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Science (New York, N.Y.) Aug 2003Membrane transport proteins that transduce free energy stored in electrochemical ion gradients into a concentration gradient are a major class of membrane proteins. We...
Membrane transport proteins that transduce free energy stored in electrochemical ion gradients into a concentration gradient are a major class of membrane proteins. We report the crystal structure at 3.5 angstroms of the Escherichia coli lactose permease, an intensively studied member of the major facilitator superfamily of transporters. The molecule is composed of N- and C-terminal domains, each with six transmembrane helices, symmetrically positioned within the permease. A large internal hydrophilic cavity open to the cytoplasmic side represents the inward-facing conformation of the transporter. The structure with a bound lactose homolog, beta-D-galactopyranosyl-1-thio-beta-D-galactopyranoside, reveals the sugar-binding site in the cavity, and residues that play major roles in substrate recognition and proton translocation are identified. We propose a possible mechanism for lactose/proton symport (co-transport) consistent with both the structure and a large body of experimental data.
Topics: Amino Acid Substitution; Binding Sites; Biological Transport; Cell Membrane; Crystallization; Crystallography, X-Ray; Escherichia coli; Escherichia coli Proteins; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Ion Transport; Lactose; Membrane Transport Proteins; Models, Molecular; Monosaccharide Transport Proteins; Mutation; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Protons; Substrate Specificity; Symporters; Thiogalactosides
PubMed: 12893935
DOI: 10.1126/science.1088196 -
Proceedings of the National Academy of... Dec 2018The lactose permease of (LacY) utilizes an alternating access symport mechanism with multiple conformational intermediates, but only inward (cytoplasmic)- or outward...
The lactose permease of (LacY) utilizes an alternating access symport mechanism with multiple conformational intermediates, but only inward (cytoplasmic)- or outward (periplasmic)-open structures have been characterized by X-ray crystallography. It is demonstrated here with sugar-binding studies that cross-linking paired-Cys replacements across the closed cytoplasmic cavity stabilize an occluded conformer with an inaccessible sugar-binding site. In addition, a nanobody (Nb) that stabilizes a periplasmic-open conformer with an easily accessible sugar-binding site in WT LacY fails to cause the cytoplasmic cross-linked mutants to become accessible to galactoside, showing that the periplasmic cavity is closed. These results are consistent with tight association of the periplasmic ends in two pairs of helices containing clusters of small residues in the packing interface between N- and C-terminal six-helix bundles of the symporter. However, after reduction of the disulfide bond, the Nb markedly increases the rate of galactoside binding, indicating unrestricted access to the Nb epitope and the galactoside-binding site from the periplasm. The findings indicate that the cross-linked cytoplasmic double-Cys mutants resemble an occluded apo-intermediate in the transport cycle.
Topics: Binding Sites; Crystallography, X-Ray; Cytoplasm; Escherichia coli; Escherichia coli Proteins; Galactosides; Membrane Transport Proteins; Monosaccharide Transport Proteins; Periplasm; Symporters
PubMed: 30478058
DOI: 10.1073/pnas.1816267115 -
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 -
Proceedings of the National Academy of... Oct 2023Bacteria produce a structural layer of peptidoglycan (PG) that enforces cell shape, resists turgor pressure, and protects the cell. As bacteria grow and divide, the...
Bacteria produce a structural layer of peptidoglycan (PG) that enforces cell shape, resists turgor pressure, and protects the cell. As bacteria grow and divide, the existing layer of PG is remodeled and PG fragments are released. Enterics such as go to great lengths to internalize and reutilize PG fragments. is estimated to break down one-third of its cell wall, yet only loses ~0 to 5% of meso-diaminopimelic acid, a PG-specific amino acid, per generation. Two transporters were identified early on to possibly be the primary permease that facilitates PG fragment recycling, i) AmpG and ii) the Opp ATP binding cassette transporter in conjunction with a PG-specific periplasmic binding protein, MppA. The contribution of each transporter to PG recycling has been debated. Here, we have found that AmpG and MppA/Opp are differentially regulated by carbon source and growth phase. In addition, MppA/Opp is uniquely capable of high-affinity scavenging of muropeptides from growth media, demonstrating that AmpG and MppA/Opp allow for different strategies of recycling PG fragments. Altogether, this work clarifies environmental contexts under which utilizes distinct permeases for PG recycling and explores how scavenging by MppA/Opp could be beneficial in mixed communities.
Topics: Membrane Transport Proteins; Escherichia coli; Peptidoglycan; Bacterial Proteins; Bacteria; Cell Wall
PubMed: 37871219
DOI: 10.1073/pnas.2308940120 -
Yeast (Chichester, England) Aug 1998The uracil permease gene of Schizosaccharomyces pombe was cloned and sequenced. The deduced protein sequence shares strong similarities with five open reading frames...
The uracil permease gene of Schizosaccharomyces pombe was cloned and sequenced. The deduced protein sequence shares strong similarities with five open reading frames from Saccharomyces cerevisiae, namely the uracil permease encoded by the FUR4 gene, the allantoin permease encoded by DAL4, a putative uridine permease (YBL042C) and two unknown ORFs YOR071c and YLR237w. A topological model retaining ten transmembrane helices, based on predictions and on experimental data established for the uracil permease of S. cerevisiae by Galan and coworkers (1996), is discussed for the four closest proteins of this family of transporters. The sequence of the uracil permease gene of S. pombe has been deposited in the EMBL data bank under Accession Number X98696.
Topics: Amino Acid Sequence; Cloning, Molecular; Fungal Proteins; Membrane Transport Proteins; Molecular Sequence Data; Nucleotide Transport Proteins; Protein Structure, Secondary; Restriction Mapping; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Schizosaccharomyces; Sequence Alignment; Sequence Analysis, DNA; Transformation, Genetic
PubMed: 9730284
DOI: 10.1002/(SICI)1097-0061(199808)14:11<1051::AID-YEA287>3.0.CO;2-1 -
Nature Reviews. Molecular Cell Biology Sep 2001Studies of Niemann-Pick C (NPC) and Tangier diseases have led to the identification of the causative genes, NPC1 and ABCA1, respectively. Characterization of their... (Review)
Review
Studies of Niemann-Pick C (NPC) and Tangier diseases have led to the identification of the causative genes, NPC1 and ABCA1, respectively. Characterization of their protein products shows that NPC1 and ABCA1 are permeases that belong to two different superfamilies of efflux pumps, which might be important in subcellular lipid and cholesterol transport.
Topics: ATP-Binding Cassette Transporters; Biological Transport; Cholesterol; Humans; Lipid Metabolism, Inborn Errors; Membrane Transport Proteins; Niemann-Pick Diseases; Subcellular Fractions; Tangier Disease
PubMed: 11533723
DOI: 10.1038/35089558 -
Journal of Bacteriology Feb 1969Growth of Pseudomonas acidovorans in the presence of l-tryptophan resulted in the appearance of a tryptophan transport system which was extremely sensitive to sodium...
Growth of Pseudomonas acidovorans in the presence of l-tryptophan resulted in the appearance of a tryptophan transport system which was extremely sensitive to sodium azide or 2,4-dinitrophenol. Asparagine-grown cells possessed no detectable tryptophan "permease" activity. Substitution of l-kynurenine for l-tryptophan in the growth medium also induced the tryptophan permease activity, along with tryptophan oxygenase and kynurenine formamidase. This is the first reported example of the product induction of a permease activity. Irrespective of whether Pseudomonas cells are grown in the presence of d- or l-tryptophan, the resulting induced tryptophan permease activity is specific for the l-isomer. In addition, the radioactive compounds l-leucine, l-phenylalanine, or dl-5-hydroxytryptophan are not transported. When dl-5-fluorotryptophan is a component of the inducing medium (with l-tryptophan), induction of tryptophan permease activity, as well as tryptophan oxygenase, is inhibited. In the permease assay system, using normally induced cells, the fluoroanalogue inhibited strikingly tryptophan transport. Therefore, this analogue may inhibit induction by blocking inducer transport into the cell. When added to the l-tryptophan-inducing medium, dl-7-azatryptophan markedly enhanced induction of tryptophan oxygenase, but the level of tryptophan permease activity was not further elevated. The mechanism of this analogue is unclear at present. Invariant tryptophan permease activity levels are found in cells grown with 5 or 15 mml-tryptophan or 5 mml-kynurenine, whereas the respective tryptophan oxygenase levels are greatly different. Together with other results, these results indicate that the synthesis of tryptophan permease activity is not coordinate with that of tryptophan oxygenase. Tryptophan transport is strongly inhibited by l-formylkynurenine and by l-kynurenine. These two metabolites were prepared in radioactive form, and they are actively transported following bacterial growth on l-tryptophan or l-kynurenine. Preliminary results suggest the tryptophan permease activity may be distinct from the permease(s) activity for l-formylkynurenine and l-kynurenine. Kynurenine, then, is capable of inducing tryptophan permease and kynurenine permease activities.
Topics: Asparagine; Azides; Biological Transport, Active; Dinitrophenols; Enzyme Induction; Feedback; Kynurenine; Membrane Transport Proteins; Phenylalanine; Pseudomonas; Tryptophan; Tryptophan Oxygenase
PubMed: 5773025
DOI: 10.1128/jb.97.2.705-714.1969 -
Journal of Molecular Biology Apr 1992Escherichia coli lac permease is a polytopic integral membrane protein with six translocated (periplasmic) domains. Individual N-terminal cytoplasmic regions and... (Review)
Review
Escherichia coli lac permease is a polytopic integral membrane protein with six translocated (periplasmic) domains. Individual N-terminal cytoplasmic regions and membrane-spanning segments adjacent to each of the periplasmic domains acted as export signals for an attached sensor protein (alkaline phosphatase). However, the export activity of one of the spanning segments was considerably lower than that of the others, and was limited by the presence of a positively charged residue (Arg302). These observations are compatible with models of membrane protein insertion in which hydrophilic domains are translocated independently. However, the results suggest that efficient translocation may sometimes require interaction between individual spanning segments.
Topics: Amino Acid Sequence; Bacterial Proteins; Biological Transport; Carrier Proteins; Escherichia coli Proteins; Membrane Proteins; Membrane Transport Proteins; Molecular Sequence Data; Monosaccharide Transport Proteins; Protein Conformation; Symporters
PubMed: 1569545
DOI: 10.1016/0022-2836(92)90542-r -
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 -
European Journal of Biochemistry Jun 1983Two distinct regulatory mechanisms are responsible for the absence of general amino-acid permease activity in cells of the wild-type strain sigma 1278b of Saccharomyces...
Two distinct regulatory mechanisms are responsible for the absence of general amino-acid permease activity in cells of the wild-type strain sigma 1278b of Saccharomyces cerevisiae grown in the presence of ammonium ions. One is a reversible inactivation process which progressively develops upon addition of ammonium ions to a proline-grown culture, and completely suppresses the permease activity within one hour. This inactivation process is absent in mutants altered at the MUT2, MUT4, or PGR genetic loci. In these mutants, a repression of the formation of active permease may clearly be observed in the presence of ammonium ions. This second regulatory mechanism is absent in mutants affected at the GDHCR locus, which might code for a repressor molecule. It is also relieved in the presence of a glnts mutation (which makes the glutamine synthetase thermosensitive) suggesting glutamine as an effector. Two other ammonia-sensitive permeases, namely the proline permease and the ureidosuccinic-acid permease, seem to be subject to the same double regulation. Mutations affecting the structural gene of the anabolic NADP-linked glutamate dehydrogenase (gdhA) seem to completely prevent repression of the general amino-acid permease, while they partially suppress its inactivation in the presence of ammonium ions.
Topics: Ammonia; Enzyme Activation; Gene Expression Regulation; Membrane Transport Modulators; Membrane Transport Proteins; Mutation; Saccharomyces cerevisiae
PubMed: 6343083
DOI: 10.1111/j.1432-1033.1983.tb07438.x