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The Journal of Biological Chemistry Apr 1994Yeast uracil permease follows the secretory pathway to the plasma membrane and is phosphorylated on serine residues in a post-Golgi compartment. The protein was found to...
Yeast uracil permease follows the secretory pathway to the plasma membrane and is phosphorylated on serine residues in a post-Golgi compartment. The protein was found to be rather stable in growing cells, but its turnover rate (half-life of about 7 h) was much faster than that of most yeast proteins. Several adverse conditions triggered the rapid degradation of uracil permease, and so a loss of uracil uptake. Turnover was rapid when yeast cells were starved of either nitrogen, phosphate, or carbon, and as they approached the stationary growth phase. Rapid permease degradation was also promoted by the inhibition of protein synthesis. The degradation of uracil permease in response to several stresses was strikingly slower in the two mutants, end3 and end4, that are deficient in the internalization step of receptor-mediated endocytosis. Thus, internalization is the first step in the permease degradative pathway. Uracil permease is degraded in the vacuole, since pep4 mutant cells lacking vacuolar protease activities accumulated large amounts of uracil permease, which was located within the vacuole by immunofluorescence. We have yet to determine whether adverse conditions enhance permease endocytosis and subsequent degradation or divert internalized uracil permease from a recycling to a degradative pathway.
Topics: Amino Acid Sequence; Biological Transport; Cell Membrane; Cycloheximide; Endocytosis; Genes, Fungal; Kinetics; Membrane Transport Proteins; Molecular Sequence Data; Mutagenesis; Nitrogen; Nucleotide Transport Proteins; Phosphorylation; Protein Processing, Post-Translational; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Time Factors; Uracil
PubMed: 8144575
DOI: No ID Found -
Biochemistry Aug 1986When Glu-325 in the lac permease of Escherichia coli is replaced with Ala, lactose/H+ symport is abolished. Thus, the altered permease catalyzes neither uphill lactose...
When Glu-325 in the lac permease of Escherichia coli is replaced with Ala, lactose/H+ symport is abolished. Thus, the altered permease catalyzes neither uphill lactose accumulation nor efflux. Remarkably, however, permease with Ala-325 catalyzes exchange and counterflow at completely normal rates. Taken together with the results presented in the accompanying paper [Püttner, I. B., Sarkar, H. K., Poonian, M. S., & Kaback, H. R. (1986) Biochemistry (preceding paper in this issue)], the findings suggest that the His-322 and Glu-325 may be components of a charge-relay system that plays an important role in the coupled translocation of lactose and H+.
Topics: Binding Sites; Escherichia coli; Escherichia coli Proteins; Glutamates; Glutamic Acid; Histidine; Kinetics; Membrane Transport Proteins; Monosaccharide Transport Proteins; Mutation; Symporters
PubMed: 2876725
DOI: 10.1021/bi00364a004 -
Proceedings of the National Academy of... Mar 1988Arg-302, His-322, and Glu-325, neighboring residues in putative helices IX and X of the lac permease (lacY gene product) of Escherichia coli, play an important role in...
Arg-302, His-322, and Glu-325, neighboring residues in putative helices IX and X of the lac permease (lacY gene product) of Escherichia coli, play an important role in lactose/H+ symport, possibly as components of a catalytic triad similar to that postulated for the serine proteases [Kaback, H. R. (1987) Biochemistry 26, 2071-2076]. By using restriction fragments of lacY genes harboring specific site-directed mutations, a fusion gene has been constructed that encodes a permease in which His-35 and His-39 are replaced with arginine, and His-205 with glutamine (RQHE permease). The resultant molecule contains a single histidine residue at position 322 and exhibits all of the properties of the wild-type permease. In addition, an analogous single-histidine permease was engineered with alanine at position 325 in place of glutamic acid (RQHA permease). This construct is defective in active transport but catalyzes exchange and counterflow normally. RQHA permease, like the single-histidine permease with Glu-325, also shows normal behavior with respect to N-ethylmaleimide inactivation, substrate protection, and binding. In addition to providing strong support for previous experiments, the engineered permease molecules should be useful for determining the apparent pK of His-322 under various conditions.
Topics: Binding, Competitive; Biological Transport, Active; DNA Mutational Analysis; Escherichia coli; Escherichia coli Proteins; Ethylmaleimide; Histidine; Lactose; Membrane Transport Proteins; Monosaccharide Transport Proteins; Nitrophenylgalactosides; Structure-Activity Relationship; Symporters; Thiogalactosides
PubMed: 3278314
DOI: 10.1073/pnas.85.5.1467 -
Molecular Microbiology Feb 1998Amino acid transporters of the yeast plasma membrane (permeases) belong to a family of integral membrane proteins with pronounced structural similarity. We present...
Amino acid transporters of the yeast plasma membrane (permeases) belong to a family of integral membrane proteins with pronounced structural similarity. We present evidence that a member of this family, encoded by the open reading frame (ORF) YDR160w (SSY1), is required for the expression of a set of transporter genes. Thus, deletion of the SSY1 gene causes loss of leucine-inducible transcription of the amino acid permease genes BAP2, TAT1 and BAP3 (ORF YDR046c) and the peptide transporter, PTR2. D-leucine can generate the signal without entering the cell. We propose that Ssy1p is situated in the plasma membrane and is involved in sensing leucine in the medium.
Topics: Amino Acid Transport Systems; Amino Acids; Carrier Proteins; Fungal Proteins; Gene Deletion; Gene Expression Regulation, Fungal; Genes, Fungal; Leucine; Membrane Proteins; Membrane Transport Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Signal Transduction; Transcription, Genetic; Transcriptional Activation
PubMed: 9489675
DOI: 10.1046/j.1365-2958.1998.00714.x -
Applied and Environmental Microbiology Aug 2007Burkholderia cepacia MBA4 is a bacterium that can utilize 2-haloacids as carbon and energy sources for growth. It has been proposed that dehalogenase-associated permease...
Burkholderia cepacia MBA4 is a bacterium that can utilize 2-haloacids as carbon and energy sources for growth. It has been proposed that dehalogenase-associated permease mediates the uptake of haloacid. In this paper, we report the first cloning and characterization of such a haloacid permease. The structural gene, designated deh4p, was found 353 bases downstream of the dehalogenase gene deh4a. Quantitative analysis of the expression of deh4p showed that it was induced by monochloroacetate (MCA), to a level similar to the MCA-induced level of deh4a. The nucleotide sequence of deh4p was determined, and an open reading frame of 1,656 bp encoding a putative peptide of 552 amino acids was identified. Deh4p has a putative molecular weight of 59,414 and an isoelectric point of 9.88. Deh4p has the signatures of sugar transport proteins and integral membrane proteins of the major facilitator superfamily. Uptake of [(14)C]MCA into the cell was Deh4p dependent. Deh4p has apparent K(m)s of 5.5 and 8.9 muM and V(max)s of 9.1 and 23.1 nmol mg(-1) min(-1) for acetate and MCA, respectively. A mutant with a transposon-inactivated haloacid operon failed to grow on MCA even when deh4a was provided in trans.
Topics: Acetates; Bacterial Proteins; Burkholderia cepacia; DNA Transposable Elements; Gene Expression Regulation, Bacterial; Hydrolases; Membrane Transport Proteins; Molecular Sequence Data; Mutagenesis, Insertional; Operon; Sequence Analysis, DNA
PubMed: 17545323
DOI: 10.1128/AEM.00576-07 -
Proceedings of the National Academy of... Sep 2003Previous experiments using intermolecular thiol cross-linking to determine surface-exposed positions in the transmembrane helices of the lactose permease suggest that...
Previous experiments using intermolecular thiol cross-linking to determine surface-exposed positions in the transmembrane helices of the lactose permease suggest that only positions accessible from the aqueous phase are susceptible to cross-linking. This approach is now extended to most of the remaining positions in the molecule. Of an additional 143 single-Cys mutants studied, homodimer formation is observed with both a 5-A- and a 21-A-long crosslinking agent containing bis-methane thiosulfonate reactive groups in 33 mutants and exclusively with the 21-A-long reagent in 43 mutants. Furthermore, intermolecular cross-linking has little or no effect on transport activity, thereby providing further support for the argument that lactose permease is functionally, as well as structurally, a monomer in the membrane. In addition, evidence is presented indicating that reentrance loops are unlikely in this polytopic membrane transport protein.
Topics: Amino Acid Sequence; Biological Transport; Escherichia coli; Escherichia coli Proteins; Lactose; Membrane Transport Proteins; Molecular Sequence Data; Monosaccharide Transport Proteins; Sulfhydryl Compounds; Symporters
PubMed: 12934015
DOI: 10.1073/pnas.1434239100 -
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 -
Biochemistry Jul 1987The Escherichia coli lactose permease has been purified on cation exchanger to contain a minimal amount of phospholipids, i.e., 4-5 mol/mol of permease, in the presence...
The Escherichia coli lactose permease has been purified on cation exchanger to contain a minimal amount of phospholipids, i.e., 4-5 mol/mol of permease, in the presence of the detergent dodecyl beta-maltoside at its critical micelle concentration. This preparation is active in galactoside binding. When the detergent level is further reduced by dialysis, the lactose permease forms filaments one molecule wide and up to several micrometers long. The filaments tend to associate laterally to form sheets. Analysis of electron micrographs of negatively stained filamentous arrays indicates an average filament spacing of 51 A and a subunit period of 26-30 A along individual filaments. These values most probably correspond to the dimensions of the lactose permease molecule measured parallel to the membrane plane. In many filaments, the subunits show a stain-penetrated cleft. It suggests that the lactose permease molecule comprises two domains, which may be correlated with internal repeats between the N- and C-terminal halves of the polypeptide sequence.
Topics: Chromatography, Ion Exchange; Escherichia coli; Escherichia coli Proteins; Kinetics; Membrane Transport Proteins; Microscopy, Electron; Monosaccharide Transport Proteins; Symporters
PubMed: 3311155
DOI: 10.1021/bi00389a032 -
Journal of Bacteriology Jun 1997A gene which was shown to be cotranscribed with the NAD+-dependent malic enzyme gene (maeE) of Streptococcus bovis ATCC 15352 was revealed to encode L-malate-specific...
A gene which was shown to be cotranscribed with the NAD+-dependent malic enzyme gene (maeE) of Streptococcus bovis ATCC 15352 was revealed to encode L-malate-specific permease (MaeP), which showed high activity at low pHs (pH 5.1 to 5.9). MaeP was strongly inhibited by the ionophores nigericin and valinomycin.
Topics: Bacterial Proteins; Base Sequence; Blotting, Northern; Genes, Bacterial; Malates; Membrane Transport Proteins; Molecular Sequence Data; Organic Anion Transporters; Streptococcus bovis
PubMed: 9190827
DOI: 10.1128/jb.179.12.4056-4060.1997 -
Biochimica Et Biophysica Acta May 1965
Topics: Alcohols; Biochemical Phenomena; Biochemistry; Biological Transport; Disaccharides; Enzymes; Escherichia coli; Galactosidases; Galactosides; Glycosides; Membrane Transport Proteins; Molecular Biology; Mutation; Research
PubMed: 14347955
DOI: 10.1016/0304-4165(65)90029-2