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FEBS Letters Nov 2003Membrane transport proteins transduce free energy stored in electrochemical ion gradients into a concentration gradient and are a major class of membrane proteins, many... (Review)
Review
Membrane transport proteins transduce free energy stored in electrochemical ion gradients into a concentration gradient and are a major class of membrane proteins, many of which play important roles in human health and disease. Recently, the X-ray structure of the Escherichia coli lactose permease (LacY), an intensively studied member of a large group of related membrane transport proteins, was solved at 3.5 A. LacY is composed of N- and C-terminal domains, each with six transmembrane helices, symmetrically positioned within the molecule. The structure represents the inward-facing conformation, as evidenced by a large internal hydrophilic cavity open to the cytoplasmic side. The structure with a bound lactose homolog reveals the sugar-binding site in the cavity, and a mechanism for translocation across the membrane is proposed in which the sugar-binding site has alternating accessibility to either side of the membrane.
Topics: Binding Sites; Biological Transport, Active; Carbohydrate Metabolism; Escherichia coli; Escherichia coli Proteins; Membrane Transport Proteins; Models, Molecular; Monosaccharide Transport Proteins; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Static Electricity; Symporters; Thermodynamics
PubMed: 14630326
DOI: 10.1016/s0014-5793(03)01087-1 -
Scientific Reports Sep 2018The sugar transporter Lactose permease (LacY) of Escherichia coli has become a prototype to understand the underlying molecular details of membrane transport. Crystal...
The sugar transporter Lactose permease (LacY) of Escherichia coli has become a prototype to understand the underlying molecular details of membrane transport. Crystal structures have trapped the protein in sugar-bound states facing the periplasm, but with narrow openings unable to accommodate sugar. Therefore, the molecular details of sugar uptake remain elusive. In this work, we have used extended simulations and metadynamics sampling to explore a putative sugar-uptake pathway and associated free energy landscape. We found an entrance at helix-pair 2 and 11, which involved lipid head groups and residues Gln 241 and Gln 359. Furthermore, the protein displayed high flexibility on the periplasmic side of Phe 27, which is located at the narrowest section of the pathway. Interactions to Phe 27 enabled passage into the binding site, which was associated with a 24 ± 4 kJ/mol binding free energy in excellent agreement with an independent binding free energy calculation and experimental data. Two free energy minima corresponding to the two possible binding poses of the lactose analog β-D-galactopyranosyl-1-thio-β-D-galactopyranoside (TDG) were aligned with the crystal structure-binding pocket. This work outlines the chemical environment of a putative periplasmic sugar pathway and paves way for understanding substrate affinity and specificity in LacY.
Topics: Cell Membrane; Lipid Bilayers; Membrane Transport Proteins; Molecular Dynamics Simulation; Periplasm; Protein Conformation; Protein Transport; Thermodynamics
PubMed: 30254312
DOI: 10.1038/s41598-018-32624-7 -
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 -
Research in Microbiology 1990The cellobiose (cel) operon of Escherichia coli was recently sequenced and shown to consist of five genes, celABCDF (Parker and Hall, 1990). We have shown that the CelA,... (Review)
Review
The cellobiose (cel) operon of Escherichia coli was recently sequenced and shown to consist of five genes, celABCDF (Parker and Hall, 1990). We have shown that the CelA, CelB and CelC proteins possess amino acid sequences which are homologous to different domains of the lactose permease of Staphylococcus aureus. CelB corresponds to the integral membrane portion of the permease (IIcel) while CelC (IIIcel) and CelA (IVcel) correspond to the two cytoplasmic domains which appear to comprise the first and second phosphorylation sites in the permease, respectively. The cellobiose permease is the only one of several homologous sequenced permeases of the phosphoenolpyruvate:sugar phosphotransferase system which has its three known functional domains residing on distinct polypeptide chains.
Topics: Amino Acid Sequence; Cellobiose; Escherichia coli; Lactose; Membrane Transport Proteins; Molecular Sequence Data; Sequence Alignment; Staphylococcus aureus
PubMed: 2092358
DOI: 10.1016/0923-2508(90)90079-6 -
Journal of Bacteriology Aug 1990
Review
Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Bacteria; Biological Transport, Active; Energy Metabolism; Membrane Transport Proteins
PubMed: 2142937
DOI: 10.1128/jb.172.8.4133-4137.1990 -
Microbiology (Reading, England) Nov 2013Permeases of the prokaryotic phosphoenolpyruvate-sugar phosphotransferase system (PTS) catalyse sugar transport coupled to sugar phosphorylation. The lipid composition... (Review)
Review
Permeases of the prokaryotic phosphoenolpyruvate-sugar phosphotransferase system (PTS) catalyse sugar transport coupled to sugar phosphorylation. The lipid composition of a membrane determines the activities of these enzyme/transporters as well as the degree of coupling of phosphorylation to transport. We have investigated mechanisms of PTS permease biogenesis and identified cytoplasmic (soluble) forms of these integral membrane proteins. We found that the catalytic activities of the soluble forms differ from those of the membrane-embedded forms. Transport via the latter is much more sensitive to lipid composition than to phosphorylation, and some of these enzymes are much more sensitive to the lipid environment than others. While the membrane-embedded PTS permeases are always dimeric, the cytoplasmic forms are micellar, either monomeric or dimeric. Scattered published evidence suggests that other integral membrane proteins also exist in cytoplasmic micellar forms. The possible functions of cytoplasmic PTS permeases in biogenesis, intracellular sugar phosphorylation and permease storage are discussed.
Topics: Cell Membrane; Cytoplasm; Lipid Metabolism; Macromolecular Substances; Membrane Transport Proteins; Phosphoenolpyruvate Sugar Phosphotransferase System; Prokaryotic Cells; Protein Multimerization
PubMed: 23985145
DOI: 10.1099/mic.0.070953-0 -
Scientific Reports Aug 2017Several yeast species catabolize hydroxyderivatives of benzoic acid. However, the nature of carriers responsible for transport of these compounds across the plasma...
Several yeast species catabolize hydroxyderivatives of benzoic acid. However, the nature of carriers responsible for transport of these compounds across the plasma membrane is currently unknown. In this study, we analyzed a family of genes coding for permeases belonging to the major facilitator superfamily (MFS) in the pathogenic yeast Candida parapsilosis. Our results revealed that these transporters are functionally equivalent to bacterial aromatic acid: H symporters (AAHS) such as GenK, MhbT and PcaK. We demonstrate that the genes HBT1 and HBT2 encoding putative transporters are highly upregulated in C. parapsilosis cells assimilating hydroxybenzoate substrates and the corresponding proteins reside in the plasma membrane. Phenotypic analyses of knockout mutants and hydroxybenzoate uptake assays provide compelling evidence that the permeases Hbt1 and Hbt2 transport the substrates that are metabolized via the gentisate (3-hydroxybenzoate, gentisate) and 3-oxoadipate pathway (4-hydroxybenzoate, 2,4-dihydroxybenzoate and protocatechuate), respectively. Our data support the hypothesis that the carriers belong to the AAHS family of MFS transporters. Phylogenetic analyses revealed that the orthologs of Hbt permeases are widespread in the subphylum Pezizomycotina, but have a sparse distribution among Saccharomycotina lineages. Moreover, these analyses shed additional light on the evolution of biochemical pathways involved in the catabolic degradation of hydroxyaromatic compounds.
Topics: Biological Transport; Candida parapsilosis; Gene Knockout Techniques; Hydroxybenzoates; Membrane Transport Proteins; Metabolic Networks and Pathways; Phylogeny; Sequence Homology
PubMed: 28827635
DOI: 10.1038/s41598-017-09408-6 -
Scientific Reports Oct 2017Lipids play key roles in Biology. Mechanical properties of the lipid bilayer influence their neighbouring membrane proteins, however it is unknown whether different...
Lipids play key roles in Biology. Mechanical properties of the lipid bilayer influence their neighbouring membrane proteins, however it is unknown whether different membrane protein properties have the same dependence on membrane mechanics, or whether mechanics are tuned to specific protein processes of the protein. We study the influence of lipid lateral pressure and electrostatic effects on the in vitro reconstitution, folding, stability and function of a representative of the ubiquitous major facilitator transporter superfamily, lactose permease. Increasing the outward chain lateral pressure in the bilayer, through addition of lamellar phosphatidylethanolamine lipids, lowers lactose permease folding and reconstitution yields but stabilises the folded state. The presence of phosphatidylethanolamine is however required for correct folding and function. An increase in headgroup negative charge through the addition of phosphatidylglycerol lipids favours protein reconstitution but is detrimental to topology and function. Overall the in vitro folding, reconstitution, topology, stability and function of lactose permease are found to have different dependences on bilayer composition. A regime of lipid composition is found where all properties are favoured, even if suboptimal. This lays ground rules for rational control of membrane proteins in nanotechnology and synthetic biology by manipulating global bilayer properties to tune membrane protein behaviour.
Topics: Lipid Bilayers; Membrane Transport Proteins; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Folding; Protein Stability
PubMed: 29026149
DOI: 10.1038/s41598-017-13290-7 -
Comptes Rendus Biologies Jun 2005More than 20% of the genes sequenced thus far appear to encode polytopic transmembrane proteins involved in a multitude of critical functions, particularly energy and... (Review)
Review
More than 20% of the genes sequenced thus far appear to encode polytopic transmembrane proteins involved in a multitude of critical functions, particularly energy and signal transduction. Many are important with regard to human disease (e.g., depression, diabetes, drug resistance), and many drugs are targeted to membrane transport proteins (e.g., fluoxetine and omeprazole). However, the number of crystal structures of membrane proteins, especially ion-coupled transporters, is very limited. Recently, an inward-facing conformer of the Escherichia coli lactose permease (LacY), a paradigm for the Major Facilitator Superfamily, which contains almost 4000 members, was solved at about 3.5 A in collaboration with Jeff Abramson and So Iwata at Imperial College London. This intensively studied membrane transport protein is composed of two pseudo-symmetrical 6-helix bundles with a large internal cavity containing bound sugar and open to the cytoplasm only. Based on the structure and a large body of biochemical and biophysical evidence, a mechanism is proposed in which the binding site is alternatively accessible to either side of the membrane.
Topics: Amino Acid Sequence; Binding Sites; Biological Transport; Escherichia coli; Escherichia coli Proteins; Membrane Transport Proteins; Models, Molecular; Molecular Sequence Data; Monosaccharide Transport Proteins; Protein Structure, Secondary; Protons; Symporters
PubMed: 15950162
DOI: 10.1016/j.crvi.2005.03.008 -
The Journal of Biological Chemistry Feb 2022Bacterial transporters are difficult to study using conventional electrophysiology because of their low transport rates and the small size of bacterial cells. Here, we...
Bacterial transporters are difficult to study using conventional electrophysiology because of their low transport rates and the small size of bacterial cells. Here, we applied solid-supported membrane-based electrophysiology to derive kinetic parameters of sugar translocation by the Escherichia coli xylose permease (XylE), including functionally relevant mutants. Many aspects of the fucose permease (FucP) and lactose permease (LacY) have also been investigated, which allow for more comprehensive conclusions regarding the mechanism of sugar translocation by transporters of the major facilitator superfamily. In all three of these symporters, we observed sugar binding and transport in real time to determine K, V, K, and k values for different sugar substrates. K and k values were attainable because of a conserved sugar-induced electrogenic conformational transition within these transporters. We also analyzed interactions between the residues in the available X-ray sugar/H symporter structures obtained with different bound sugars. We found that different sugars induce different conformational states, possibly correlating with different charge displacements in the electrophysiological assay upon sugar binding. Finally, we found that mutations in XylE altered the kinetics of glucose binding and transport, as Q175 and L297 are necessary for uncoupling H and d-glucose translocation. Based on the rates for the electrogenic conformational transition upon sugar binding (>300 s) and for sugar translocation (2 s - 30 s for different substrates), we propose a multiple-step mechanism and postulate an energy profile for sugar translocation. We also suggest a mechanism by which d-glucose can act as an inhibitor for XylE.
Topics: Carbohydrate Metabolism; Electrophysiology; Escherichia coli; Escherichia coli Proteins; Glucose; Kinetics; Membrane Transport Proteins; Monosaccharide Transport Proteins; Sugars; Symporters
PubMed: 34929170
DOI: 10.1016/j.jbc.2021.101505