Article

Expression of the rat GLUT1 glucose transporter in the yeast Saccharomyces cerevisiae

May 1996
Biochemical Journal 315 ( Pt 1)(Pt 1):177-82

May 1996
315 ( Pt 1)(Pt 1):177-82

Source
PubMed

Authors:

We expressed the rat GLUT1 facilitative glucose transporter in the yeast Saccharomyces cerevisiae with the use of a galactose-inducible expression system. Confocal immunofluorescence microscopy indicated that a majority of this protein is retained in an intracellular structure that probably corresponds to endoplasmic reticulum. Yeast cells expressing GLUT1 exhibited little increase in glucose-transport activity. We prepared a crude membrane fraction from these cells and made liposomes with this fraction using the freeze-thaw/sonication method. In this reconstituted system, D-glucose-transport activity was observed with a Km for D-glucose of 3.4 +/- 0.2 mM (mean +/- S.E.M.) and was inhibited by cytochalasin B (IC50= 0.44 +/- 0.03 microM), HgCl2 (IC50)= 3.5 +/- 0.5 microM), phloretin (IC50= 49 +/- 12 microM) and phloridzin (IC50= 355 +/- 67 microM). To compare these properties with native GLUT1 we made reconstituted liposomes with a membrane fraction prepared from human erythrocytes, in which the Km of D-glucose transport and ICs of these inhibitors were approximately equal to those obtained with GLUT1 made by yeast. When the relative amounts of GLUT1 in the crude membrane fractions were measured by quantitative immunoblotting, the specific activity of the yeast-made GLUT1 was 110% of erythrocyte GLUT1, indicating that GLUT1 expressed in yeast is fully active in glucose transport.

Tyrosine kinase inhibitors reduce glucose uptake by binding to an exofacial site on hGLUT‐1: Effects on 18 F‐FDG PET uptake

Article

Full-text available

May 2021
CTS-CLIN TRANSL SCI

Positron emission tomography (PET) using 2‐deoxy‐2‐[18F]fluoro‐D‐glucose ([18F]FDG), a marker of energy metabolism and cell proliferation, is routinely used in the clinic to assess patient response to chemotherapy and to monitor tumor growth. Treatment with some tyrosine kinase inhibitors (TKIs) causes changes in blood glucose levels in both non‐diabetic and diabetic patients. We evaluated the interaction of several classes of TKIs with human glucose transporter‐1 (hGLUT‐1) in FaDu and GIST‐1 cells by measuring [3H]2‐deoxy‐D‐glucose ([3H]2‐DG) and [3H]FDG uptake. Uptake of both was inhibited to varying extents by the TKIs, and representative TKIs from each class showed competitive inhibition of [3H]2‐DG uptake. In GIST‐1 cells, [3H]FDG uptake inhibition by temsirolimus and nilotinib was irreversible, whereas inhibition by imatinib, gefitinib, and pazopanib was reversible. Molecular modeling studies showed that TKIs form multiple hydrogen bonds with polar residues of the sugar binding site (i.e., Q161, Q282, Q283, N288, N317, and W388), and van der Waals interactions with the H‐pocket site. Our results showed interaction of TKIs with amino acid residues at the glucose binding site to inhibit glucose uptake by hGLUT‐1. We showed that inhibition of hGLUT‐1 by TKIs could alter glucose levels in patients treated with TKIs, leading to hypoglycemia and fatigue, although further studies are required to evaluate roles of other SLC2 and SLC5 members. In addition, TKIs could affect tumor [18F]FDG uptake, increasingly used as a marker of tumor response. hGLUT‐1 inhibition by TKIs may have implications for routine [18F]FDG‐PET monitoring of tumor response in patients.

Molekulargenetische Untersuchungen zur Funktion des FGY1-Gens bei der Expression heterologer Glukosetransporter in Saccharomyces cerevisiae Diplomarbeit

Article

Dörthe Voss

Funktionsanalyse des Saccharomyces cerevisiae Proteins Fgy1 und dessen Einfluss auf die heterologe Expression der Glukosetransporter GLUT1 und GLUT4 aus Säugetieren

Article

Dörthe Voss

Im Rahmen der vorliegenden Dissertation sollte die Funktion des bislang unbekannten Proteins Fgy1/Efr3 aus der Hefe S. cerevisiae charakterisiert werden. Die Funktionsanalyse des Proteins wurde für unterschiedliche zelluläre Prozesse der Hefe durchgeführt. Eine Klärung der Funktion von Fgy1 sollte helfen, seinen inhibitorischen Effekt auf die Funktionalität der heterolog in Hefe exprimierten Glukosetransporter GLUT1 und GLUT4 aus Säugetieren zu verstehen. Saccharosedichtegradientenzentrifugationsexperimente und Nachweis eines Fgy1-GFP Fusions-proteins deuteten auf eine Lokalisierung von Fgy1 an der Plasmamembran bzw. in endosomalen Vesikeln hin. Phänotyp-Microarray-Analysen eines fgy1-Deletionsstammes zeigten eine pleiotrophe Funktion des Proteins an. In verschiedenen Experimenten konnte ein genereller Einfluss von Fgy1 auf den intrazellulären Transport von Plasmamembranproteinen oder die Phospholipidzusammensetzung der Plasmamembran ausgeschlossen werden. Untersuchungen des PHO-Signalweges deuteten auf eine Funktion von Fgy1 bei der Phosphataufnahme hin. Mittels einer Transposongenbank wurden mit HTA1 und CTR9 weitere Gene gefunden, deren Deletion eine funktionelle Expression von GLUT1 in der Hefe erlauben. Die Produkte beider Gene spielen eine Rolle in der Transkription. Aber weder für die hta1-Deletion noch die fgy1-1 Mutation konnte ein Effekt auf den Expressionslevel des GLUT1-Proteins nachgewiesen werden. Einen ebenfalls positiven Effekt auf die funktionelle Expression von GLUT1 zeigte eine Deletion von ERG6, das für die C-24 Sterol-Methyltransferase im Ergosterolbiosyntheseweg kodiert. Untersuchungen mit Sekretionsmutanten der Hefe deuteten auf eine Funktion von Fgy1 bei der Fusion von sekretorischen Vesikeln mit der Plasmamembran nach der Bildung des SNARE-Komplexes. Auch der Nachweis einer fehlenden Zugänglichkeit des GLUT1-Proteins von ausserhalb der Zelle über Modifizierung mit TNBS und anschließende Detektion von GLUT1 ließ darauf schließen, dass das Fgy1 Protein eine korrekte Insertion von GLUT1 in die Plasmamembran der Zellen verhindert. Rekonstitution des Transporters in Liposomen ergab jedoch eine von Fgy1 unabhängige Glukosetransportaktivität. Eine Blockierung der Endozytose in rsp5-Mutantenstämmen der Hefe führte nicht zu einer funktionellen Expression von GLUT1. Zusammengefasst deuten die Ergebnisse auf eine Beteiligung von Fgy1 bei der Fusion von sekretorischen Vesikeln mit der Plasmamembran. Es wurden einige Hinweise erhalten, die dafür sprechen, dass Fgy1, nachdem post-Golgi Vesikel über den SNARE-Komplex an die Membran gebunden wurden, eine regulatorische Funktion dahingehend ausübt, welche Proteine in die Plasmamembran inseriert werden.

Tryptophan 388 in Putative Transmembrane Segment 10 of the Rat Glucose Transporter Glut1 Is Essential for Glucose Transport

Article

Full-text available

Nov 1998

The molecular mechanism of substrate recognition in membrane transport is not well understood. Two amino acid residues, Tyr446 and Trp455 in transmembrane segment 10 (TM10), have been shown to be important for galactose recognition by the yeast Gal2 transporter; Tyr446 was found to be essential in that its replacement by any of the other 19 amino acids abolished transport activity (Kasahara, M., Shimoda, E., and Maeda, M. (1997) J. Biol. Chem. 272, 16721-16724). The Glut1 glucose transporter of animal cells belongs to the same Glut transporter family as does Gal2 and thus might be expected to show a similar mechanism of substrate recognition. The role of the two amino acids, Phe379 and Trp388, in rat Glut1 corresponding to Tyr446 and Trp455 of Gal2 was therefore studied. Phe379 and Trp388 were individually replaced with each of the other 19 amino acids, and the mutant Glut1 transporters were expressed in yeast. The expression level of most mutants was similar to that of the wild-type Glut1, as revealed by immunoblot analysis. Glucose transport activity was assessed by reconstituting a crude membrane fraction of the yeast cells in liposomes. No significant glucose transport activity was observed with any of Trp388 mutants, whereas the Phe379 mutants showed reduced or no activity. These results indicate that the two aromatic amino acids in TM10 of Glut1 are important for glucose transport. However, unlike Gal2, the residue at the cytoplasmic end of TM10 (Trp388, corresponding to Trp455 of Gal2), rather than that in the middle of TM10 (Phe379, corresponding to Tyr446 of Gal2), is essential for transport activity.

Inhibition of human GLUT1 and GLUT5 by plant carbohydrate products; insights into transport specificity

Article

Full-text available

Aug 2015

Glucose transporters GLUT1 (transports glucose) and GLUT5 (transports fructose), in addition to their functions in normal metabolism, have been implicated in several diseases including cancer and diabetes. While GLUT1 has several inhibitors, none have been described for GLUT5. By transport activity assays we found two plant products, rubusoside (from Rubus suavissimus) and astragalin-6-glucoside (a glycosylated derivative of astragalin, from Phytolacca americana) that inhibited human GLUT5. These plants are utilized in traditional medicine: R. suavissimus for weight loss and P. americana for cancer treatment, but the molecular interactions of these products are unknown. Rubusoside also inhibited human GLUT1, but astragalin-6-glucoside did not. In silico analysis of rubusoside:protein interactions pinpointed a major difference in substrate cavity between these transporters, a residue that is a tryptophan in GLUT1 but an alanine in GLUT5. Investigation of mutant proteins supported the importance of this position in ligand specificity. GLUT1W388A became susceptible to inhibition by astragalin-6-glucoside and resistant to rubusoside. GLUT5A396W transported fructose and also glucose, and maintained inhibition by rubusoside and astragalin-6-glucoside. Astragalin-6-glucoside can serve as a starting point in the design of specific inhibitors for GLUT5. The application of these studies to understanding glucose transporters and their interaction with substrates and ligands is discussed.

Characterization of rat Glut4 glucose transporter expressed in the yeast Saccharomyces cerevisiae: comparison with Glut1 glucose transporter 1 The nucleotide sequence data of rat GLUT4 reported in this paper have been submitted to the GenBank/DDBJ/EMBL database under the accession number D28561. 1

Article

Feb 1997
BBA-BIOMEMBRANES

Rat Glut4 glucose transporter was expressed in the yeast Saccharomyces cerevisiae, but was retained in an intracellular membranous compartment and did not contribute to glucose uptake by intact cells. A crude membrane fraction was prepared and reconstituted in liposome with the use of the freeze–thaw/sonication method. d-glucose-specific, cytochalasin B inhibitable glucose transport activity was observed. Kinetic analysis of d-glucose transport was performed by an integrated rate equation approach. The Km under zero-trans influx condition was 12±1 mM (mean±S.E., n=3) and that under equilibrium exchange condition was 22±3 mM (n=4). d-glucose transport was inhibited by 2-deoxy-d-glucose or 3-O-methyl-d-glucose, but not by d-allose, d-fructose or l-glucose. Cytochalasin B, phloretin and phlorizin inhibited d-glucose transport, but neither p-chloromercuribenzoic acid (pCMB) (0–0.1 mM) nor p-chloromercuribenzene sulfonic acid (pCMBS) (0–1.0 mM) inhibited this activity. High concentrations of HgCl2 were required to inhibit d-glucose transport (IC50, 370 μM). Comparing these properties to those of rat Glut1, we found two notable differences; (1) in Glut1, Km under zero-trans influx was significantly smaller than that under equilibrium exchange but in Glut4 less than two-fold difference was seen between these two Km values; and (2) Glut1 was inhibited with pCMB, pCMBS and low concentrations of HgCl2 (IC50, 3.5 μM), whereas Glut4 was almost insensitive to SH reagents. To examine the role of the exofacial cysteine, we replaced Met-455 of Glut4 (corresponding to Cys-429 of Glut1) with cysteine. The mutated Glut4 was inhibited by pCMB or pCMBS and the IC50 of HgCl2 decreased to 47 μM, whereas Km, substrate specificity and the sensitivity to cytochalasin B were not significantly changed, indicating that the existence of exofacial cysteine contributed only to increase SH sensitivity in Glut4.

Tricks of the trade used to accelerate high-resolution structure determination of membrane proteins

Article

Jun 2010
FEBS LETT

The rate at which X-ray structures of membrane proteins are solved is on a par with that of soluble proteins in the late 1970s. There are still many obstacles facing the membrane protein structural community. Recently, there have been several technical achievements in the field that have started to dramatically accelerate structural studies. Here, we summarize these so-called 'tricks-of-thetrade' and include case studies of several mammalian transporters. (C) 2010 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Functional Expression of the Human Glucose Transporters GLUT2 and GLUT3 in Yeast Offers Novel Screening Systems for GLUT-Targeting Drugs

Article

Full-text available

Feb 2021

Human GLUT2 and GLUT3, members of the GLUT/SLC2 gene family, facilitate glucose transport in specific tissues. Their malfunction or misregulation is associated with serious diseases, including diabetes, metabolic syndrome, and cancer. Despite being promising drug targets, GLUTs have only a few specific inhibitors. To identify and characterize potential GLUT2 and GLUT3 ligands, we developed a whole-cell system based on a yeast strain deficient in hexose uptake, whose growth defect on glucose can be rescued by the functional expression of human transporters. The simplicity of handling yeast cells makes this platform convenient for screening potential GLUT2 and GLUT3 inhibitors in a growth-based manner, amenable to high-throughput approaches. Moreover, our expression system is less laborious for detailed kinetic characterization of inhibitors than alternative methods such as the preparation of proteoliposomes or uptake assays in Xenopus oocytes. We show that functional expression of GLUT2 in yeast requires the deletion of the extended extracellular loop connecting transmembrane domains TM1 and TM2, which appears to negatively affect the trafficking of the transporter in the heterologous expression system. Furthermore, single amino acid substitutions at specific positions of the transporter sequence appear to positively affect the functionality of both GLUT2 and GLUT3 in yeast. We show that these variants are sensitive to known inhibitors phloretin and quercetin, demonstrating the potential of our expression systems to significantly accelerate the discovery of compounds that modulate the hexose transport activity of GLUT2 and GLUT3.

Small‐Molecule Inhibition of Glucose Transporters GLUT‐1–4

Article

Full-text available

Nov 2019
CHEMBIOCHEM

Glucose addiction is observed in cancer and other diseases that are associated with hyperproliferation. The development of compounds that restrict glucose supply and decrease glycolysis has great potential for the development of new therapeutic approaches. Addressing facilitative glucose transporters (GLUTs), which are often upregulated in glucose‐dependent cells, is therefore of particular interest. This article reviews a selection of potent, isoform‐selective GLUT inhibitors and their biological characterization. Potential therapeutic applications of GLUT inhibitors in oncology and other diseases that are linked to glucose addiction are discussed.

Sugar flux and signaling in plant-microbe interactions

Article

Nov 2017

Plant breeders have developed crop plants that are resistant to pests, but the continual evolution of pathogens creates the need to iteratively develop new control strategies. Molecular tools have allowed us to gain deep insights into disease responses, allowing for more efficient, rational engineering of crops that are more robust or resistant to a greater number of pathogen variants. Here we describe the roles in disease progress of SWEET and STP transporters, which are membrane proteins that mediate transport of sugars across the plasma membrane. We discuss how these transporters may enhance or restrict disease through controlling the level of nutrients provided to pathogens and if the transporters play a role in sugar signaling for disease resistance. This review indicates open questions that require further research and proposes the use of genome editing technologies for engineering disease resistance.

Establishing a yeast-based screening system for discovery of human GLUT5 inhibitors and activators

Article

Full-text available

Dec 2017

Human GLUT5 is a fructose-specific transporter in the glucose transporter family (GLUT, SLC2 gene family). Its substrate-specificity and tissue-specific expression make it a promising target for treatment of diabetes, metabolic syndrome and cancer, but few GLUT5 inhibitors are known. To identify and characterize potential GLUT5 ligands, we developed a whole-cell system based on a yeast strain deficient in fructose uptake, in which GLUT5 transport activity is associated with cell growth in fructose-based media or assayed by fructose uptake in whole cells. The former method is convenient for high-throughput screening of potential GLUT5 inhibitors and activators, while the latter enables detailed kinetic characterization of identified GLUT5 ligands. We show that functional expression of GLUT5 in yeast requires mutations at specific positions of the transporter sequence. The mutated proteins exhibit kinetic properties similar to the wild-type transporter and are inhibited by established GLUT5 inhibitors N-[4-(methylsulfonyl)-2-nitrophenyl]-1,3-benzodioxol-5-amine (MSNBA) and (−)-epicatechin-gallate (ECG). Thus, this system has the potential to greatly accelerate the discovery of compounds that modulate the fructose transport activity of GLUT5.

Expression of SGLT1 in Human Hearts and Impairment of Cardiac Glucose Uptake by Phlorizin during Ischemia-Reperfusion Injury in Mice

Article

Full-text available

Jun 2015
PLOS ONE

Objective: Sodium-glucose cotransporter 1 (SGLT1) is thought to be expressed in the heart as the dominant isoform of cardiac SGLT, although more information is required to delineate the subtypes of SGLTs in human hearts. Moreover, the functional role of SGLTs in the heart remains to be fully elucidated. We herein investigated whether SGLT1 is expressed in human hearts and whether SGLTs significantly contribute to cardiac energy metabolism during ischemia-reperfusion injury (IRI) via enhanced glucose utilization in mice. Methods and results: We determined that SGLT1 was highly expressed in both human autopsied hearts and murine perfused hearts, as assessed by immunostaining and immunoblotting with membrane fractionation. To test the functional significance of the substantial expression of SGLTs in the heart, we studied the effects of a non-selective SGLT inhibitor, phlorizin, on the baseline cardiac function and its response to ischemia-reperfusion using the murine Langendorff model. Although phlorizin perfusion did not affect baseline cardiac function, its administration during IRI significantly impaired the recovery in left ventricular contractions and rate pressure product, associated with an increased infarct size, as demonstrated by triphenyltetrazolium chloride staining and creatine phosphokinase activity released into the perfusate. The onset of ischemic contracture, which indicates the initiation of ATP depletion in myocardium, was earlier with phlorizin. Consistent with this finding, there was a significant decrease in the tissue ATP content associated with reductions in glucose uptake, as well as lactate output (indicating glycolytic flux), during ischemia-reperfusion in the phlorizin-perfused hearts. Conclusions: Cardiac SGLTs, possibly SGLT1 in particular, appear to provide an important protective mechanism against IRI by replenishing ATP stores in ischemic cardiac tissues via enhancing availability of glucose. The present findings provide new insight into the significant role of SGLTs in optimizing cardiac energy metabolism, at least during the acute phase of IRI.

Accumulation of Ascorbate by Endocrine-Regulated and Glucose-Sensitive Transport of Dehydroascorbic Acid in Luteinized Rat Ovarian Cells1

Article

Feb 1998

P H Kodaman

The corpus luteum is notable for very high levels of ascorbic acid. In luteal cells, ascorbic acid depletion occurs as a result of consumption during radical scavenging, inhibition of ascorbic acid uptake, and stimulation of its secretion. Oxidation of ascorbic acid generates dehydroascorbic acid (DHAA). Although levels of DHAA in blood are much lower than those of ascorbic acid, DHAA serves as the major transportable form of ascorbate for certain cell types. The aim of the present studies was to investigate whether DHAA transport is a potential mechanism for conserving ascorbic acid in the corpus luteum. DHAA uptake by rat luteal cells precultured for 24 h was linear for up to 30 min. Kinetics studies showed that uptake of DHAA was a concentration-dependent and saturable process with an estimated Michaelis constant (Km) of 830 microM and a maximum velocity (Vmax) of 700 pmol/min per 10(6) cells, a rate 50 times that of ascorbate transport. More than 90% of DHAA was reduced to ascorbic acid within 2 h of cellular uptake. DHAA uptake was energy- and microfilament-dependent, as transport was inhibited by 2,4-dinitrophenol (1 mM) and cytochalasin B (10 microM). Menadione (50 microM), an intracellular generator of reactive oxygen species, also markedly reduced DHAA uptake. In contrast to ascorbic acid transport, DHAA uptake was potently inhibited by glucose and phloretin, an inhibitor of glucose transporters, with IC50s of approximately 5 mM and 10 microM, respectively. DHAA uptake appears to occur via an insulin-insensitive transporter, as insulin (10 nM) had no effect on uptake. However, 24-h preincubation with insulin-like growth factor (IGF)-I dose-dependently (10-100 ng/ml) stimulated DHAA uptake; similar concentrations of IGF-II had no effect. The secretion of radioactivity by cells preloaded with radiolabeled DHAA was significantly increased by prostaglandin F2alpha (1 microM). The ability of luteal cells to transport DHAA in a regulated manner may serve to maintain vital levels of ascorbic acid within the corpus luteum.

VEGF secretion by adipose tissue-derived regenerative cells is impaired under hyperglycemic conditions via glucose transporter activation and ROS increase

Article

Full-text available

Dec 2014

Transplantation of cultured adipose-derived regenerative cells (ADRCs) into ischemic tissues promotes neovascularization and blood perfusion recovery. These effects are attenuated in diabetes patients. We examined the effects of hyperglycemia on the angiogenic capacity of ADRCs derived from Wistar rats both in vivo and in vitro. Cultured ADRCs were predominantly composed of CD90 positive cells; prevalence of CD90 positive cells was not affected by hyperglycemia. mRNA and protein levels of vascular endothelial growth factor (VEGF) were significantly decreased in ADRCs under hyperglycemic conditions independent of osmolarity, whereas mRNA levels of hepatocyte growth factor and fibroblast growth factor were unaffected. Since ADRCs express glucose transporter proteins GLUT1, 3 and 4, we examined the effects of the glucose transporter inhibitor phloretin on reactive oxygen species (ROS) and angiogenic factors. Phloretin decreased the glucose uptake rate, reduced ROS, and increased VEGF mRNA in ADRCs exposed to a hyperglycemic condition. In vivo transplantation of ADRCs cultured under hyperglycemic conditions into mouse ischemic limbs resulted in significantly decreased blood perfusion and capillary density in ischemic regions compared with transplantation of ADRCs cultured under normoglycemic conditions. These results suggest that hyperglycemia impaired VEGF production in ADRCs via an increase of ROS, impairing the angiogenic capacity of ADRCs transplanted into ischemic limbs.

Effect of D-Fructose on Sugar Transport Systems in Trichoplusia ni Cells and Photolabeling of the Trichoplusia ni Cell-Expressed Human HepG2 Type Glucose Transport Protein

Article

Jan 2014

Chong-Kee Lee

Trichoplusia ni cells are used as a host permissive cell line in the baculovirus expression system, which is useful for large-scale production of human sugar transport proteins. However, the activity of endogenous sugar transport systems in insect cells is extremely high. Therefore, the transport activity resulting from the expression of exogenous transporters is difficult to detect. Furthermore, very little is known about the nature of endogenous insect transporters. To exploit the expression system further, the effect of D-fructose on 2-deoxy-D-glucose (2dGlc) transport by T. ni cells was investigated, and T. ni cell-expressed human transporters were photolabeled with [] cytochalasin B to develop a convenient method for measuring the biological activity of insect cell-expressed transporters. The uptake of 1 mM 2dGlc by uninfected- and recombinant AcMPV-GTL infected cells was examined in the presence and absence of 300 mM of D-fructose, with and without of cytochalasin B. The sugar uptake in the uninfected cells was strongly inhibited by fructose but only poorly inhibited by cytochalasin B. Interestingly, the AcMPV-GTL-infected cells showed an essentially identical pattern of transport inhibition, and the rate of 2dGlc uptake was somewhat less than that seen in the non-infected cells. In addition, a sharply labeled peak was produced only in the AcMPV-GTL-infected membranes labeled with [] cytochalasin B in the presence of L-glucose. No peak of labeling was seen in the membranes prepared from the uninfected cells. Furthermore, photolabeling of the expressed protein was completely inhibited by the presence of D-glucose, demonstrating the stereoselectivity of labeling.

Crystal structure of a glucose/H+ symporter and its mechanism of action

Article

Oct 2013
P NATL ACAD SCI USA

Significance Glucose transporters mediate the exchange of glucose and related hexoses in living cells. In humans, these transporters (known as GLUT) are involved in several diseases, including cancer and diabetes. The glucose transporter from Staphylococcus epidermidis (GlcP Se ) has high sequence homology to human GLUT, is specific for glucose, and is inhibited by human GLUT inhibitors. The crystal structure of GlcP Se , along with site-directed mutagenesis and transport-activity studies, provide insight into the mechanism of glucose transport.

Phloretin differentially inhibits volume-sensitive and cyclic AMP-activated, but not Ca-activated, Cl− channels

Article

Full-text available

Aug 2001
BRIT J PHARMACOL

Some phenol derivatives are known to block volume‐sensitive Cl ⁻ channels. However, effects on the channel of the bisphenol phloretin, which is a known blocker of glucose uniport and anion antiport, have not been examined. In the present study, we investigated the effects of phloretin on volume‐sensitive Cl ⁻ channels in comparison with cyclic AMP‐activated CFTR Cl ⁻ channels and Ca ²⁺ ‐activated Cl ⁻ channels using the whole‐cell patch‐clamp technique. Extracellular application of phloretin (over 10 μ M ) voltage‐independently, and in a concentration‐dependent manner (IC 50 ∼30 μ M ), inhibited the Cl ⁻ current activated by a hypotonic challenge in human epithelial T84, Intestine 407 cells and mouse mammary C127/CFTR cells. In contrast, at 30 μ M phloretin failed to inhibit cyclic AMP‐activated Cl ⁻ currents in T84 and C127/CFTR cells. Higher concentrations (over 100 μ M ) of phloretin, however, partially inhibited the CFTR Cl ⁻ currents in a voltage‐dependent manner. At 30 and 300 μ M , phloretin showed no inhibitory effect on Ca ²⁺ ‐dependent Cl ⁻ currents induced by ionomycin in T84 cells. It is concluded that phloretin preferentially blocks volume‐sensitive Cl ⁻ channels at low concentrations (below 100 μ M ) and also inhibits cyclic AMP‐activated Cl ⁻ channels at higher concentrations, whereas phloretin does not inhibit Ca ²⁺ ‐activated Cl ⁻ channels in epithelial cells. British Journal of Pharmacology (2001) 133 , 1096–1106; doi: 10.1038/sj.bjp.0704159

Radioligand Binding Analysis as a Tool for Quality Control of GPCR Production for Structural Characterization: Adenosine A 2a R as a Template for Study

Article

Feb 2012
Curr Protoc Protein Sci

Functional characterization of G protein-coupled receptors is essential to ascertain the suitability of a protein target for downstream studies and to help develop optimal expression and isolation procedures. Radioligand binding analysis is a well-established technique, which allows direct measurement of the amount of functional receptor in a sample. It can be readily applied to both membrane-bound and soluble receptor samples and is an ideal method for monitoring the amount of functional protein at each stage in the expression and isolation process. This unit presents protocols for the radioligand binding analysis of the human adenosine A(2a) receptor and provides examples of how these assays can be used at several stages to help optimize expression, solubilization, and isolation procedures.

Reduction of Glucose Uptake through Inhibition of Hexose Transporters and Enhancement of Their Endocytosis by Methylglyoxal in Saccharomyces cerevisiae

Article

Full-text available

Nov 2011
J BIOL CHEM

Diabetes mellitus is characterized by an impairment of glucose uptake even though blood glucose levels are increased. Methylglyoxal is derived from glycolysis and has been implicated in the development of diabetes mellitus, because methylglyoxal levels in blood and tissues are higher in diabetic patients than in healthy individuals. However, it remains to be elucidated whether such factors are a cause, or consequence, of diabetes. Here, we show that methylglyoxal inhibits the activity of mammalian glucose transporters using recombinant Saccharomyces cerevisiae cells genetically lacking all hexose transporters but carrying cDNA for human GLUT1 or rat GLUT4. We found that methylglyoxal inhibits yeast hexose transporters also. Glucose uptake was reduced in a stepwise manner following treatment with methylglyoxal, i.e. a rapid reduction within 5 min, followed by a slow and gradual reduction. The rapid reduction was due to the inhibitory effect of methylglyoxal on hexose transporters, whereas the slow and gradual reduction seemed due to endocytosis, which leads to a decrease in the amount of hexose transporters on the plasma membrane. We found that Rsp5, a HECT-type ubiquitin ligase, is responsible for the ubiquitination of hexose transporters. Intriguingly, Plc1 (phospholipase C) negatively regulated the endocytosis of hexose transporters in an Rsp5-dependent manner, although the methylglyoxal-induced endocytosis of hexose transporters occurred irrespective of Plc1. Meanwhile, the internalization of hexose transporters following treatment with methylglyoxal was delayed in a mutant defective in protein kinase C.

Experimental Pharmacology of Glucosamine Sulfate

Article

Full-text available

Jan 2011

Several clinical studies demonstrated that glucosamine sulfate (GS) is effective in controlling osteoarthritis (OA), showing a structure-modifying action. However, little is known about the molecular mechanism(s) by which GS exerts such action and about the effects of GS at a tissue level on osteoarthritic cartilage and other joint structures. Here we provide mechanistic evidence suggesting that in vitro GS attenuates NF-κB activation at concentrations in the range of those observed after GS administration to volunteers and patients, thus strengthening previous findings. Furthermore, we describe the effects of GS at a tissue level on the progression of the disease in a relevant model of spontaneous OA, the STR/ort mouse. In this model, the administration of GS at human corresponding doses was associated with a significant decrease of OA scores. Histomorphometry showed that the lesion surface was also significantly decreased, while the number of viable chondrocytes within the matrix was significantly increased. GS improved the course of OA in the STR/Ort mouse, by delaying cartilage breakdown as assessed histologically and histomorphometrically.

Facilitative plasma membrane transporters function during ER transit

Article

Full-text available

Mar 2010
FASEB J

Although biochemical studies suggested a high permeability of the endoplasmic reticulum (ER) membrane for small molecules, proteomics identified few specialized ER transporters. To test functionality of transporters during ER passage, we tested whether glucose transporters (GLUTs, SGLTs) destined for the plasma membrane are active during ER transit. HepG2 cells were characterized by low-affinity ER transport activity, suggesting that ER uptake is protein mediated. The much-reduced capacity of HEK293T cells to take up glucose across the plasma membrane correlated with low ER transport. Ectopic expression of GLUT1, -2, -4, or -9 induced GLUT isoform-specific ER transport activity in HEK293T cells. In contrast, the Na(+)-glucose cotransporter SGLT1 mediated efficient plasma membrane glucose transport but no detectable ER uptake, probably because of lack of a sufficient sodium gradient across the ER membrane. In conclusion, we demonstrate that GLUTs are sufficient for mediating ER glucose transport en route to the plasma membrane. Because of the low volume of the ER, trace amounts of these uniporters contribute to ER solute import during ER transit, while uniporters and cation-coupled transporters carry out export from the ER, together potentially explaining the low selectivity of ER transport. Expression levels and residence time of transporters in the ER, as well as their coupling mechanisms, could be key determinants of ER permeability.

Vitamin C transporters

Article

Full-text available

Jan 2009

Vitamin C is a wide spectrum antioxidant essential for humans, which are unable to synthesize the vitamin and must obtain it from dietary sources. There are two biologically important forms of vitamin C, the reduced form, ascorbic acid, and the oxidized form, dehydroascorbic acid. Vitamin C exerts most of its biological functions intracellularly and is acquired by cells with the participation of specific membrane transporters. This is a central issue because even in those species capable of synthesizing vitamin C, synthesis is restricted to the liver (and pancreas) from which is distributed to the organism. Most cells express two different transporter systems for vitamin C; a transporter system with absolute specificity for ascorbic acid and a second system that shows absolute specificity for dehydroascorbic acid. The dehydroascorbic acid transporters are members of the GLUT family of facilitative glucose transporters, of which at least three isoforms, GLUT1, GLUT3 and GLUT4, are dehydroascorbic acid transporters. Ascorbic acid is transported by the SVCT family of sodium-coupled transporters, with two isoforms molecularly cloned, the transporters SVCT1 y SVCT2, that show different functional properties and differential cell and tissue expression. In humans, the maintenance of a low daily requirement of vitamin C is attained through an efficient system for the recycling of the vitamin involving the two families of vitamin C transporters.

Identification of a key residue determining substrate affinity in the human glucose transporter GLUT1

Article

Mar 2009
Biochim Biophys Acta

Asn(331) in transmembrane segment 7 of the yeast Saccharomyces cerevisiae transporter Hxt2 has been identified as a single key residue for high-affinity glucose transport by comprehensive chimera approach. The glucose transporter GLUT1 of mammals belongs to the same major facilitator superfamily as Hxt2 and may therefore show a similar mechanism of substrate recognition. The functional role of Ile(287) in human GLUT1, which corresponds to Asn(331) in Hxt2, was studied by its replacement with each of the other 19 amino acids. The mutant transporters were individually expressed in a recently developed yeast expression system for GLUT1. Replacement of Ile(287) generated transporters with various affinities for glucose that correlated well with those of the corresponding mutants of the yeast transporter. Residues exhibiting high affinity for glucose were medium-sized, non-aromatic, uncharged and irrelevant to hydrogen-bond capability, suggesting an important role of van der Waals interaction. Sensitivity to phloretin, a specific inhibitor for the presumed exofacial glucose binding site, was decreased in two mutants, whereas that to cytochalasin B, a specific inhibitor for the presumed endofacial glucose binding site, was unchanged in the mutants. These results suggest that Ile(287) is a key residue for maintaining high glucose affinity in GLUT1 as revealed in Hxt2 and is located at or near the exofacial glucose binding site.

Accumulation of ascorbate by endocrine-regulated and glucose-sensitive transport of dehydroascorbic acid in luteinized rat ovarian cells

Article

Mar 1998

The underlying causes, treatment options of gut microbiota and food habits in type 2 diabetes mellitus: a narrative review

Article

May 2024
J Basic Clin Physiol Pharmacol

Type 2 diabetes mellitus is a long-lasting endocrine disorder characterized by persistent hyperglycaemia, which is often triggered by an entire or relative inadequacy of insulin production or insulin resistance. As a result of resistance to insulin (IR) and an overall lack of insulin in the body, type 2 diabetes mellitus (T2DM) is a metabolic illness that is characterized by hyperglycaemia. Notably, the occurrence of vascular complications of diabetes and the advancement of IR in T2DM are accompanied by dysbiosis of the gut microbiota. Due to the difficulties in managing the disease and the dangers of multiple accompanying complications, diabetes is a chronic, progressive immune-mediated condition that plays a significant clinical and health burden on patients. The frequency and incidence of diabetes among young people have been rising worldwide. The relationship between the gut microbiota composition and the physio-pathological characteristics of T2DM proposes a novel way to monitor the condition and enhance the effectiveness of therapies. Our knowledge of the microbiota of the gut and how it affects health and illness has changed over the last 20 years. Species of the genus Eubacterium, which make up a significant portion of the core animal gut microbiome, are some of the recently discovered ‘generation’ of possibly helpful bacteria. In this article, we have focused on pathogenesis and therapeutic approaches towards T2DM, with a special reference to gut bacteria from ancient times to the present day.

The role of salt bridge networks in the stability of the yeast hexose transporter 1

Article

Oct 2023
BBA-GEN SUBJECTS

Endothelial GLUTs and vascular biology

Article

Feb 2023
BIOMED PHARMACOTHER

Endothelial metabolism is a promising target for vascular functional regulation and disease therapy. Glucose is the primary fuel for endothelial metabolism, supporting ATP generation and endothelial cell survival. Multiple studies have discussed the role of endothelial glucose catabolism, such as glycolysis and oxidative phosphorylation, in vascular functional remodeling. However, the role of the first gatekeepers of endothelial glucose utilization, glucose transporters, in the vasculature has long been neglected. Here, this review summarizes glucose transporter studies in vascular research. We mainly focus on GLUT1 and GLUT3 because they are the most critical glucose transporters responsible for most endothelial glucose uptake. Some interesting topics are also discussed, intending to provide directions for endothelial glucose transporter research in the future.

Isoginkgetin, a potential CDK6 inhibitor, suppresses SLC2A1/GLUT1 enhancer activity to induce AMPK-ULK1-mediated cytotoxic autophagy in hepatocellular carcinoma

Article

Sep 2022

Isoginkgetin (ISO), a natural biflavonoid, exhibited cytotoxic activity against several types of cancer cells. However, its effects on hepatocellular carcinoma (HCC) cells and mechanism remain unclear. Here, we revealed that ISO effectively inhibited HCC cell proliferation and migration in vitro. LC3-II expression and autophagosomes were increased under ISO treatment. In addition, ISO-induced cell death was attenuated by treatment with chloroquine or knockdown of autophagy-related genes (ATG5 or ULK1). ISO significantly suppressed SLC2A1/GLUT1 (solute carrier family 2 member 1) expression and glucose uptake, leading to activation of the AMPK-ULK1 axis in HepG2 cells. Overexpression of SLC2A1/GLUT1 abrogated ISO-induced autophagy. Combining molecular docking with thermal shift analysis, we confirmed that ISO directly bound to the N terminus of CDK6 (cyclin dependent kinase 6) and promoted its degradation. Overexpression of CDK6 abrogated ISO-induced inhibition of SLC2A1/GLUT1 transcription and induction of autophagy. Furthermore, ISO treatment significantly decreased the H3K27ac, H4K8ac and H3K4me1 levels on the SLC2A1/GLUT1 enhancer in HepG2 cells. Finally, ISO suppressed the hepatocarcinogenesis in the HepG2 xenograft mice and the diethylnitrosamine+carbon tetrachloride (DEN+CCl4)-induced primary HCC mice and we confirmed SLC2A1/GLUT1 and CDK6 as promising oncogenes in HCC by analysis of TCGA data and human HCC tissues. Our results provide a new molecular mechanism by which ISO treatment or CDK6 deletion promotes autophagy; that is, ISO targeting the N terminus of CDK6 for degradation inhibits the expression of SLC2A1/GLUT1 by decreasing the enhancer activity of SLC2A1/GLUT1, resulting in decreased glucose levels and inducing the AMPK-ULK1 pathway.

An Unrevealed Molecular Function of Corannulene Buckybowl Glycoconjugates in Selective Tumor Annihilation by Targeting the Cancer‐Specific Warburg Effect

Article

Full-text available

Mar 2022

The biomedical application of corannulene π‐bowls is historically limited by low solubility and bioavailability despite the potential in their unique electronic properties for new functional materials. Herein, the unexpected role and molecular mechanism of Corranulene π‐bowls are uncovered in biomedical applications as an effective anticancer agent for Warburg effect mediated selective tumor targeting. The corannulene triazolyl monosaccharides Cor‐sugars exhibit highly potent cytotoxicity against human cancer cells and effectively inhibit xenograft growth of hyperglycolytic tumors. Particularly, the galactose‐conjugated Cor‐gal exhibits superior in vivo anticancer efficacy in A549 tumor models with outstanding safety profile compared to doxorubicin. Moreover, the combined treatment of Cor‐gal with immune checkpoint inhibitor results in an effective synergy in treating H460 human lung carcinoma. An uptake mechanism study reveals that Cor‐sugars exploit tumor‐specific glucose transporter glucose transporter 1 (GLUT1) for targeted cell delivery and intra‐tumoral accumulation through the cancer‐specific Warburg effect. Their significant anticancer activity is attributed to multiphasic DNA‐binding and cell cycle alteration effects. This study uncovers new molecular properties of corannulene buckybowl and enabling their potential new applications in biomedical engineering. Sugar‐conjugated corannulene π‐bowls possess a new molecular function in DNA interactive anticancer therapy. Corannulene‐sugar conjugates can be transported through glucose transporter 1 (GLUT1) for cancer cell uptake and effectively annihilate xenograft tumors by leveraging the Warburg effect. The anticancer activity relies on a unique multiphasic DNA binding mechanism of the curved corannulene molecule and selectively induces cell cycle arrest in tumor cells.

Sodium Glucose Cotransporter Inhibitors for the Treatment of Diabetes

Chapter

Apr 2021

Worldwide, the incidence of diabetes mellitus, a chronic metabolic disease that results in four million deaths from it and associated complications annually, is increasing and its treatment is an enormous public health concern. Type 1 diabetes is treated by insulin, but a variety of drugs are used to treat the more prevalent type 2 diabetes. Inhibitors of sodium glucose cotransporters (SGLTs) are the newest drugs to reach the market to achieve the blood‐glucose control needed to treat type 2 diabetes. SGLT1 and 2 carry out renal reabsorption of more than 90% of glucose from urine against a concentration gradient using the electrochemical energy from the cotransport of sodium ions. Blocking SGLTs causes excess glucose to be excreted through the urine, reducing blood sugar. The first known SGLT inhibitor was the O‐glycosylated flavonoid natural product phlorizin, which enabled the discovery of SGLTs and served as the initial lead compound for medicinal chemistry. O‐ and C‐Glycosylated flavonoid and dihydrochalcone natural products also inhibit SGLTs. The drugs on the market today, dapagliflozin, canagliflozin, empagliflozin, ertugliflozin, and others, are all aryl C‐glycosides. They are selective for SGLT2 over SGLT1 to avoid adverse side effects associated with SGLT1 inhibition. Studies have suggested that SGLT inhibitors have a renal and cardiovascular protective effect that is advantageous, although their use increases the risk of urinary tract and genital infections and ketoacidosis. SGLT inhibitors are in trials as an adjuvant to insulin for the treatment of type 1 diabetes.

Functional characterization and tissue localization of the facilitative glucose transporter GLUT12

Article

Aug 2020
J BIOCHEM

Facilitative glucose transporters play crucial roles in glucose utilization and homeostasis. GLUT12 was initially isolated as a novel GLUT4-like transporter involved in insulin-dependent glucose transport. However, tissue distribution and biochemical properties of GLUT12 are not well understood. In this study, we investigated the basic kinetic properties and tissue distribution of GLUT12. Human GLUT12 and GLUT1 were overexpressed and purified using Ni-NTA column chromatography. Reconstituted proteoliposomes showed time-dependent D-glucose transport activity, which was inhibited by phloretin and dehydroascorbate. Dose dependence of glucose transport revealed a KM and Vmax values of 6.4 mM and 1.2 μmol/mg/min, respectively, indicating that GLUT12 is a high-affinity type glucose transporter. Glucose transport by GLUT12 was inhibited by ATP and glucose-1-phosphate, glucose-6-phosphate, and disaccharides (properties similar to those of GLUT1). Indirect immunohistochemistry revealed the distribution of mouse GLUT12 in the apical region of distal tubules and collecting ducts in the kidney and epithelial cells of the jejunum. In addition to these cells, GLUT12 was present in chromaffin cells in the adrenal medulla, the anterior pituitary lobe, as well as the thyroid and pyloric glands. These tissue distributions suggest a unique function of GLUT12, besides that of an insulin-dependent glucose transport.

Heterogeneity of Glucose Transport in Lung Cancer

Article

Full-text available

Jun 2020

Increased glucose uptake is a known hallmark of cancer. Cancer cells need glucose for energy production via glycolysis and the tricarboxylic acid cycle, and also to fuel the pentose phosphate pathway, the serine biosynthetic pathway, lipogenesis, and the hexosamine pathway. For this reason, glucose transport inhibition is an emerging new treatment for different malignancies, including lung cancer. However, studies both in animal models and in humans have shown high levels of heterogeneity in the utilization of glucose and other metabolites in cancer, unveiling a complexity that is difficult to target therapeutically. Here, we present an overview of different levels of heterogeneity in glucose uptake and utilization in lung cancer, with diagnostic and therapeutic implications.

Development of Glucose Transporter (GLUT) Inhibitors

Article

Full-text available

Nov 2019
EUR J ORG CHEM

The discovery of novel compound classes endowed with biological activity is at the heart of chemical biology and medicinal chemistry research. This enables novel biological insights and inspires new approaches to the treatment of diseases. Cancer cells frequently exhibit altered glycolysis and glucose metabolism and an increased glucose demand. Thus, targeting glucose uptake and metabolism may open up novel opportunities for the discovery of compounds that differentiate between normal and malignant cells. This review discusses the different chemical approaches to the development of novel inhibitors of glucose uptake through facilitative glucose transporters (GLUTs), and focusses on the most advanced and potent inhibitor classes known to date. GLUT inhibitors may find application not only in the treatment of cancer, but also of other proliferative diseases that exhibit glucose addiction.

Ligand Screening Systems for Human Glucose Transporters as Tools in Drug Discovery

Article

Full-text available

May 2018

Hexoses are the major source of energy and carbon skeletons for biosynthetic processes in all kingdoms of life. Their cellular uptake is mediated by specialized transporters, including glucose transporters (GLUT, SLC2 gene family). Malfunction or altered expression pattern of GLUTs in humans is associated with several widespread diseases including cancer, diabetes and severe metabolic disorders. Their high relevance in the medical area makes these transporters valuable drug targets and potential biomarkers. Nevertheless, the lack of a suitable high-throughput screening system has impeded the determination of compounds that would enable specific manipulation of GLUTs so far. Availability of structural data on several GLUTs enabled in silico ligand screening, though limited by the fact that only two major conformations of the transporters can be tested. Recently, convenient high-throughput microbial and cell-free screening systems have been developed. These remarkable achievements set the foundation for further and detailed elucidation of the molecular mechanisms of glucose transport and will also lead to great progress in the discovery of GLUT effectors as therapeutic agents. In this mini-review, we focus on recent efforts to identify potential GLUT-targeting drugs, based on a combination of structural biology and different assay systems.

A Growth-Based Screening System for Hexose Transporters in Yeast

Chapter

Jan 2018
Meth Mol Biol

As the simplest eukaryotic model system, the unicellular yeast Saccharomyces cerevisiae is ideally suited for quick and simple functional studies as well as for high-throughput screening. We generated a strain deficient for all endogenous hexose transporters, which has been successfully used to clone, characterize, and engineer carbohydrate transporters from different source organisms. Here we present basic protocols for handling this strain and characterizing sugar transporters heterologously expressed in it.

Glucose Transport and Homeostasis in Lung Epithelia

Chapter

Dec 2017

Glucose transporters in the lung epithelium play a critical role in maintaining a healthy lung. Glucose in airway secretions is normally maintained at concentrations ~12.5 times lower than plasma by homeostatic processes including epithelial glucose transport and tight junction barrier function. These mechanisms are disrupted by chronic lung disease and hyperglycemia, increasing airway glucose concentrations. In cell culture and animal models, disruption of airway glucose homeostasis increases luminal bacterial growth and infection. Mechanisms include direct stimulation of bacterial growth due to increased nutrient availability and inhibition of local innate immunity. The effect of glucose on luminal bacterial growth and infection can be reversed by drugs that restore airway glucose homeostasis. These findings have implications for the pathogenesis and treatment of respiratory infection in humans with hyperglycemia and chronic lung disease.

Inhibitor Discovery for the Human GLUT1 from Homology Modeling and Virtual Screening

Article

Full-text available

Apr 2016
ACS CHEM BIOL

The human Glucose Transporter 1 (hGLUT1 or SLC2A1) is a facilitative membrane transporter found in the liver, intestines, kidney, and brain, where it transports sugars such as D-glucose and D-galactose. Genetic variations in hGLUT1 are associated with a broad range of diseases and metabolic disorders. For example, hGLUT1 is upregulated in various cancer types (e.g., breast carcinoma) to support the increased anaerobic glycolysis and the Warburg Effect. Thus, hGLUT1 is an emerging therapeutic target, which also transports commonly used cancer biomarkers (e.g., (18)F-DG). In this study, we use computational prediction followed by experimental testing, to characterize hGLUT1. We construct homology models of hGLUT1 in a partially occluded outward open ('occluded') conformation based on the X-ray structure of the E. coli xylose transporter, XylE. Comparison of the binding site of the occluded models to experimentally determined hGLUT structures revealed a hydrophobic pocket adjacent to the sugar-binding site, which was tested experimentally via site-directed mutagenesis. Virtual screening of various libraries of purchasable compounds against the occluded models, followed by experimental testing with cellular assays revealed seven previously unknown hGLUT1 ligands with IC50 values ranging from 0.45 μM to 59 μM. These ligands represent three unique chemotypes that are chemically different from any other known hGLUT1 ligands. The newly characterized hydrophobic pocket can potentially be utilized by the new ligands for increased affinity. Furthermore, the previously unknown hGLUT1 ligands can serve as chemical tools to further characterize hGLUT1 function or lead molecules for future drug development.

Differential response to sulfur nutrition of two common bean genotypes differing in storage protein composition

Article

Full-text available

Feb 2015

It has been hypothesized that the relatively low concentration of sulfur amino acids in legume seeds might be an ecological adaptation to nutrient poor, marginal soils. SARC1 and SMARC1N-PN1 are genetically related lines of common bean (dry bean, Phaseolus vulgaris) differing in seed storage protein composition. In SMARC1N-PN1, the lack of phaseolin and major lectins is compensated by increased levels of sulfur-rich proteins, resulting in an enhanced concentration of cysteine and methionine, mostly at the expense of the abundant non-protein amino acid, S-methylcysteine. To identify potential effects associated with an increased concentration of sulfur amino acids in the protein pool, the response of the two genotypes to low and high sulfur nutrition was evaluated under controlled conditions. Seed yield was increased by the high sulfate treatment in SMARC1N-PN1. The seed concentrations of sulfur, sulfate, and S-methylcysteine were altered by the sulfur treatment in both genotypes. The concentration of total cysteine and extractible globulins was increased specifically in SMARC1N-PN1. Proteomic analysis identified arcelin-like protein 4, lipoxygenase-3, albumin-2, and alpha amylase inhibitor beta chain as having increased levels under high sulfur conditions. Lipoxygenase-3 accumulation was sensitive to sulfur nutrition only in SMARC1N-PN1. Under field conditions, both SARC1 and SMARC1N-PN1 exhibited a slight increase in yield in response to sulfur treatment, typical for common bean.

Glucose Transport Machinery Reconstituted in Cell Models

Article

Dec 2014
CHEM COMMUN

Here we demonstrate the production of a functioning cell model by formation of giant vesicles reconstituted with the GLUT1 glucose transporter and a glucose oxidase and hydrogen peroxidase linked fluorescent reporter internally. Hence, a simplified artificial cell is formed that is able to take up glucose and process it.

[The role of glucose transporters in human metabolic regulation]

Article

Full-text available

Jul 2013
Postepy Biochem

Glucose is one of the most important sources of energy in human metabolizm. Cells absorb it by active transport (with SGLT transporters) or by facilitated diffusion (with GLUT transporters). GLUT family consists of 14 proteins grouped in 3 subclasses based on similarities in their architecture. They differ from one another in affinity to glucose, tissue distribution and type of signals that cause their translocaton to the cell membrane what results in different levels of sugar transport into the tissues. SGLT proteins cotransport glucose with Na+ ions. Energy required to this transport is acquired from gradient of Na+ ions that is maintained by Na+/K(+)-ATPase. SGLT family consists of 12 proteins which include sugar cotransporters of anions, vitamins and short-chain fatty acids. Some of them also have a function of glucose sensors as well as water and urea channels.

Recombinant Microorganisms as Tools for High Throughput Screening for Nonantibiotic Compounds

Article

Full-text available

Feb 1997
J BIOMOL SCREEN

Microorganisms were among the first tools used for the discovery of biologically active compounds. Their utility reached a zenith during the era of antibiotic development in the 1950s and 1960s, then declined. Subsequently, a substantial role for microorganisms in the pharmaceutical industry developed with the realization that microbial fermentations were intriguing sources of nonantibiotic natural products. From recombinant DNA technology emerged another important role for microorganisms in pharmaceutical research: the expression of heterologous proteins for therapeutic products or for in vitro high throughput screens (HTSs). Recent developments in cloning, genetics, and expression systems have opened up new applications for recombinant microorganisms in screening for nonantibiotic compounds in HTSs. These screens employ microorganisms that depend upon the function of a heterologous protein for survival under defined nutritional conditions. Compounds that specifically target the heterologous protein can be identified by measuring viability of the microorganism under different nutrient selection. Advantages of this approach include a built-in selection for target selectivity, an easily measured end point that can be used for a multitude of different targets, and compatibility with automation required for HTSs. Mechanism-based HTSs using recombinant microorganisms can also address drug targets that are not readily approachable in other HTS formats, including certain enzymes; ion channels and transporters; and protein::protein, protein::DNA, and protein::RNA interactions.

GLUT1 Structure Function; Context, Ligand Cooperativity, and Mutagenesis Studies: A Dissertation

Article

Full-text available

Jul 2008

Trista Robichaud

Carrier mediated nutrient import is vital for cell and tissue homeostasis. Structural insights of carrier mediated transport, particularly the human glucose transporter GLUT1, are essential for understanding the mechanisms of human metabolic disease, and provide model systems for cellular processes as a whole. GLUT1 function and expression is characterized by a complexity unexplained by the current hypotheses for carrier-mediated sugar transport (9). It is possible that the operational properties of GLUT1 are determined by host cell environment. A glucose transport-null strain of Saccharomyces cerevisiae (RE700A) was transfected with the p426 GPD yeast expression vector containing DNA encoding the wild-type human glucose transport protein (GLUT1) to characterize its functional properties. Identical protein sequences generated different kinetic parameters when expressed in RE700A yeast, erythrocytes, and HEK293 cells. These findings support the hypothesis that red cell sugar transport complexity is host cell-specific. Cytochalasin B (CB) and forskolin (FSK) inhibit GLUT1-mediated sugar transport in red cells by binding at or close to the GLUT1 sugar export site. Paradoxically, very low concentrations of these inhibitors produce a modest stimulation of sugar transport (16). This result is consistent with the hypothesis that the glucose transporter contains multiple, interacting, intracellular binding sites for e1 ligands CB and FSK. The present study tests this hypothesis directly and, by screening a library of cytochalasin and forskolin analogs, asks what structural features of exit site ligands determine binding site affinity and cooperativity. Our findings are explained by a carrier that presents at least two interacting endofacial binding sites for CB or FSK. We discuss this result within the context of GLUT1 quaternary structure and evaluate the major determinants of ligand binding affinity and cooperativity. Cytochalasin B (CB) inhibits GLUT1 substrate transport at or near the endofacial sugar binding site. N-bromosuccinamide analysis combined with 3H-CB photolabeling implicates the region between Trp388 and Trp412 in ligand binding. Although its structure has been modeled(5), the specific residues comprising the sugar binding site are unknown. A series of alanine point mutants were made, and mutant protein 2-deoxy glucose transport was tested in the presence of increasing [CB]. Arg126Ala and Cys421Ala GLUT1 mutations altered CB affinity but were determined not to be in the e1 site. The Arg400Ala mutation decreased binding affinity for CB, and may comprise part of the e1 binding site. Because point mutations were individually insufficient to abrogate CB binding, Trp388 to Trp412 chimeras were made. GLUT1/GLUT4388-412/GLUT1 and GLUT1/GLUT5388-412/GLUT1 chimeras showed moderately less sensitivity to CB inhibition of transport; these amino acids likely comprise regions determinant of CB binding affinity. Furthermore GLUT1/GLUT5388-412/GLUT1 shows enhancement of 2-DG uptake at 50nM CB, but an overall dose response indistinguishable from WT GLUT1. A multisite fit of the data suggested GLUT1/GLUT5388-412/GLUT1 chimera possesses strong first site affinity for CB but slight negative second-site cooperativity. We conclude that point mutants were insufficient to abrogate CB binding and that the Trp388 to Trp412 sequence is necessary for CB binding affinity but is not the sole determinant of inhibition of 2 deoxyglucose uptake by CB. We discuss these results with their implications for structure-function sequence localization of the CB binding site, and by extension, the e1 sugar binding site.

Expression of Human Glucose Transporter Type 1 and Rat Hexokinase Type II Complementary DNAs in Rainbow Trout Embryos: Effects on Glucose Metabolism

Article

Feb 1999

Sugars are utilized poorly in fish mainly because of low rates of transport across plasma membrane and phosphorylation. To evaluate whether it is possible to augment carbohydrate metabolism in fish using heterologous genes, expression of human glucose transporter type 1 (hGLUT1) and rat hexokinase type II (rHKII) complementary DNAs cloned with cytomegalovirus promoter was followed in rainbow trout embryos. Both genes were transcribed. Hexokinase activity, undetectable in control, was found in transformed blastulas. Increased rates of 14C-methylglucose uptake and sensitivity to cytochalasin B indicated the presence of facilitative hexose transport due to hGLUT1 expression. Effect of hGLUT1 on production of 14CO2 from glucose was greater than that of rHKII. Coexpression of the genes did not increase the rate of glucose oxidation compared with expression of hGLUT1 alone.

Ancillary proteins in membrane targeting of transporters

Chapter

Mar 2004

The integral protein components of the eukaryotic plasma membrane (PM) are targeted to the PM via the secretory pathway. Movement between the compartments of the secretory pathway occurs via small transport vesicles that form and bud from donor compartments, and that specifically target to and fuse with distinct acceptor compartments. The sorting and concentration of cargo proteins, including the proteins required for vesicle targeting, occurs concomitantly with the formation of transport vesicles. A detailed knowledge of the events facilitating the formation of transport vesicles is key to understanding the vectorial transport of proteins through the secretory pathway. A class of ancillary proteins, all integral components of the endoplasmic reticulum (ER) membrane, is required for the incorporation of discrete sets of metabolite transporters into ER-derived COPII coated transport vesicles. These ancillary proteins, also called packaging chaperones, exert highly specific effects and function in a manner that only contributes to the packaging of a very limited set of related transport proteins - their cognate substrates. In cells lacking a particular packaging chaperone only its cognate cargo accumulates within the ER. Here we review the current state of understanding of how packaging chaperones facilitate the specific selection of metabolite transporters as cargo during the formation of COPII vesicles.

Crucial Effects of Amino Acid Side Chain Length in Transmembrane Segment 5 on Substrate Affinity in Yeast Glucose Transporter Hxt7

Article

Sep 2011
BIOCHEMISTRY-US

We previously identified Asp(340) in transmembrane segment 7 (TM7) as a key determinant of substrate affinity in Hxt7, a high-affinity facilitative glucose transporter of Saccharomyces cerevisiae. To gain further insight into the structural basis of substrate recognition by Hxt7, we performed cysteine-scanning mutagenesis of 21 residues in TM5 of a Cys-less form of Hxt7. Four residues were sensitive to Cys replacement, among which Gln(209) was found to be essential for high-affinity glucose transport activity. The 17 remaining sites were examined further for the accessibility of cysteine to the hydrophilic sulfhydryl reagent p-chloromercuribenzenesulfonate (pCMBS). Among the Cys mutants, T213C was the only one whose transport activity was completely inhibited by 0.5 mM pCMBS. Moreover, this mutant was protected from pCMBS inhibition by the substrate d-glucose and by 2-deoxy-D-glucose but not by L-glucose, indicating that Thr(213) is situated at or close to a substrate recognition site. The functional role of Thr(213) was further examined with its replacement with each of the other 19 amino acids in wild-type Hxt7. Such replacement generated seven functional transporters with various affinities for glucose. Only three mutants, those with Val, Cys, and Ser at position 213, exhibited high-affinity glucose transport activity. All of these residues possess a side chain length similar to that of Thr, indicating that side chain length at this position is a key determinant of substrate affinity. A working homology model of Hxt7 indicated that Gln(209) and Thr(213) face the central cavity and that Thr(213) is located within van der Waals distance of Asp(340) (TM7).

Structure and Function of Plasma Membrane Amino Acid, Oligopeptide and Sucrose Transporters from Higher Plants

Article

Apr 1998

Functional analysis of the glucose transporters-1 , -6, and -13.1 expressed by zebrafish epithelial cells

Article

Full-text available

Feb 2011
AM J PHYSIOL-REG I

The hexose supply and subsequent metabolism are crucial for the operations of the iono- and osmoregulatory mechanisms in fish, but how hexose is transported and supplied to cells of the ionoregulatory epithelia is unknown. Three zebrafish glucose transporters (zGLUTs), zGLUT1a, -13.1, and -6, were previously found to respectively be expressed by ionocytes (Na(+)-K(+)-ATPase-rich, Na(+)-Cl(-) cotransporter-expressing, and H(+)-ATPase-rich cells) and adjacent energy-depositing cells [glycogen-rich (GR) cells] in zebrafish skin and gills (32). The present study aimed to test if the transport kinetics of these three zGLUTs differ, and if the transport functional differences are of physiological relevance to the respective functions of epithelial cells. The three zGLUTs expressed by Xenopus laevis oocytes revealed different d-glucose transport kinetics; zGLUT13.1 showed the lowest Michaelis constant (K(m)), whereas zGLUT6 had the highest K(m) and maximal velocity. In morpholino injection experiments, translational knockdown of zGLUT1a and -13.1, respectively, impaired Cl(-)/Ca(2+) and Na(+)/Ca(2+) uptake, but loss-of-function of zGLUT6 did not cause a significant effect on ion uptake functions in zebrafish. Based on these results, zGLUT1a and -13.1 appear to be superior to zGLUT6 in competing for glucose under a situation of low blood glucose due to extensive energy consumption, whereas, in a high blood glucose situation, zGLUT6 is able to absorb the excess glucose for energy deposition. The timely and sufficient supply of energy to ionocytes so that they can carry out ion regulation is definitely a more important event than storing energy in GR cells, particularly when acute environmental change disturbs the ion balance in zebrafish.

Identification of a key residue determining substrate affinity in the yeast glucose transporter Hxt7: A two-dimensional comprehensive study

Article

Full-text available

Aug 2010
J BIOL CHEM

We previously identified Asn(331) in transmembrane segment 7 (TM7) as a key residue determining substrate affinity in Hxt2, a moderately high-affinity facilitative glucose transporter of Saccharomyces cerevisiae. To gain further insight into the structural basis of substrate recognition by yeast glucose transporters, we have now studied Hxt7, whose affinity for glucose is the highest among the major hexose transporters. The functional role of Asp(340) in Hxt7, the residue corresponding to Asn(331) of Hxt2, was examined by replacing it with each of the other 19 amino acids. Such replacement of Asp(340) generated transporters with various affinities for glucose, with the affinity of the Cys(340) mutant surpassing that of the wild-type Hxt7. To examine the structural role of Asp(340) in the substrate translocation pathway, we performed cysteine-scanning mutagenesis of the 21 residues in TM7 of a functional Cys-less Hxt7 mutant in conjunction with exposure to the hydrophilic sulfhydryl reagent p-chloromercuribenzenesulfonate (pCMBS). The transport activity of the D340C mutant of Cys-less Hxt7, in which Asp(340) is replaced with Cys, was completely inhibited by pCMBS, indicating that Asp(340) is located in a water-accessible position. This D340C mutant showed a sensitivity to pCMBS that was approximately 70 times that of the wild-type Hxt7, and it was protected from pCMBS inhibition by the substrates d-glucose and 2-deoxy-d-glucose but not by l-glucose. These results indicate that Asp(340) is situated at or close to a substrate recognition site and is a key residue determining high-affinity glucose transport by Hxt7, supporting the notion that yeast glucose transporters share a common mechanism for substrate recognition.

The Protein Family of Glucose Transport Facilitators: It's Not Only About Glucose After All

Article

May 2010
IUBMB LIFE

Robert Augustin

The protein family of facilitative glucose transporters comprises 14 isoforms that share common structural features such as 12 transmembrane domains, N- and C-termini facing the cytoplasm of the cell, and a N-glycosylation side either within the first or fifth extracellular loop. Based on their sequence homology, three classes can be distinguished: class I includes GLUT1-4 and GLUT14, class II the "odd transporters" GLUT5, 7, 9, 11, and class III the "even transporters" GLUT6, 8, 10, 12 and the proton driven myoinositol transporter HMIT (or GLUT13). With the cloning and characterization of the more recent class II and III isoforms, it became apparent that despite their structural similarities, the different isoforms not only show a distinct tissue-specific expression pattern but also show distinct characteristics such as alternative splicing, specific (sub)cellular localization, and affinities for a spectrum of substrates. This review summarizes the current understanding of the physiological role for the various transport facilitators based on human genetically inherited disorders or single-nucleotide polymorphisms and knockout mice models. The emphasis of the review will be on the potential functional role of the more recent isoforms.

GLUT1 as a therapeutic target in hepatocellular carcinoma

Article

Oct 2009
EXPERT OPIN THER TAR

Primary hepatocellular carcinoma (HCC) is one of the most fatal cancers in humans with rising incidence in many regions around the world. Currently, no satisfactory curative pharmacological treatment is available, and the outcome is mostly poor. Recently, we have shown that the glucose transporter GLUT1 is increased in a subset of patients with HCC and functionally affects tumorigenicity. GLUT1 is a rate-limiting transporter for glucose uptake, and its expression correlates with anaerobic glycolysis. This phenomenon is also known as the Warburg effect and recently became of great interest, since it affects not only glucose uptake and utilization but also has an influence on tumorigenic features like metastasis, chemoresistance and escape from immune surveillance. Consistent with this, RNA-interference-mediated inhibition of GLUT1 expression in HCC cells resulted in reduced tumorigenicity. Together, these findings indicate that GLUT1 is a novel and attractive therapeutic target for HCC. This review summarizes our current knowledge on the expression and function of GLUT1 in HCC, available drugs/strategies to inhibit GLUT1 expression or function, and potential side effects of such therapeutic strategies.

Amino acid substitutions at tryptophan 388 and tryptophan 412 of the HepG2 (Glut1) glucose transporter inhibit transport activity and targeting to the plasma membrane in Xenopus oocytes

Article

Full-text available

May 1992

All 6 tryptophan residues in the human HepG2-type glucose transporter (Glut1) were individually altered by site-directed mutagenesis to investigate the role of these residues in transport function. Tryptophan residues in positions 48, 65, 186, 363, 388, and 412 of Glut1 were changed to either a glycine or leucine residue. Mutant mRNAs were synthesized and injected into Xenopus laevis oocytes. Transporter function as assessed by uptake of 2-deoxy-D-[3H]glucose or transport of 3-O-[3H]methylglucose was decreased in the 388 and 412 mutants but was unaltered in all other mutants. The amount of the mutant transporters expressed in total membrane and plasma membrane fractions was measured using Glut1-specific antibodies. Calculation of the intrinsic transport activity of each of the mutants using these data demonstrated that the reduced transport activity of the 412 mutants was caused entirely by a dramatic decrease in the intrinsic activity of the mutant proteins whereas the reduced activity of the 388 mutants was a result of a decreased level of the protein in oocytes, decreased targeting to the plasma membrane, and a modest decrease in the intrinsic activity. Protease/glycosidase mapping of in vitro translation products indicated that the effects of the 388 and 412 point mutations could not be attributed to a disruption in the ability of the mutant proteins to insert properly into the membrane. The ID50 for cytochalasin B inhibition of 2-deoxyglucose uptake was increased from 5 x 10(-7) M for the wild-type Glut1 to 4 x 10(-6) M in the 388 mutants but was unaltered in the 412 mutants. These observations suggest that 1) Trp-412 may comprise part of a hexose binding site or is involved in maintaining a local tertiary structure critical for transport function; 2) Trp-388 is involved in stabilizing the equilibrium binding of cytochalasin B to the transporter. Trp-388 may therefore lie near a substrate binding site and also appears to participate in stabilization of local tertiary structure important for full catalytic activity and efficient targeting to the Xenopus plasma membrane.

Characterization of functional human erythrocyte-type glucose transporter (GLUT1) expressed in insect cells using a recombinant baculovirus

Article

Full-text available

Jun 1992

The human erythrocyte-type glucose transporter (GLUT1) has been abundantly expressed in insect cells by using a recombinant baculovirus. At 4 days after infection with the virus, the insect cell-surface and intracellular membranes were found to contain greater than 200 pmol of D-glucose-sensitive binding sites for the transport inhibitor cytochalasin B per mg of protein. The characteristics of binding were identical with those of the erythrocyte transporter, although the two proteins differed substantially in apparent Mr, probably as a result of glycosylation differences.

The HXT2 gene of Saccharomyces cerevisiae is required for higha Ynity glucose transport

Article

Full-text available

Dec 1990

The HXT2 gene of the yeast Saccharomyces cerevisiae was identified on the basis of its ability to complement the defect in glucose transport of a snf3 mutant when present on the multicopy plasmid pSC2. Analysis of the DNA sequence of HXT2 revealed an open reading frame of 541 codons, capable of encoding a protein of Mr 59,840. The predicted protein displayed high sequence and structural homology to a large family of procaryotic and eucaryotic sugar transporters. These proteins have 12 highly hydrophobic regions that could form transmembrane domains; the spacing of these putative transmembrane domains is also highly conserved. Several amino acid motifs characteristic of this sugar transporter family are also present in the HXT2 protein. An hxt2 null mutant strain lacked a significant component of high-affinity glucose transport when under derepressing (low-glucose) conditions. However, the hxt2 null mutation did not incur a major growth defect on glucose-containing media. Genetic and biochemical analyses suggest that wild-type levels of high-affinity glucose transport require the products of both the HXT2 and SNF3 genes; these genes are not linked. Low-stringency Southern blot analysis revealed a number of other sequences that cross-hybridize with HXT2, suggesting that S. cerevisiae possesses a large family of sugar transporter genes.

Insulin-stimulated Translocation of the HepG2/Erythrocyte-type Glucose Transporter Expressed in 3T3-L1 Adipocytes

Article

Full-text available

Mar 1989

Insulin stimulates glucose transport into adipocytes, at least in part, via the translocation of intracellular transporters to the plasma membrane. The human HepG2-type transporter, which is not insulin-responsive in its native cell type, was expressed in 3T3-L1 adipocytes by infection with recombinant retrovirus harboring the HepG2 transporter cDNA in order to determine whether glucose transporter translocation in adipocytes is restricted to a distinct insulin-sensitive transporter species. The distributions of the endogenous murine and the HepG2 transporters were estimated by quantitative immunoblot analysis of subcellular fractions probed with either a monoclonal antibody that recognized only the human transporter or a polyclonal antibody that recognized both transporter species. In the basal state, the intracellular membrane fraction comprised approximately 50% of the total of each transporter type. Insulin decreased the content of both transporter species in the intracellular membranes by approximately 50% and increased the plasma membrane content of both species by approximately 1.5-2-fold. The similar insulin-mediated increase in the plasma membrane content of endogenous murine and HepG2 glucose transporters was verified by labeling of cell surface glycoproteins with [3H]NaBH4 followed by immunoprecipitation with glucose transporter antibodies. These data indicate that insulin-mediated translocation in 3T3-L1 adipocytes is not restricted to a tissue-specific insulin-responsive glucose transporter species and suggest that other tissue-specific factors regulate the translocation process.

Recycling of the glucose transporter, the insulin receptor, and insulin in rat adipocytes. Effect of acidtropic agents

Article

Full-text available

Apr 1986

The notion of an insulin-dependent translocation of the glucose transporter in rat adipocytes was confirmed by immunoblotting and reconstitution of glucose transport activity of subcellular fractions. Quantitatively, however, significantly different results were obtained with these two techniques; when compared with reconstitution, immunoblotting detected translocation of a larger amount of the transporter from a low density microsome fraction to a plasma membrane fraction. The acidtropic agents chloroquine and dibucaine, which have been reported to inhibit the recycling of various receptors, were utilized to study the detailed translocation mechanism of the glucose transporter and the insulin receptor. These acidtropic agents caused accumulation of 125I-insulin in a subcellular fraction probably corresponding to lysosomes. They did not, however, significantly affect either the insulin-induced activation of glucose transport or the recycling of the transporter and the insulin receptor as detected by immunoblotting. About 50% of radioactivity released from adipocytes which were allowed to internalize insulin was due to intact insulin, and chloroquine did not change the release rate of intact insulin, raising the possibility of receptor-mediated exocytosis of insulin. The release of degraded insulin decreased with chloroquine treatment. The results are consistent with the idea that these acidtropic agents mainly act to inhibit degradation of insulin in lysosomes, and their effect on the recycling of the glucose transporter and the insulin receptor is minimal, indicating that the recycling of these membrane proteins proceeds irrespective of organelle acidification. Electron micrographs showed vesicles underneath the plasma membranes, with sizes similar to those of the low density microsome fraction where the internalized glucose transporter and the insulin receptor were located.

Random distribution of the glucose transporter of human erythrocytes in reconstituted liposomes

Article

Full-text available

Oct 1982

The glucose transporter of human erythrocytes was reconstituted with soybean phospholipids by the freeze-thaw/sonication method and the distribution of the transporter molecules in liposomes was studied. The steady state level of glucose transport in reconstituted liposomes showed saturation when increased amounts of the transporter were used for reconstitution. The saturation curve fitted well to a theoretical curve which was derived assuming a Poisson distribution of the transporter. Freeze-fracture electron micrographs showed random distribution of intramembraneous particles on liposomes, irrespective of liposome size or amount of the transporter added. A detailed study showed a parameter of the distribution (the ratio of transporter to liposome) obtained from the transport measurement can be used for the analysis of the distribution of intramembraneous particles, indicating that most of the molecules seen as particles were active in transport.

Substrate Recognition Domain of the Gal2 Galactose Transporter in Yeast Saccharomyces cerevisiae as Revealed by Chimeric Galactose-Glucose Transporters

Article

Full-text available

Mar 1995

The Gal2 galactose transporter takes up galactose in yeast. A homologous glucose transporter from the same organism, Hxt2, was selected, and various chimeras between these two transporters were constructed by making use of homologous recombination in Escherichia coli. Comparison of the galactose transport activities of three series of chimeras enabled us to positively identify a crucial substrate recognition region of 101 amino acids that lies close to the carboxyl terminus of the Gal2 transporter.

Sorting of membrane protein in the yeast secretory pathway

Article

Full-text available

May 1994

Structure and function of mammalian facilitative sugar transporters

Article

Full-text available

Oct 1993

Complementation in situ of the yeast plasma-membrane H+-ATPase gene Pma1 by an H+-ATPase gene from a heterologous species

Article

Full-text available

Mar 1993

In plants and fungi, the transport of solutes across the plasma membrane (pm) is driven by a proton pump (H(+)-ATPase) that produces an electric potential and a pH gradient. We expressed AHA2, a member of the Arabidopsis thaliana pm H(+)-ATPase gene family, in yeast cells in which transcription of the endogenous pm H(+)-ATPase gene (pma1) had been turned off. AHA2 was expressed mainly in intracellular membranes and only supported very slow growth of transformed yeast cells. Removal of the last 92 C-terminal amino acids from the plant H(+)-ATPase produced an enzyme with 2-3-fold higher specific ATPase activity than the wild-type plant enzyme. Surprisingly, the truncated H(+)-ATPase was now targetted to the yeast pm and fully supported normal yeast growth.

Methods In Yeast Genetics: Laboratory Manual

Article

Jan 1983

Methods in Yeast Genetics: A Laboratory Course Manual

Book

Jan 1990

Mark D Rose

Membrane transport proteins: Implications of sequence comparisons

Article

Sep 1992
CURR OPIN CELL BIOL

Analyses of the sequences and structures of many transport proteins that differ in substrate specificity, direction of transport and mechanism of transport suggest that they form a family of related proteins. Their sequence similarities imply a common mechanism of action. This hypothesis provides an objective basis for examining their mechanisms of action and relationships to other transporters.

Subcellular distribution and activity of glucose transporter isoforms GLUT1 and GLUT4 transiently expressed in COS-7 cells

Article

Aug 1992
Biochim Biophys Acta

In adipose and muscle cells, the glucose transporter isoform GLUT4 is mainly located in an intracellular, vesicular compartment from which it is translocated to the plasma membrane in response to insulin. In order to test the hypothesis that this preferential targeting of a glucose transporter to an intracellular storage site is conferred only by its primary sequence, we compared the subcellular distribution of the fat/muscle glucose transporter GLUT4 with that of the erythrocyte/brain-type glucose transporter GLUT1 after transient expression in COS-7 cells. Full-length cDNA was ligated into the expression vector pCMV that is driven by the cytomegalovirus promoter, and introduced into COS cells by the DEAE-dextran method. Cells were homogenized and fractionated by differential centrifugation to yield plasma membranes and a Golgi-enriched fraction of intracellular membranes (low-density microsomes). In these membrane fractions, the abundance of glucose transporters was assessed by immunoblotting with specific antibodies against GLUT1 and GLUT4, and their transport activity was assayed after solubilization and reconstitution into lecithin liposomes. Uptake rates of 2-deoxyglucose assayed in parallel samples were higher in cells expressing GLUT1 or GLUT4 as compared with control cells (transfection of pCMV without transporter cDNA). Reconstituted glucose transport activity in plasma membranes was about 5-fold higher after expression of GLUT1 and GLUT4 as compared with control cells. The relative amount of GLUT4 in the low-density microsomes as detected by reconstitution and immunoblotting exceeded that of the GLUT1, but was much lower than that observed in typical insulin-sensitive cells, e.g., rat fat cells or 3T3-L1 adipocytes. These data indicate that COS-7 cells transfected with glucose transporter cDNA express the active transport proteins and can be used for functional studies.

Immnofluorescence methods for yeast

Article

Feb 1991
METHOD ENZYMOL

This chapter provides protocols for the application of immunofluorescence procedures to yeast. It should perhaps be stressed that immunofluorescence and other light microscopic techniques play a role that is separate from but equal to the role of electron microscopy. Although in some situations the greater resolving power of the electron microscope is clearly essential to obtain the needed structural information, in other situations the necessary information can be obtained more easily, more reliably, or both, by light microscopy. The potential advantages of light microscopic approaches derive from various facts: (1) they can be applied to lightly processed or (in some cases) living cells, (2) Much larger numbers of cells can be examined than by electron microscopy (note especially the great labor involved in visualizing the structure of whole cells by serial-section methods), and (3) Some structures (for example, the cytoplasmic microtubules) have simply been easier to see by light microscopy than by electron microscopy.

Cloning genes by complementation in yeast

Article

Feb 1991
METHOD ENZYMOL

This chapter describes the use of genomic and cDNA banks to isolate specific genes by complementation in Saccharomyces cerevisiae. The most straightforward approach to cloning genes from plasmidborne banks is complementation of a recessive marker. A recipient strain is constructed that carries a recessive mutation in the gene of interest as well as a nonreverting null allele of the chromosomal cognate of the selectable marker carried on the plasmid vector, This strain is then transformed with pools of plasmids from a bank constructed from wild-type genomic DNA. Transformants are recovered by selecting for eomplementation by the vector-borne selectable marker. Cloning genes that are defined by dominant alleles is a straightforward extension of cloning by complementation of recessive alleles. The only difference is that the clone bank has to be constructed de novo from genomic or cDNA prepared from the strain carrying the dominant mutation. In the absence of any direct information about the identity of a gene or its gene product, one recourse is to isolate a set of genes whose regulation fulfills some interesting set of criteria. One approach to achieving this end has been to clone random genomic fragments into a plasmid carrying an enhancerless promoter that drives expression of a readily scored gene, such as lacZ. Random transformants are then examined for conditional expression of lacZ in response to the desired signal.

Erythrocyte/HEPG2-type glucose transporter is concentrated in cells of blood-tissue barriers

Article

Dec 1990

In search of possible diverse roles of glucose transporters (GT's), we examined whether any GT's are present in blood-tissue barriers where selective flow of glucose from blood to tissue cells is critically important. We found in rat that the erythrocyte/HepG2-type GT is localized in all the limiting plasma membranes known to serve as blood-tissue barriers, whether the barriers are endothelial type (brain, iris, inner retina, peripheral nerve) or epithelial type (choroid plexus, ciliary body, outer retina, peripheral nerve, placenta), except for plasma membranes in testis and thymus where no appreciable amount of the GT was found. The erythrocyte/HepG2-type GT may play a vital role for the entry of glucose into these firmly guarded tissues.

Yeast galactose permease is related to yeast and mammalian glucose transporters

Article

Jan 1990
GENE

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.

The kinetic of glucose transport in human red blood cells

Article

Jun 1986
Biochim Biophys Acta

A quenched-flow apparatus and a newly developed automated syringe system have been used to measure initial rates of D-[14C]glucose transport into human red blood cells at temperatures ranging from 0 degrees to 53 degrees C. The Haldane relationship is found to be obeyed satisfactorily at both 0 and 20 degrees C, but Arrhenius plots of maximum D-[14C]glucose transport rates are non-linear under conditions of both equilibrium exchange and zero trans influx. Fitting of the data by non-linear regression to the conventional model for glucose transport gives values at 0 degrees C of 0.726 +/- 0.0498 s-1 and 12.1 +/- 0.98 s-1 for the rate constants governing outward and inward movements of the unloaded carrier molecule and 90.3 +/- 3.47 s-1 and 1113 +/- 494 s-1 for outward and inward movements of the carrier-glucose complex. Activation energies for these four rate constants are respectively 173 +/- 3.10, 127 +/- 4.78, 88.0 +/- 6.17 and 31.7 +/- 5.11 kJ X mol-1. These parameters indicate that at low temperatures the marked asymmetry of the transport mechanism arises mainly from the high proportion of inward-facing carriers and carrier-glucose complexes, and that there is a relatively small difference between the affinities of the carrier for glucose in the inward and outward-facing conformations. At high (physiological) temperatures the carrier is fairly evenly distributed between outward- and inward-facing conformations and the affinity for glucose is about 2.5-times greater outside than inside.

Distinction of three types of D-glucose transport systems in animal cells

Article

Nov 1985

Immunoblotting of plasma membrane fractions from rat kidney cortex with antibody to human erythrocyte glucose transporter showed a single major cross-reacting material of 48K in basolateral membrane fractions possessing a facilitated diffusion system for D-glucose, but not in brush border membrane fractions which have a Na-dependent active transport system. Cytochalasin B inhibited D-glucose uptake in basolateral membrane vesicles but not in brush border vesicles. Cross-reacting materials of 44-55K were detected in several animal cells exhibiting facilitated diffusion systems, including a hormone dependent system. These results indicate molecular difference between glucose transporters of facilitated diffusion systems and active transport systems.

Kinetics of glucose uptake in erythrocytes. Effect of trans-concentration

Article

Mar 1972

Mueckler MFacilitative glucose transporters. Eur J Biochem 219: 713-725

Article

Mar 1994

Mike Mueckler

Facilitative glucose transport is mediated by members of the Glut protein family that belong to a much larger superfamily of 12 transmembrane segment transporters. Six members of the Glut family have been described thus far. These proteins are expressed in a tissue- and cell-specific manner and exhibit distinct kinetic and regulatory properties that reflect their specific functional roles. Glut1 is a widely expressed isoform that provides many cells with their basal glucose requirement. It also plays a special role in transporting glucose across epithelial and endothelial barrier tissues. Glut2 is a high-Km isoform expressed in hepatocytes, pancreatic beta cells, and the basolateral membranes of intestinal and renal epithelial cells. It acts as a high-capacity transport system to allow the uninhibited (non-rate-limiting) flux of glucose into or out of these cell types. Glut3 is a low-Km isoform responsible for glucose uptake into neurons. Glut4 is expressed exclusively in the insulin-sensitive tissues, fat and muscle. It is responsible for increased glucose disposal in these tissues in the postprandial state and is important in whole-body glucose homeostasis. Glut5 is a fructose transporter that is abundant in spermatozoa and the apical membrane of intestinal cells. Glut7 is the transporter present in the endoplasmic reticulum membrane that allows the flux of free glucose out of the lumen of this organelle after the action of glucose-6-phosphatase on glucose 6-phosphate. This review summarizes recent advances concerning the structure, function, and regulation of the Glut proteins.

Yeast Sugar Transporters

Article

Feb 1993

Transport of sugars is a fundamental property of all eukaryotic cells. Of particular importance is the uptake of glucose, a preferred carbon and energy source. The rate of glucose utilization in yeast is often dictated by the activity and concentration of glucose transporters in the plasma membrane. Given the importance of transport as a site of control of glycolytic flux, the regulation of glucose transporters is necessarily complex. The molecular analysis of these transporters in Saccharomyces has revealed the existence of a multigene family of sugar carriers. Recent data have raised the question of the actual role of all of these proteins in sugar catabolism, as some appear to be lowly expressed, and point mutations of these genes may confer pleiotropic phenotypes, inconsistent with a simple role as catabolic transporters. The transporters themselves appear to be intimately involved in the process of sensing glucose, a model for which there is growing support.

The role of N-glycosylation in the targeting and stability of GLUT1 glucose transporter

Article

Jul 1993

The cDNAs encoding the GLUT1 glucose transporter protein were altered by site-directed mutagenesis at consensus sites for the addition of N-linked glycosylation. These cDNAs were transfected into CHO cells with an expression vector and the subcellular distribution and stability of the expressed glycosylation-defective GLUT1 protein were analyzed. Immunohistochemical analysis with a specific antibody demonstrated that a significant portion of glycosylation-defective GLUT1 protein remained in the intracellular compartment. By contrast, most of the wild-type GLUT1 protein expressed with the same procedures resided in the plasma membranes. Metabolic labeling studies revealed that the half-life of the glycosylation-defective GLUT1 protein was significantly shorter than that of wild-type GLUT1 protein. These results indicate that N-glycosylation of the glucose transporter affects its intracellular targeting and protein stability.

A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport

Article

Feb 1993
TRENDS BIOCHEM SCI

Many transport proteins of bacteria and eukaryotes are thought to possess a common structural motif of 12 transmembrane-spanning alpha-helical segments. In this report we use statistical methods to establish that five families or clusters of these facilitators comprise a single superfamily. The five clusters include: (1) drug-resistance proteins, (2) sugar facilitators, (3) facilitators for Krebs cycle intermediates, (4) phosphate ester-phosphate antiporters and (5) a distinct group of oligosaccharide-H+ symporters. Over 50 transporters of bacteria, lower eukaryotes, plants and animals, and one putative bacterial transcriptional regulatory protein are members of this superfamily, which we term the 'major facilitator superfamily' (MFS).

Mammalian passive glucose transporters: members of an ubiquitous family of active and passive transport proteins

Article

Jul 1993
Biochim Biophys Acta

Stephen A Baldwin

Sugar transport in the red blood cell: structure-activity relationships in substrates and antagonists

Article

Apr 1961

Paul G. LeFevre

Jan 1990
67-73

K Takata
T Kasahara
M Kasahara
O Ezaki
H Hirano

Takata, K., Kasahara, T., Kasahara, M., Ezaki, O. and Hirano, H. (1990) Biochem. Biophys. Res. Commun. 173, 67–73

Jan 1993
13-20

M D Marger
M H Saier
Jr

Marger, M. D. and Saier, M. H., Jr. (1993) Trends Biochem. Sci. 18, 13–20

Jan 1993
258-261

T Asano
K Takata
H Katagiri
H Ishihara
K Inukai
M Anai
H Hirano
Y Yazaki
Y Oka

Asano, T., Takata, K., Katagiri, H., Ishihara, H., Inukai, K., Anai, M., Hirano, H., Yazaki, Y. and Oka, Y. (1993) FEBS Lett. 324, 258–261

Jan 1994
713-725

M Mueckler

Mueckler, M. (1994) Eur. J. Biochem. 219, 713–725

Jan 1989
313-319

J O Nehlin
M Carlberg
H Ronne

Nehlin, J. O., Carlberg, M. and Ronne, H. (1989) Gene 85, 313–319

Jan 1985
490-496

M Kasahara
K Inui
M Takano
R Hori

Kasahara, M., Inui, K., Takano, M. and Hori, R. (1985) Biochem. Biophys. Res. Commun. 132, 490–496

Jan 1990
5903-5913

A L Kruckeberg
L F Bisson

Kruckeberg, A. L. and Bisson, L. F. (1990) Mol. Cell. Biol. 10, 5903–5913

Jan 1993
216-222

M G Palmgen
G Christensen

Palmgen, M. G. and Christensen, G. (1993) FEBS Lett. 317, 216–222

Expression of the rat GLUT1 glucose transporter in the yeast Saccharomyces cerevisiae

Abstract

No full-text available

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