alpha-glucoside) and diminished recognition for...
In the present study, lactose permease mutants were isolated which have an enhanced recognition toward maltose (an alpha-glucoside) and diminished recognition for cellobiose (a beta-glucoside). Nine mutants were isolated from a strain encoding a wild-type permease (pTE18) and nine from a strain encoding a mutant permease which recognizes maltose (pB15). All 18 mutants were subjected to DNA sequencing, and it was found that all mutations are single base substitutions within the lac Y gene effecting single amino acid substitutions within the protein. From the pTE18 parent, substitutions involved Tyr-236 to Phe or His; Ser-306 to Thr; and six independent mutants in which Ala-389 was changed to Pro. From pB15, Tyr-236 was changed to Phe or Asn, Ser-306 to Thr or Leu, Lys-319 to Asn, and His-322 to Tyr, Asn, or Gln. All 18 mutants exhibited enhanced recognition for maltose (compared with the pTE18 strain) and a diminished recognition for cellobiose. In addition, all mutants showed a diminished recognition toward beta-galactosides as well. The Phe-236, His-236, Leu-306, Asn-319, Tyr-322, Asn-322, and Gln-322 mutants were completely defective in the uphill accumulation of methyl-beta-D-thiogalactopyranoside whereas the Asn-236, Thr-306, and Pro-389 mutants could effectively accumulate methyl-beta-D-thiogalactopyranoside against a concentration gradient. The mutants obtained in this study, together with previous lactose permease mutants, tend to be found on transmembrane segments, and those which are on the same transmembrane segment are often found three or four amino acids away from each other. This pattern is consistent with a protein structure in which important amino acid side chains project from several transmembrane segments in such a way as to form a hydrophilic channel for the recognition and transport of H+ and galactosides. It is proposed that the mechanism for H+/lactose cotransport is consistent with a "flanking gate" model in which the protein contains a single recognition site for galactosides within the channel which is flanked on either side by gates.
Topics: Base Sequence; Biological Transport; Cellobiose; DNA, Bacterial; Disaccharides; Escherichia coli; Escherichia coli Proteins; Galactosides; Lactose; Maltose; Membrane Transport Proteins; Monosaccharide Transport Proteins; Mutation; Plasmids; Symporters
PubMed: 2670925
DOI: No ID Found -
Journal of Bacteriology Jul 2017synthesizes the high-affinity ABC transporters CysTWA and ModABC to specifically import the chemically related oxyanions sulfate and molybdate, respectively. In...
synthesizes the high-affinity ABC transporters CysTWA and ModABC to specifically import the chemically related oxyanions sulfate and molybdate, respectively. In addition, has the low-affinity permease PerO acting as a general oxyanion transporter, whose elimination increases tolerance to molybdate and tungstate. Although PerO-like permeases are widespread in bacteria, their function has not been examined in any other species to date. Here, we present evidence that PerO permeases from the alphaproteobacteria , , , and and the gammaproteobacterium functionally substitute for PerO in sulfate uptake and sulfate-dependent growth, as shown by assimilation of radioactively labeled sulfate and heterologous complementation. Disruption of genes in , , and increased tolerance to tungstate and, in the case of , to molybdate, suggesting that heterometal oxyanions are common substrates of PerO permeases. This study supports the view that bacterial PerO permeases typically transport sulfate and related oxyanions and, hence, form a functionally conserved permease family. Despite the widespread distribution of PerO-like permeases in bacteria, our knowledge about PerO function until now was limited to one species, In this study, we showed that PerO proteins from diverse bacteria are functionally similar to the prototype, suggesting that PerO permeases form a conserved family whose members transport sulfate and related oxyanions.
Topics: Anions; Bacterial Proteins; Biological Transport; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Membrane Transport Proteins; Mutation; Rhodobacter capsulatus; Sulfates
PubMed: 28461447
DOI: 10.1128/JB.00183-17 -
The Journal of Biological Chemistry Dec 1987Purified lac permease, the 46.5-kDa product of the lac Y gene that catalyzes lactose/H+ symport, or purified cytochrome o, a terminal oxidase of the Escherichia coli...
Purified lac permease, the 46.5-kDa product of the lac Y gene that catalyzes lactose/H+ symport, or purified cytochrome o, a terminal oxidase of the Escherichia coli respiratory chain composed of four subunits with a composite molecular mass of 140 kDa, was reconstituted into proteoliposomes individually or in combination. The preparations were then examined by freeze-fracture electron microscopy employing conventional platinum/carbon replicas or by means of a new technique using thin tantalum replicas. In nonenergized proteoliposomes, both proteins appear to reconstitute as monomers based on (i) the variation of intramembrane particle density with protein concentration; (ii) the ratio of particles corresponding to each protein in proteoliposomes reconstituted with a known ratio of permease to oxidase; and (iii) the dimensions of the particles observed in tantalum replicas. The intramembrane particle diameters in tantalum replicas are about 20-25% smaller than those observed in conventional platinum/carbon replicas, indicating that the dimensions of the particles revealed with tantalum more accurately reflect the sizes of lac permease and cytochrome o. The diameters and heights of the permease and cytochrome o in tantalum replicas are 5.1 nm X 2.8 nm and 7.4 nm X 4.2 nm, respectively. Furthermore, a higher percentage of lac permease molecules exhibits a notch or cleft in tantalum replicas relative to platinum/carbon replicas. Importantly, the initial rate of lactose/H+ symport in proteoliposomes varies linearly with the ratio of lac permease to phospholipid, and no change is observed in either the size or distribution of lac permease molecules when the proteoliposomes are energized. The results taken as a whole provide a strong indication that both lac permease and cytochrome o reconstitute into proteoliposomes as monomers, that the permease does not dimerize in the presence of the H+ electrochemical gradient, and that both molecules are completely functional as monomers.
Topics: Carbon; Electrochemistry; Electron Transport Complex IV; Escherichia coli; Escherichia coli Proteins; Freeze Fracturing; Lactose; Macromolecular Substances; Membrane Transport Proteins; Microscopy, Electron; Monosaccharide Transport Proteins; Platinum; Proteolipids; Symporters; Tantalum
PubMed: 2824513
DOI: No ID Found -
Biochemistry Dec 2011The sucrose permease (CscB) and lactose permease (LacY) of Escherichia coli belong to the oligosaccharide/H(+) symporter subfamily of the major facilitator superfamily,... (Comparative Study)
Comparative Study
The sucrose permease (CscB) and lactose permease (LacY) of Escherichia coli belong to the oligosaccharide/H(+) symporter subfamily of the major facilitator superfamily, and both catalyze sugar/H(+) symport across the cytoplasmic membrane. Thus far, there is no common substrate for the two permeases; CscB transports sucrose, and LacY is highly specific for galactopyranosides. Determinants for CscB sugar specificity are unclear, but the structural organization of key residues involved in sugar binding appears to be similar in CscB and LacY. In this study, several sugars containing galactopyranosyl, glucopyranosyl, or fructofuranosyl moieties were tested for transport with cells overexpressing either CscB or LacY. CscB recognizes not only sucrose but also fructose and lactulose, but glucopyranosides are not transported and do not inhibit sucrose transport. The findings indicate that CscB exhibits practically no specificity with respect to the glucopyranosyl moiety of sucrose. Inhibition of sucrose transport by CscB tested with various fructofuranosides suggests that the C(3)-OH group of the fructofuranosyl ring may be important for recognition by CscB. Lactulose is readily transported by LacY, where specificity is directed toward the galactopyranosyl ring, and the affinity of LacY for lactulose is similar to that observed for lactose. The studies demonstrate that the substrate specificity of CscB is directed toward the fructofuranosyl moiety of the substrate, while the specificity of LacY is directed toward the galactopyranosyl moiety.
Topics: Alkylation; Anilino Naphthalenesulfonates; Binding Sites; Binding, Competitive; Biological Transport; Cysteine; Disaccharides; Escherichia coli; Escherichia coli Proteins; Fructose; Galactosides; Glucosides; Glycosides; Kinetics; Lactulose; Membrane Transport Proteins; Models, Molecular; Molecular Conformation; Monosaccharide Transport Proteins; Recombinant Proteins; Sulfhydryl Reagents; Symporters
PubMed: 22106930
DOI: 10.1021/bi201592y -
European Journal of Biochemistry May 1987The proline permease gene PUT4 has been cloned. Nitrogen-source regulation ('ammonia sensitivity') of this and at least two other amino-acid permeases is believed to...
The proline permease gene PUT4 has been cloned. Nitrogen-source regulation ('ammonia sensitivity') of this and at least two other amino-acid permeases is believed to occur at two distinct levels, i.e. permease synthesis and permease activity. Therefore, PUT4 transcription/messenger stability was examined in the ammonia- and proline-grown wild type as well as in mutant strains supposedly affected at only one or at both of these levels. We report transcript-level repression of proline permease synthesis in ammonia-grown cells. Repression is lifted at this level in gdhCR, gln1ts and gdhA mutants which exhibit pleiotropically derepressed permease and catabolic enzyme activities. On the other hand, the npi1 and npi2 mutations, formerly called mut2 and mut4, relieve an inactivation process which seems only to affect permeases. These mutations do not affect the detected PUT4 RNA level. The only known positive factor in proline permease regulation, the nitrogen permease reactivator protein Npr1, is believed to counteract the inactivation process on derepressing media. This protein appears to have an additional, indirect effect on PUT4 transcription/messenger stability: it would actually mediate repression via its activating effect on ammonia uptake.
Topics: Amino Acid Transport Systems, Neutral; Cloning, Molecular; Genes; Genes, Fungal; Membrane Transport Proteins; Mutation; Nitrogen; Proline; RNA, Fungal; Saccharomyces cerevisiae; Transformation, Genetic
PubMed: 3552672
DOI: 10.1111/j.1432-1033.1987.tb11169.x -
FEMS Microbiology Letters Aug 2020Organic acids such as monocarboxylic acids, dicarboxylic acids or even more complex molecules such as sugar acids, have displayed great applicability in the industry as... (Review)
Review
Organic acids such as monocarboxylic acids, dicarboxylic acids or even more complex molecules such as sugar acids, have displayed great applicability in the industry as these compounds are used as platform chemicals for polymer, food, agricultural and pharmaceutical sectors. Chemical synthesis of these compounds from petroleum derivatives is currently their major source of production. However, increasing environmental concerns have prompted the production of organic acids by microorganisms. The current trend is the exploitation of industrial biowastes to sustain microbial cell growth and valorize biomass conversion into organic acids. One of the major bottlenecks for the efficient and cost-effective bioproduction is the export of organic acids through the microbial plasma membrane. Membrane transporter proteins are crucial elements for the optimization of substrate import and final product export. Several transporters have been expressed in organic acid-producing species, resulting in increased final product titers in the extracellular medium and higher productivity levels. In this review, the state of the art of plasma membrane transport of organic acids is presented, along with the implications for industrial biotechnology.
Topics: Acids; Bacteria; Biotechnology; Fungi; Industrial Microbiology; Membrane Transport Proteins
PubMed: 32681640
DOI: 10.1093/femsle/fnaa118 -
Journal of Microbiology and... Dec 2012Iron plays a key role in host-pathogen interactions. Microbial pathogens require iron for survival and virulence, whereas mammalian hosts sequester and withhold iron as...
Iron plays a key role in host-pathogen interactions. Microbial pathogens require iron for survival and virulence, whereas mammalian hosts sequester and withhold iron as a means of nutritional immunity. We previously identified two paralogous genes, CFT1 and CFT2, which encode homologs of a fungal iron permease, Cft1 and Cft2, respectively, in the human fungal pathogen Cryptococcus neoformans. Cft1 was shown to play a role in the high-affinity reductive iron uptake system, and was required for transferrin utilization and full virulence in mammalian hosts. However, no role of Cft2 has been suggested yet. Here, we identified the third gene, CFT3, that produces an additional fungal iron permease homolog in C. neoformans, and we also generated the cft3 mutant for functional characterization. We aimed to reveal distinct functions of Cft1, Cft2 and Cft3 by analyzing phenotypes of the mutants lacking CFT1, CFT2 and CFT3, respectively. The endogenous promoter of CFT1, CFT2 and CFT3 was replaced with the inducible GAL7 promoter in the wildtype strain or in the cft1 mutant for gain-of-function analysis. Using these strains, we were able to find that CFT2 is required for growth in low-iron conditions in the absence of CFT1 and that overexpression of CFT2 compensates for deficiency of the cft1 mutant in iron uptake and various cellular stress conditions. However, unlike CFT2, no clear phenotypic characteristic of the cft3 mutant and the strain overexpressing CFT3 was observed. Overall, our data suggested a redundant role of Cft2 in the high-affinity iron uptake and stress responses in C. neoformans.
Topics: Amino Acid Sequence; Cryptococcosis; Cryptococcus neoformans; Fungal Proteins; Humans; Iron; Membrane Transport Proteins; Molecular Sequence Data; Mutation; Phenotype; Sequence Alignment
PubMed: 23221526
DOI: 10.4014/jmb.1209.09019 -
Proceedings of the National Academy of... Aug 1993A simplified approach for purification of functional lactose permease from Escherichia coli is described that is based on the construction of chimeras between the...
A simplified approach for purification of functional lactose permease from Escherichia coli is described that is based on the construction of chimeras between the permease and a 100-amino acid residue polypeptide containing the biotin acceptor domain from the oxaloacetate decarboxylase of Klebsiella pneumoniae [Cronan, J. E., Jr. (1990) J. Biol. Chem. 265, 10327-10333]. Chimeras were constructed with a factor Xa protease site and the biotin acceptor domain in the middle cytoplasmic loop (loop 6) or at the C terminus of the permease. Each construct catalyzes active lactose transport in cells and right-side-out membrane vesicles. Moreover, the constructs are biotinylated in vivo, and in both chimeras, the factor Xa protease site is accessible from the cytoplasmic surface of the membrane. Both biotinylated permeases bind selectively to immobilized monomeric avidin and are eluted with free biotin in a high state of purity, and the loop 6 chimera catalyzes active transport after reconstitution into proteoliposomes. The methodology described should be applicable to other membrane proteins.
Topics: Bacterial Proteins; Biological Transport; Biotin; Chromatography, Affinity; Escherichia coli; Escherichia coli Proteins; Lactose; Membrane Proteins; Membrane Transport Proteins; Monosaccharide Transport Proteins; Recombinant Fusion Proteins; Symporters
PubMed: 8346199
DOI: 10.1073/pnas.90.15.6934 -
The Journal of Biological Chemistry Jan 1994Uptake of inositol by Saccharomyces cerevisiae is regulated through transcriptional control of the gene that encodes the major inositol permease, ITR1 (Nikawa, J.,...
Uptake of inositol by Saccharomyces cerevisiae is regulated through transcriptional control of the gene that encodes the major inositol permease, ITR1 (Nikawa, J., Tsukagoshi, Y., and Yamashita, S. (1991) J. Biol. Chem. 266, 11184-11191). ITR1 mRNA abundance decreases when cells are transferred from medium without inositol to medium with inositol. Here we demonstrate that the mechanism of transcriptional regulation of ITR1 is through the action of the INO2, INO4 and OPI1 genes. INO2 and INO4 are required for derepressed levels of ITR1 mRNA, and OPI1 is necessary for repression of transcript levels in response to inositol. The INO2, INO4, and OPI1 genes thus coordinate uptake of inositol to endogenous inositol biosynthesis and to phospholipid biosynthesis. Repression of transcription of ITR1 also requires ongoing synthesis of phosphatidylcholine, defining an additional link between synthesis of phospholipids and regulation of inositol uptake. Analysis showed that the INO1 gene, encoding a key enzyme in the inositol biosynthetic pathway, responded to decreases in permease activity with a graduated increase in the level of INO1 mRNA. We also found that, in addition to the transcriptional regulation, inositol permease activity is regulated by irreversible inactivation. Inactivation of the ITR1 permease occurs in response to the presence of inositol and involves a change in the functional half-life of the protein.
Topics: Biological Transport; Carrier Proteins; Enzyme Repression; Fungal Proteins; Gene Deletion; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Fungal; Genes, Fungal; Inositol; Kinetics; Membrane Transport Modulators; Membrane Transport Proteins; Models, Genetic; Monosaccharide Transport Proteins; Mutagenesis, Insertional; Plasmids; Polymerase Chain Reaction; RNA, Messenger; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcription, Genetic
PubMed: 8294482
DOI: No ID Found