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Journal of Bacteriology Nov 1989Saccharomyces yeasts ferment several alpha-glucosides including maltose, maltotriose, turanose, alpha-methylglucoside, and melezitose. In the utilization of these sugars...
Saccharomyces yeasts ferment several alpha-glucosides including maltose, maltotriose, turanose, alpha-methylglucoside, and melezitose. In the utilization of these sugars transport is the rate-limiting step. Several groups of investigators have described the characteristics of the maltose permease (D. E. Kroon and V. V. Koningsberger, Biochim. Biophys. Acta 204:590-609, 1970; R. Serrano, Eur. J. Biochem. 80:97-102, 1977). However, Saccharomyces contains multiple alpha-glucoside transport systems, and these studies have never been performed on a genetically defined strain shown to have only a single permease gene. In this study we isolated maltose-negative mutants in a MAL6 strain and, using a high-resolution mapping technique, we showed that one class of these mutants, the group A mutants, mapped to the MAL61 gene (a member of the MAL6 gene complex). An insertion into the N-terminal-coding region of MAL61 resulted in the constitutive production of MAL61 mRNA and rendered the maltose permease similarly constitutive. Transformation by high-copy-number plasmids containing the MAL61 gene also led to an increase in the maltose permease. A deletion-disruption of MAL61 completely abolished maltose transport activity. Taken together, these results prove that this strain has only a single maltose permease and that this permease is the product of the MAL61 gene. This permease is able to transport maltose and turanose but cannot transport maltotriose, alpha-methylglucoside, or melezitose. The construction of strains with only a single permease will allow us to identify other maltose-inducible transport systems by simple genetic tests and should lead to the identification and characterization of the multiple genes and gene products involved in alpha-glucoside transport in Saccharomyces yeasts.
Topics: Cloning, Molecular; Genes, Fungal; Genotype; Kinetics; Maltose; Membrane Transport Proteins; Monosaccharide Transport Proteins; Plasmids; Restriction Mapping; Saccharomyces; Species Specificity
PubMed: 2808304
DOI: 10.1128/jb.171.11.6148-6154.1989 -
Microbiology (Reading, England) Apr 1998To elucidate the importance of oligopeptide permease for Borrelia burgdorferi, the agent of Lyme disease, a chromosomal locus in B. burgdorferi that encodes homologues...
To elucidate the importance of oligopeptide permease for Borrelia burgdorferi, the agent of Lyme disease, a chromosomal locus in B. burgdorferi that encodes homologues of all five subunits of oligopeptide permease has been identified and characterized. B. burgdorferi has multiple copies of the gene encoding the peptide-binding component, OppA; three reside at the chromosomal locus and two are on plasmids. Northern analyses indicate that each oppA gene is independently transcribed, although the three chromosomal oppA genes are also expressed as bi- and tri-cistronic messages. Induction of one of the plasmid-encoded oppA genes was observed following an increase in temperature, which appears to be an important cue for adaptive responses in vivo. The deduced amino acid sequences suggest that all five borrelial oppA homologues are lipoproteins, but the protease-resistance of at least one of them in intact bacteria is inconsistent with outer-surface localization. Insertional inactivation of a plasmid-encoded oppA gene demonstrates that it is not essential for growth in culture.
Topics: Amino Acid Sequence; Bacterial Proteins; Blotting, Northern; Blotting, Southern; Borrelia burgdorferi Group; Chromosomal Proteins, Non-Histone; Immunoblotting; Membrane Transport Proteins; Molecular Sequence Data; Plasmids; Sequence Alignment
PubMed: 9579077
DOI: 10.1099/00221287-144-4-1033 -
Proceedings of the National Academy of... Sep 1995Biotinylated lactose permease from Escherichia coli containing a single-cysteine residue at position 330 (helix X) or at position 147, 148, or 149 (helix V) was purified...
Biotinylated lactose permease from Escherichia coli containing a single-cysteine residue at position 330 (helix X) or at position 147, 148, or 149 (helix V) was purified by avidin-affinity chromatography and derivatized with 5-(alpha-bromoacetamido)-1,10-phenanthroline-copper [OP(Cu)]. Studies with purified, OP(Cu)-labeled Leu-330 --> Cys permease in dodecyl-beta-D-maltopyranoside demonstrate that after incubation in the presence of ascorbate, cleavage products of approximately 19 and 6-8 kDa are observed on immunoblots with anti-C-terminal antibody. Remarkably, the same cleavage products are observed with permease embedded in the native membrane. Comparison with the C-terminal half of the permease expressed independently as a standard indicates that the 19-kDa product results from cleavage near the cytoplasmic end of helix VII, whereas the 6- to 8-kDa fragment probably results from fragmentation near the cytoplasmic end of helix XI. Results are entirely consistent with a tertiary-structure model of the C-terminal half of the permease derived from earlier site-directed fluorescence and site-directed mutagenesis studies. Similar studies with OP(Cu)-labeled Cys-148 permease exhibit cleavage products at approximately 19 kDa and at 15-16 kDa. The larger fragment probably reflects cleavage at a site near the cytoplasmic end of helix VII, whereas the 15- to 16-kDa fragment is consistent with cleavage near the cytoplasmic end of helix VIII. When OP(Cu) is moved 100 degrees to position 149 (Val-149 --> Cys permease), a single product is observed at 19 kDa, suggesting fragmentation at the cytoplasmic end of helix VII. However, when the reagent is moved 100 degrees in the other direction to position 147 (Gly-147 --> Cys permease), cleavage is not observed. The results suggest that helix V is in close proximity to helices VII and VIII with position 148 in the interface between the helices, position 149 facing helix VII, and position 147 facing the lipid bilayer.
Topics: Amino Acid Sequence; Antibodies; Cell Membrane; Chromatography, Affinity; Copper; Copper Sulfate; Cysteine; Escherichia coli; Escherichia coli Proteins; Immunoblotting; Membrane Transport Proteins; Models, Structural; Molecular Sequence Data; Monosaccharide Transport Proteins; Mutagenesis, Site-Directed; Organometallic Compounds; Peptide Fragments; Phenanthrolines; Point Mutation; Protein Structure, Secondary; Recombinant Proteins; Symporters
PubMed: 7568098
DOI: 10.1073/pnas.92.20.9186 -
FEMS Microbiology Letters Feb 1995Competition experiments revealed that adenine and guanine were transported by a purine permease in both Candida glabrata 4 and a C. glabrata 4 cytosine permease negative... (Comparative Study)
Comparative Study
Competition experiments revealed that adenine and guanine were transported by a purine permease in both Candida glabrata 4 and a C. glabrata 4 cytosine permease negative mutant. The C. glabrata 4 cytosine permease negative mutant was isolated using 5-fluorocytosine selection. This mutant no longer transported cytosine, but transported adenine and guanine. A transport system for hypoxanthine was not detected. Hence, in addition to the cytosine permease, a purine permease exists in C. glabrata. This differs from the purine cytosine permeases in Saccharomyces cerevisiae and Candida albicans which transport adenine, cytosine, guanine and hypoxanthine.
Topics: Adenine; Candida; Cytosine; Membrane Transport Proteins; Mutation; Nucleobase Transport Proteins; Purines; Saccharomyces cerevisiae; Species Specificity
PubMed: 7896084
DOI: 10.1111/j.1574-6968.1995.tb07397.x -
Gene Dec 1989We have cloned and sequenced the GAL2 gene of Saccharomyces cerevisiae, which encodes galactose permease. The GAL2 protein is related to the yeast glucose transporter... (Comparative Study)
Comparative Study
We have cloned and sequenced the GAL2 gene of Saccharomyces cerevisiae, which encodes galactose permease. The GAL2 protein is related to the yeast glucose transporter encoded by the SNF3 gene, and also to mammalian and bacterial sugar permeases. Like the other members of this protein family, GAL2 has twelve hydrophobic segments that are separated by loops of charged amino acids. A comparison of different members of this protein family shows that those parts of the polypeptides thought to be on the cytoplasmic side of the cell membrane, are more conserved than other parts of the molecules.
Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; Genes; Genes, Fungal; Humans; Membrane Transport Proteins; Molecular Sequence Data; Monosaccharide Transport Proteins; Restriction Mapping; Saccharomyces cerevisiae; Sequence Homology, Nucleic Acid
PubMed: 2697639
DOI: 10.1016/0378-1119(89)90423-x -
Proceedings of the National Academy of... Jan 1990Oligonucleotide-directed, site-specific mutagenesis has been utilized to modify the melB gene of Escherichia coli such that each of the seven His residues in the...
Oligonucleotide-directed, site-specific mutagenesis has been utilized to modify the melB gene of Escherichia coli such that each of the seven His residues in the melibiose permease has been replaced with Arg. Replacement of His-213, His-442, or His-456 has no significant effect on permease activity, while permease with Arg in place of His-198, His-318, or His-357 retains more than 70% of wild-type activity. In striking contrast, replacement of His-94 with Arg causes a complete loss of sugar binding and transport, although the cells contain a normal complement of permease molecules. Thus, as shown previously with lac permease, only a single His residue is important for activity, but, in the case of mel permease, the critical His residue is present in the 3rd putative transmembrane helix rather than the 10th.
Topics: Arginine; Base Sequence; Escherichia coli; Genes, Bacterial; Histidine; Membrane Transport Proteins; Molecular Sequence Data; Mutation; Oligonucleotide Probes; Plasmids; Protein Conformation; Symporters
PubMed: 2404282
DOI: 10.1073/pnas.87.1.468 -
Research in Microbiology 1990
Topics: Biological Transport, Active; Carbohydrate Metabolism; Escherichia coli; Escherichia coli Proteins; Membrane Proteins; Membrane Transport Proteins; Models, Molecular; Monosaccharide Transport Proteins; Mutation; Protein Conformation; Protons; Substrate Specificity; Symporters
PubMed: 2177910
DOI: 10.1016/0923-2508(90)90004-a -
Advances in Experimental Medicine and... 2008Kinetoplastid protozoa express hundreds of membrane transport proteins that allow them to take up nutrients, establish ion gradients, efflux metabolites, translocate... (Review)
Review
Kinetoplastid protozoa express hundreds of membrane transport proteins that allow them to take up nutrients, establish ion gradients, efflux metabolites, translocate compounds from one intracellular compartment to another, and take up or export drugs. The combination of molecular cloning, genetic approaches, and the completed genome projects for Trypanosoma brucei, Leishmania major, and Trypanosoma cruzi have allowed detailed functional analysis of various transporters and predictions about the likely functions of others. Thus many opportunities exist to define the biological and pharmacological properties of parasite transporters whose genes were often difficult to identify in the pregenomic era. A subset of these transporters that are essential for parasite viability could serve as targets for novel drug therapies by identifying compounds that interfere with their uptake functions. Other permeases provide routes for uptake of selectively cytotoxic compounds and can thus be useful for delivery of drugs. Drug resistance may develop in strains where such drug uptake transporters are nonfunctional or in parasites that over-express other permeases that export a drug. A summary of recent work on Leishmania transporters for glucose and for purines is provided as an example of permeases that are being studied in molecular detail.
Topics: Animals; Membrane Transport Proteins; Protozoan Proteins; Trypanocidal Agents; Trypanosomatina
PubMed: 18365656
DOI: 10.1007/978-0-387-77570-8_3 -
European Journal of Biochemistry Dec 1983Escherichia coli strains have been isolated in which 3, 39 or 805 5'-end codons of lacZ, the gene for the cytoplasmic enzyme beta-galactosidase are fused to codon 9 of...
Escherichia coli strains have been isolated in which 3, 39 or 805 5'-end codons of lacZ, the gene for the cytoplasmic enzyme beta-galactosidase are fused to codon 9 of lacY, the gene for lactose permease. Lactose-permease-deficient cells, carrying the lacZ-Y fusions on F' lac pro episomes, are phenotypically positive on eosin/methylene blue/lactose or on melibiose plates, demonstrating that the beta-galactosidase--lactose-permease chimaeras transport lactose and melibiose in vivo. The apparent affinity for beta-D-galactopypanosyl 1-thio-beta-D-galactopyranoside (GalSGal) in cells is similar to that of the wild-type gene product. The maximum velocity of active GalSGal transport is reduced in all three fusion strains. Both lactose and p-nitrophenyl alpha-D-galactopyranoside inhibit GalSGal uptake. As demonstrated by immunoblot experiments the chimaeras cross-react with polyclonal antibodies directed against native lactose permease and they are present in the cell envelope fraction of homogenates. Their apparent molecular weights upon electrophoresis in NaDodSO4/polyacrylamide gels correspond to those expected from their respective primary sequences, taking into account the migration properties of wild-type lactose permease. It is proposed that substitution of eight N-terminal lactose permease residues by N-terminal beta-galactosidase residues neither prevents membrane incorporation of permease nor completely impairs the ability to transport galactosides actively. Alternative interpretations of the experimental results are discussed.
Topics: Biological Transport, Active; Chemical Phenomena; Chemistry; Escherichia coli; Escherichia coli Proteins; Galactosidases; Immunochemistry; Kinetics; Membrane Transport Proteins; Molecular Weight; Monosaccharide Transport Proteins; Phenotype; Symporters
PubMed: 6363063
DOI: 10.1111/j.1432-1033.1983.tb07863.x -
Journal of Molecular Biology Mar 1998Although missense mutations that inactivate integral membrane proteins cause a variety of diseases, the mechanisms by which they act are poorly understood. To establish...
Although missense mutations that inactivate integral membrane proteins cause a variety of diseases, the mechanisms by which they act are poorly understood. To establish a model for investigating this issue, we identified 51 missense mutations arising in vivo that inactivate Escherichia coli lac permease, a well-characterized membrane transport protein. The mutants were isolated using a genetic screening procedure which eliminates mutations that block expression of the lac permease gene, such as nonsense and frameshift mutations. The majority of the 51 missense mutations caused highly non-conservative changes in membrane-spanning sequences, such as the introduction of charged residues. Nevertheless, the greatest clustering of substitutions occurred in the two regions of lac permease thought to be most important for transport function. The existence of this clustering indicates that even highly non-conservative substitutions may cause relatively localized structural defects. Conservative inactivating substitutions were scattered throughout lac permease and may affect residues that make contacts required for normal folding. Two unexpected phenotypes were observed in the collection of mutants: about 20% of the substitutions led to cold-sensitive lactose utilization, and one substitution made the mutant lac permease toxic to cells. This relatively unbiased collection of mutants should provide a resource for further studies of how missense mutations inactivate membrane proteins in vivo.
Topics: Base Sequence; Biological Transport; Escherichia coli; Escherichia coli Proteins; Lactose; Membrane Transport Modulators; Membrane Transport Proteins; Models, Biological; Monosaccharide Transport Proteins; Mutagenesis, Site-Directed; Mutation; Symporters
PubMed: 9514765
DOI: 10.1006/jmbi.1998.1627