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Characterisation of Mammalian GLUT Glucose Transporters in a Heterologous Yeast Expression System
Abstract
We have developed a new heterologous expression system for mammalian glucose transporters. The system is based on a Saccharomyces cerevisiae strain completely deleted for all its endogenous hexose transporters and unable to take up and to grow on hexoses. To target the heterologous glucose transporters into the yeast plasma membrane in a fully active form, additional mutations had to be introduced into the hexose transport-deficient strain. Although GLUT1 was localized at the cell surface already in the parent strain, it supported uptake of glucose only in an deltaHXT FGY1-1 mutant strain. Moreover, various mutations within the first half of the second predicted transmembrane helix converted GLUT1 into a form able to support uptake of glucose into yeast cells. GLUT4 was trapped in intracellular structures but became functionally expressed in the plasma membrane in deltaHXT FGY1-1 FGY4X mutant strains. Glucose transport kinetics were determined with intact yeast cells by zero-trans influx measurements with a Km of 3.2 mM for human GLUT1 and of 12.6 mM for human GLUT4. Cytochalasin B inhibited these activities. Growth tests revealed that both transporter proteins are able to mediate uptake of glucose, mannose and galactose, but not of fructose. The new heterologous expression system should be a valuable tool to develop cell based high-throughput screening assays for identifying pharmaceutical compounds influencing the transporters.
... Deletion of histidinol dehydrogenase [63] S. cerevisiae EBY.S7 MATα hxt1-17∆gal2∆agt1∆stl1∆leu2-3,112 ura3-52 trp1-289 his3-∆1 MAL2-8c SUC2 hxt∆fgy1 Deletion of hexose transporters [64] S. cerevisiae EBY.F4-1 MATα hxt1-17∆gal2∆agt1∆stl1∆leu2-3,112 ura3-52 trp1-289 his3-∆1 MAL2-8c SUC2 hxt∆fgy1 fgy41 Deletion of hexose transporters [64] S. cerevisiae EBY.VW4000 MATa leu2-3,112 ura3-52 trp1-289 his3-1 MAL2-8c SUC2 ∆hxt1-17 ∆gal2 ∆stl1::loxP ∆agt1::loxP ∆mph2::loxP ∆mph3::loxP Deletion of hexose transporters [65] S. cerevisiae SDY.022 MATa leu2-3,112 ura3-52 trp1-289 his3-∆1 MAL2-8C SUC2 ∆hxt1-17 ∆gal2 ∆agt1 ∆stl1 fgy1-1 erg4::kanMX Deletion of hexose transporters [66] S. cerevisiae BJ5457 MATα ura3-52 trp1 lys2-801 leu2-∆1 his3-∆200 pep4:HIS3 prb1-delta1.6R can1 GAL Protease deficient [67] S. cerevisiae RE700A MATa hxt1::HIS3::hxt4 hxt5::LEU2 hxt2::HIS3hxt3::LEU2::hxt6 hxt7::HIS3 Deletion of hexose transporters [68] S. cerevisiae BY4742 MATα his3∆1 leu2∆0 lys2∆0 ura3∆ Minimize homologous recombination [59] S. cerevisiae BY4742 GEV MATa, (PGAL10+gal1)∆::loxP, leu2∆0::PACT1-GEV-NatMX, gal4∆::LEU2, HAP1+ Minimize homologous recombination [69] P. pastoris SMD1168H pep4 Protease A deficiency [59] S. cerevisiae FAB158 MATa his3-∆200 leu2-∆1 lys2-801 trp1-∆1 ade2-101 ura3-52 tat2 ∆::HIS3 Deletion of tryptophan transporter [70] S. cerevisiae TMY203 MATa his3-∆200 leu2-∆1 lys2-801 trp1-∆1 ade2-101 ura3-52 tat1 ∆::kanMX4 tat2∆::LEU2 Deletion of tryptophan transporters [70] S. cerevisiae FAY18A MATa his3-∆200 leu2-∆1 lys2-801 trp1-∆1 ade2-101 ura3-52 HPG1-1, Rsp5 P514T Deletion of Rsp5 ubiquitin ligase [70] S. cerevisiae XPY1263a MATa thi3∆::natMX thi7D::kanMX Deletion of thiamine transporter [71] S. cerevisiae BY4741mp MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; mir1∆; pic2∆ Deletion of phosphate and copper transporter [72] S. cerevisiae BY4741 pic2∆ MATa, leu2,met15, ura3, his3, PIC2::KANMX Deletion of copper transporter [73] S. cerevisiae WB-12 MATα ade2-1 trp1-1 ura3-1 can1-100 aac1::LEU2 aac2::HIS3 Deletion of adenine nucleotide carriers [74] ...
... Deletion of histidinol dehydrogenase [63] S. cerevisiae EBY.S7 MATα hxt1-17∆gal2∆agt1∆stl1∆leu2-3,112 ura3-52 trp1-289 his3-∆1 MAL2-8c SUC2 hxt∆fgy1 Deletion of hexose transporters [64] S. cerevisiae EBY.F4-1 MATα hxt1-17∆gal2∆agt1∆stl1∆leu2-3,112 ura3-52 trp1-289 his3-∆1 MAL2-8c SUC2 hxt∆fgy1 fgy41 Deletion of hexose transporters [64] S. cerevisiae EBY.VW4000 MATa leu2-3,112 ura3-52 trp1-289 his3-1 MAL2-8c SUC2 ∆hxt1-17 ∆gal2 ∆stl1::loxP ∆agt1::loxP ∆mph2::loxP ∆mph3::loxP Deletion of hexose transporters [65] S. cerevisiae SDY.022 MATa leu2-3,112 ura3-52 trp1-289 his3-∆1 MAL2-8C SUC2 ∆hxt1-17 ∆gal2 ∆agt1 ∆stl1 fgy1-1 erg4::kanMX Deletion of hexose transporters [66] S. cerevisiae BJ5457 MATα ura3-52 trp1 lys2-801 leu2-∆1 his3-∆200 pep4:HIS3 prb1-delta1.6R can1 GAL Protease deficient [67] S. cerevisiae RE700A MATa hxt1::HIS3::hxt4 hxt5::LEU2 hxt2::HIS3hxt3::LEU2::hxt6 hxt7::HIS3 Deletion of hexose transporters [68] S. cerevisiae BY4742 MATα his3∆1 leu2∆0 lys2∆0 ura3∆ Minimize homologous recombination [59] S. cerevisiae BY4742 GEV MATa, (PGAL10+gal1)∆::loxP, leu2∆0::PACT1-GEV-NatMX, gal4∆::LEU2, HAP1+ Minimize homologous recombination [69] P. pastoris SMD1168H pep4 Protease A deficiency [59] S. cerevisiae FAB158 MATa his3-∆200 leu2-∆1 lys2-801 trp1-∆1 ade2-101 ura3-52 tat2 ∆::HIS3 Deletion of tryptophan transporter [70] S. cerevisiae TMY203 MATa his3-∆200 leu2-∆1 lys2-801 trp1-∆1 ade2-101 ura3-52 tat1 ∆::kanMX4 tat2∆::LEU2 Deletion of tryptophan transporters [70] S. cerevisiae FAY18A MATa his3-∆200 leu2-∆1 lys2-801 trp1-∆1 ade2-101 ura3-52 HPG1-1, Rsp5 P514T Deletion of Rsp5 ubiquitin ligase [70] S. cerevisiae XPY1263a MATa thi3∆::natMX thi7D::kanMX Deletion of thiamine transporter [71] S. cerevisiae BY4741mp MATa; his3∆1; leu2∆0; met15∆0; ura3∆0; mir1∆; pic2∆ Deletion of phosphate and copper transporter [72] S. cerevisiae BY4741 pic2∆ MATa, leu2,met15, ura3, his3, PIC2::KANMX Deletion of copper transporter [73] S. cerevisiae WB-12 MATα ade2-1 trp1-1 ura3-1 can1-100 aac1::LEU2 aac2::HIS3 Deletion of adenine nucleotide carriers [74] ...
... Later, engineered S. cerevisiae strains, incapable of growing on glucose or related monosaccharides, were created generating useful assay systems for ligand screening of the class I GLUTs and of the class II member, GLUT5 [202,204,236]. The first generated strain was the EBY.18ga (∆hxt) which was deleted for all the endogenous hexose transporters HXTs, the homologous of the GLUTs; the EBY.VW4000 strain (∆hxt ∆mph) was then obtained by further deletion of genes coding maltose transporters (MPH2, MPH3) through the loxP-Cre recombinase system in a CEN.PK2-1C strain background [64,65]. Detailed protocols are available for handling this strain and characterizing expressed sugar transporters [237]. ...
For more than 20 years, yeast has been a widely used system for the expression of human membrane transporters. Among them, more than 400 are members of the largest transporter family, the SLC superfamily. SLCs play critical roles in maintaining cellular homeostasis by transporting nutrients, ions, and waste products. Based on their involvement in drug absorption and in several human diseases, they are considered emerging therapeutic targets. Despite their critical role in human health, a large part of SLCs’ is ‘orphans’ for substrate specificity or function. Moreover, very few data are available concerning their 3D structure. On the basis of the human health benefits of filling these knowledge gaps, an understanding of protein expression in systems that allow functional production of these proteins is essential. Among the 500 known yeast species, S. cerevisiae and P. pastoris represent those most employed for this purpose. This review aims to provide a comprehensive state-of-the-art on the attempts of human SLC expression performed by exploiting yeast. The collected data will hopefully be useful for guiding new attempts in SLCs expression with the aim to reveal new fundamental data that could lead to potential effects on human health.
... EFR3 was identified from a screen for regulators of GLUT function when heterologously expressed in yeast [9]. EFR3 is a palmitoylated protein responsible for PM localisation of phosphatidylinositol 4-kinase type IIIα (PI4K-IIIα) [10][11][12][13]. ...
... When heterologously expressed in the yeast Saccharomyces cerevisiae the mammalian glucose transporter GLUT4 is retained intracellularly, underscoring the conservation of molecular machinery required for regulated trafficking [14]. Even when localized to the cell surface, GLUT4 does not efficiently transport glucose suggesting that regulation of GLUT4 within the PM might be important [9,15]. We carried out a genetic screen to select mutations in the yeast genome that enable mammalian glucose transporters to support uptake of glucose into yeast cells lacking their own endogenous hexose transporters [9]. ...
... Even when localized to the cell surface, GLUT4 does not efficiently transport glucose suggesting that regulation of GLUT4 within the PM might be important [9,15]. We carried out a genetic screen to select mutations in the yeast genome that enable mammalian glucose transporters to support uptake of glucose into yeast cells lacking their own endogenous hexose transporters [9]. Expression of GLUT4 was unable to support growth on glucose of yeast lacking endogenous hexose transporters unless they also carry the recessive mutant fgy1-1 allele [9]. ...
Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localisation of phosphatidylinositol 4-kinase type IIIa to the cell surface. Here we show that knockdown of EFR3 or phosphatidylinositol 4-kinase type IIIa impairs insulin-stimulated glucose transport in adipocytes. Using direct stochastic reconstruction microscopy, we also show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. We propose that EFR3 plays a previously unidentified role in controlling insulin-stimulated glucose transport by facilitating dispersal of GLUT4 within the plasma membrane.
... Establishing the ligand selectivity among closely related GLUT isoforms requires systems that assay a single GLUT. Such GLUT-specific assay systems have been established in hexose transporter-deficient yeast strains engineered to express a single GLUT and are available for GLUT1-5 [65][66][67] . Their application to GLUT ligand discovery and selectivity assessment has been recently demonstrated by Schmidl et al. 63 , who have reported eleven novel GLUT2 inhibitors, among which nine are GLUT2-specific. ...
... For this, we used the recently established assay system 67 in which GLUT3 is expressed in a yeast strain devoid of endogenous hexose transporters (hxt 0 ) so that glucose uptake into cells relies solely on the activity of GLUT3. Similar assay systems are available for GLUT1 65 , GLUT2 67 , GLUT4 65 , and GLUT5 66 , enabling identification and selectivity assessment of their inhibitors 63 . We determined GLUT3 transport activity by measuring the accumulation of radioactive glucose inside whole hxt 0 yeast cells (see "Materials and methods" for details). ...
... Its value of 3.11 ± 0.42 mM ( Supplementary Fig. S1) is higher than that for 2-deoxy-glucose reported for GLUT3 expressed in Xenopus laevis oocytes (K M, 2-deoxy-glucose ~ 1.4 mM 12 ), the discrepancy possibly stemming from the different structure of the substrates or the changed lipid environment. Indeed, functional expression of human GLUTs in hxt 0 cells often requires mutations in genes related to yeast lipid composition 65,73 as well as single-site mutations that may favor the outward-facing conformation of the transporters [65][66][67] . As previously proposed 69 , this suggests that the transporter conformational dynamics depends on the lipid environment and, in the yeast membrane, GLUT's function is 'nativized' by facilitating the outward-facing conformation. ...
The passive transport of glucose and related hexoses in human cells is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT3 is a high-affinity glucose transporter primarily responsible for glucose entry in neurons. Changes in its expression have been implicated in neurodegenerative diseases and cancer. GLUT3 inhibitors can provide new ways to probe the pathophysiological role of GLUT3 and tackle GLUT3-dependent cancers. Through in silico screening of an ~ 8 million compounds library against the inward- and outward-facing models of GLUT3, we selected ~ 200 ligand candidates. These were tested for in vivo inhibition of GLUT3 expressed in hexose transporter-deficient yeast cells, resulting in six new GLUT3 inhibitors. Examining their specificity for GLUT1-5 revealed that the most potent GLUT3 inhibitor (G3iA, IC 50 ~ 7 µM) was most selective for GLUT3, inhibiting less strongly only GLUT2 (IC 50 ~ 29 µM). None of the GLUT3 inhibitors affected GLUT5, three inhibited GLUT1 with equal or twofold lower potency, and four showed comparable or two- to fivefold better inhibition of GLUT4. G3iD was a pan-Class 1 GLUT inhibitor with the highest preference for GLUT4 (IC 50 ~ 3.9 µM). Given the prevalence of GLUT1 and GLUT3 overexpression in many cancers and multiple myeloma’s reliance on GLUT4, these GLUT3 inhibitors may discriminately hinder glucose entry into various cancer cells, promising novel therapeutic avenues in oncology.
... system that expresses human GLUT2 38 . Similar GLUT-specific hxt 0 yeast systems are available for all Class I GLUTs and GLUT5 38,[40][41][42] , providing a convenient assay platform for these transporters' ligands 16 . For GLUT2, the applied yeast strain EBY.S7 is devoid of all its endogenous hexose transporters (hxt 0 ) and carries the fgy1 mutation 36 in the EFR3 gene, proven to be beneficial for the heterologous expression of human GLUTs 16 . ...
... Therefore, to determine the selectivity of the identified GLUT2 inhibitors, we tested them for their effect on the GLUT homologs GLUT1, 3, 4, and 5. For this, hxt 0 yeast cells actively expressing the respective transporter 38,[40][41][42] were incubated with 100 µM of the tested compound, and the transport activity was assayed in the same manner as for GLUT2 but at substrate concentrations close to the K M in the respective GLUT (i.e., 5 mM glucose for GLUT1 44 and GLUT4 44 , 1.5 mM glucose for GLUT3 43 , 10 mM fructose for GLUT5 41 ) (Fig. 3A). GLUT2 is more closely Table S1) are identified by the ChemNavigator structure ID. ...
... Yeast cell culturing was done at 30 ºC with shaking (180-220 rpm). The plasmids containing the functional constructs of GLUT1-5 (GLUT1, GLUT2 ∆loopS_Q455R , GLUT3 S66Y , GLUT4, GLUT5 S72Y ) were transformed in the corresponding hxt 0 strains (EBY.VW4000 for GLUT5, EBY.S7 for GLUT1-3, and EBY.S7 Δerg4 for GLUT4) 38,[40][41][42] and grown on 2% (w/v) agar plates of the respective media supplemented with 1% (w/v) maltose. An initial culture of ~ 10 ml was started with a few colonies and grown for 2-3 days if the media was SC-uracil with 1% (w/v) maltose (for GLUT1, GLUT3, and GLUT4) or 1-2 days if the media was YEP with 1% (w/v) maltose and 100 µg/ml G418 (for GLUT2 and GLUT5). ...
Glucose is an essential energy source for cells. In humans, its passive diffusion through the cell membrane is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT2 transports both glucose and fructose with low affinity and plays a critical role in glucose sensing mechanisms. Alterations in the function or expression of GLUT2 are involved in the Fanconi–Bickel syndrome, diabetes, and cancer. Distinguishing GLUT2 transport in tissues where other GLUTs coexist is challenging due to the low affinity of GLUT2 for glucose and fructose and the scarcity of GLUT-specific modulators. By combining in silico ligand screening of an inward-facing conformation model of GLUT2 and glucose uptake assays in a hexose transporter-deficient yeast strain, in which the GLUT1-5 can be expressed individually, we identified eleven new GLUT2 inhibitors (IC50 ranging from 0.61 to 19.3 µM). Among them, nine were GLUT2-selective, one inhibited GLUT1-4 (pan-Class I GLUT inhibitor), and another inhibited GLUT5 only. All these inhibitors dock to the substrate cavity periphery, close to the large cytosolic loop connecting the two transporter halves, outside the substrate-binding site. The GLUT2 inhibitors described here have various applications; GLUT2-specific inhibitors can serve as tools to examine the pathophysiological role of GLUT2 relative to other GLUTs, the pan-Class I GLUT inhibitor can block glucose entry in cancer cells, and the GLUT2/GLUT5 inhibitor can reduce the intestinal absorption of fructose to combat the harmful effects of a high-fructose diet.
... When heterologously expressed in the yeast S. cerevisiae the mammalian glucose transporter GLUT4 is retained intracellularly, underscoring the conservation of molecular machinery required for regulated trafficking (Shewan et al., 2013). Even when localised to the cell surface, GLUT4 does not efficiently transport glucose suggesting that regulation of GLUT4 within the PM might be important (Kasahara and Kasahara, 1997;Wieczorke et al., 2003). We carried out a genetic screen to select mutations in the yeast genome that enable mammalian glucose transporters to support uptake of glucose into yeast cells lacking their own endogenous hexose transporters (Wieczorke et al., 2003). ...
... Even when localised to the cell surface, GLUT4 does not efficiently transport glucose suggesting that regulation of GLUT4 within the PM might be important (Kasahara and Kasahara, 1997;Wieczorke et al., 2003). We carried out a genetic screen to select mutations in the yeast genome that enable mammalian glucose transporters to support uptake of glucose into yeast cells lacking their own endogenous hexose transporters (Wieczorke et al., 2003). ...
... Expression of GLUT4 was unable to support growth on glucose of yeast lacking endogenous hexose transporters unless they also carry the recessive mutant fgy1-1 allele (Wieczorke et al., 2003). fgy1-1 is a mutant allele of EFR3 (Wieczorke and Boles, personal communication; (Schmidl et al., 2020). ...
Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localisation of phosphatidylinositol 4-kinase type IIIα to the cell surface. Here we show that knockdown of EFR3 or phosphatidylinositol 4-kinase type IIIα impairs insulin-stimulated glucose transport in adipocytes. Using direct stochastic reconstruction microscopy, we also show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. We propose that EFR3 plays a previously unidentified role in controlling insulin-stimulated glucose transport by facilitating dispersal of GLUT4 within the plasma membrane.
... February 2021 | Volume 7 | Article 598419 transformation of EBY.VW4000 cells with native rat GLUTs did not yield cell growth on glucose (for GLUT1 and GLUT4) (Kasahara and Kasahara, 1996;Kasahara and Kasahara, 1997) or fructose (for GLUT5) (Tripp et al., 2017). Nevertheless, single point mutations in the TM2 of GLUT1 and GLUT5 enabled their activity in EBY.VW4000 (Wieczorke et al., 2002;Tripp et al., 2017). Also, wild-type GLUT1 was functionally expressed in a hxt 0 strain harboring the additional fgy1 ("functional expression of GLUT1 in yeast") mutation (Wieczorke et al., 2002) that affects the scaffold protein Efr3 (Wieczorke and Boles, personal communication). ...
... Nevertheless, single point mutations in the TM2 of GLUT1 and GLUT5 enabled their activity in EBY.VW4000 (Wieczorke et al., 2002;Tripp et al., 2017). Also, wild-type GLUT1 was functionally expressed in a hxt 0 strain harboring the additional fgy1 ("functional expression of GLUT1 in yeast") mutation (Wieczorke et al., 2002) that affects the scaffold protein Efr3 (Wieczorke and Boles, personal communication). Efr3 is essential for recruiting the Stt4 phosphatidylinositol-4-kinase to the plasma membrane and, consequently, builds a prerequisite for normal membrane phosphatidylinositol-4-phosphate levels (Wu et al., 2014). ...
... Efr3 is essential for recruiting the Stt4 phosphatidylinositol-4-kinase to the plasma membrane and, consequently, builds a prerequisite for normal membrane phosphatidylinositol-4-phosphate levels (Wu et al., 2014). The corresponding strain was named EBY.S7 (Wieczorke et al., 2002). Murine GLUT4 was only active in a strain named SDY.022 ...
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.
... Preventing energy input for extracellular transport (nutrient uptake and product excretion) Transport of substrates and products over the microbial plasma membrane is an integral part of fermentation processes. In many studies homologous and heterologous transporters have been expressed to modify substrate specificity, growth and product formation (Zaslavskaia et al., 2001;Hernández-Montalvo et al., 2003;Wieczorke et al., 2003;De Anda et al., 2006;Doebbe et al., 2007;Subtil and Boles, 2011;Young et al., 2011;Wang et al., 2015a;Shin et al., 2018). Several transport mechanisms are available which differ with respect to the requirement of metabolic energy input (Jahreis et al., 2008). ...
... Because PTS systems require the conversion of phosphoenolpyruvate into pyruvate, their application for products relying on phosphoenolpyruvate but not pyruvate is limited (Floras et al., 1996;Hernández-Montalvo et al., 2003;Nakamura and Whited, 2003;De Anda et al., 2006;Shin et al., 2018; Yang et al., 2018). Sugar facilitator transporters have been identified in mammals, yeasts and bacteria (Wieczorke et al., 2003;Jahreis et al., 2008;Leandro et al., 2011). Several groups have successfully expressed the glucose facilitator of Zymomonas mobilis in E. coli strains (Snoep et al., 1994;Parker et al., 1995;Weisser et al., 1995) and human glucose facilitators in S. cerevisiae (Wieczorke et al., 2003). ...
... Sugar facilitator transporters have been identified in mammals, yeasts and bacteria (Wieczorke et al., 2003;Jahreis et al., 2008;Leandro et al., 2011). Several groups have successfully expressed the glucose facilitator of Zymomonas mobilis in E. coli strains (Snoep et al., 1994;Parker et al., 1995;Weisser et al., 1995) and human glucose facilitators in S. cerevisiae (Wieczorke et al., 2003). PTS systems occur in eubacteria, a few archaebacteria but not in plants and animals (Jeckelmann and Erni, 2019). ...
Microbial production of bulk chemicals and biofuels from carbohydrates competes with low‐cost fossil‐based production. To limit production costs, high titres, productivities and especially high yields are required. This necessitates metabolic networks involved in product formation to be redox‐neutral and conserve metabolic energy to sustain growth and maintenance. Here, we review the mechanisms available to conserve energy and to prevent unnecessary energy expenditure. First, an overview of ATP production in existing sugar‐based fermentation processes is presented. Substrate‐level phosphorylation (SLP) and the involved kinase reactions are described. Based on the thermodynamics of these reactions, we explore whether other kinase‐catalysed reactions can be applied for SLP. Generation of ion‐motive force is another means to conserve metabolic energy. We provide examples how its generation is supported by carbon‐carbon double bond reduction, decarboxylation and electron transfer between redox cofactors. In a wider perspective, the relationship between redox potential and energy conservation is discussed. We describe how the energy input required for coenzyme A (CoA) and CO2 binding can be reduced by applying CoA‐transferases and transcarboxylases. The transport of sugars and fermentation products may require metabolic energy input, but alternative transport systems can be used to minimize this. Finally, we show that energy contained in glycosidic bonds and the phosphate‐phosphate bond of pyrophosphate can be conserved. This review can be used as a reference to design energetically efficient microbial cell factories and enhance product yield.
... We checked the effect of compounds G1-G17 on the transport activity of an individual GLUT by using a GLUT-specific assay method, which utilizes hexose transporter deficient yeast cells (hxt 0 ) expressing a particular human GLUT (Tripp et al., 2017;Wieczorke et al., 2003). In hxt 0 strains, yeast cell growth in hexose-based media relies solely on the transport activity of the expressing human GLUT for bringing the carbon source into cells. ...
... In hxt 0 strains, yeast cell growth in hexose-based media relies solely on the transport activity of the expressing human GLUT for bringing the carbon source into cells. Thus, GLUT transport activity is determined as the C 14 -hexose uptake into whole cells (Tripp et al., 2017;Wieczorke et al., 2003). The primary inhibition screening was carried out at 100 μM compound concentration on three glucose transporters: GLUT1, GLUT4, and GLUT5 ( Fig. 3 ...
... Compounds G1-G17 were analyzed for their effect on the transport activity of GLUT1 (Wieczorke et al., 2003), GLUT4 (Wieczorke et al., 2003), and GLUT5 (Tripp et al., 2017) expressed in hexose transporter null yeast cells (hxt 0 ). The assay was carried out as described previously . ...
Cancer is a heterogeneous disease, and its treatment requires the identification of new ways to thwart tumor cells. Amongst such emerging targets are glucose transporters (GLUTs, SLC2 family), which are overexpressed by almost all types of cancer cells; their inhibition provides a strategy to disrupt tumor metabolism selectively, leading to antitumor effects. Here, novel Thiazolidinedione (TZD) derivatives were designed, synthesized, characterized, and evaluated for their GLUT1, GLUT4, and GLUT5 inhibitory potential, followed by in vitro cytotoxicity determination in leukemic cell lines. Compounds G5, G16, and G17 inhibited GLUT1, with IC50 values of 5.4 ± 1.3, 26.6 ± 1.8, and 12.6 ± 1.2 μM, respectively. G17 was specific for GLUT1, G16 inhibited GLUT4 (IC50=21.6 ± 4.5 μM) comparably but did not affect GLUT5. The most active compound, G5, inhibited all three GLUT types, with GLUT4 IC50 = 9.5 ± 2.8 μM, and GLUT5 IC50 = 34.5 ± 2.4 μM. Docking G5, G16, and G17 to the inward- and outward-facing structural models of GLUT1 predicted ligand binding affinities consistent with the kinetic inhibition data and implicated E380 and W388 of GLUT1 vs. their substitutions in GLUT5 (A388 and A396, respectively) in inhibitor preference for GLUT1. G5 inhibited the proliferation of leukemia CEM cells at low micromolar range (IC50 = 13.4 μM) while being safer for normal blood cells. Investigation of CEM cell cycle progression after treatment with G5 showed that cells accumulated in the G2/M phase. Flow cytometric apoptosis studies revealed that compound G5 induced both early and late-stage apoptosis in CEM cells.
... To assay the transport activity and determine the effect of designed compounds on a singular GLUT, we used GLUT-specific assay systems: hexose transporter deficient yeast cells (hxt 0 ) expressing a particular human GLUT [47]. In hxt 0 strains, yeast cell growth in hexose-based media relies solely on the transport activity of the expressing human GLUT for bringing the carbon source into cells. ...
... In hxt 0 strains, yeast cell growth in hexose-based media relies solely on the transport activity of the expressing human GLUT for bringing the carbon source into cells. Thus, GLUT transport activity is determined as the C 14hexose uptake into whole cells [47]. The primary inhibition screening was carried out at 100 mM compound concentration on three glucose transporters: GLUT1, GLUT4, and GLUT5. ...
... Synthesized compounds were examined for their effect on transport activity of human GLUT1 [47], GLUT4 [47], and GLUT5 ...
Cancer cells increase their glucose uptake and glycolytic activity to meet the high energy requirements of proliferation. Glucose transporters (GLUTs), which facilitate the transport of glucose and related hexoses across the cell membrane, play a vital role in tumor cell survival and are overexpressed in various cancers. GLUT1, the most overexpressed GLUT in many cancers, is emerging as a promising anti-cancer target. To develop GLUT1 inhibitors, we rationally designed, synthesized, structurally characterized, and biologically evaluated in-vitro and in-vivo a novel series of furyl-2-methylene thiazolidinediones (TZDs). Among 25 TZDs tested, F18 and F19 inhibited GLUT1 most potently (IC50 11.4 and 14.7 μM, respectively). F18 was equally selective for GLUT4 (IC50 6.8 μM), while F19 was specific for GLUT1 (IC50 152 μM in GLUT4). In-silico ligand docking studies showed that F18 interacted with conserved residues in GLUT1 and GLUT4, while F19 had slightly different interactions with the transporters. In in-vitro antiproliferative screening of leukemic/lymphoid cells, F18 was most lethal to CEM cells (CC50 of 1.7 μM). Flow cytometry analysis indicated that F18 arrested cell cycle growth in the subG0-G1 phase and lead to cell death due to necrosis and apoptosis. Western blot analysis exhibited alterations in cell signaling proteins, consistent with cell growth arrest and death. In-vivo xenograft study in a CEM model showed that F18 impaired tumor growth significantly.
... Considering that HDAC inhibitors also interfere with GLUT1 expression and to assess their inhibitory potential on GLUTs, we screened all target compounds (P1-P25) for their effect on the transport activity of GLUT 1, 4, and 5 at 50 μM concentration (Fig. 5). For the transport assay, we used the GLUT-specific systems provided by the hexose transport null (hxt 0 ) yeast strains engineered to express a particular human GLUT [47,48]; the only glucose or fructose uptake in these cells is through the recombinant human GLUTs. Among all tested compounds, P19 showed significant inhibition of GLUT1 (IC 50 = 28.2 ± 1.8 μM) and remained ineffective on the other two GLUT isoforms (Fig. 5). ...
... Synthesized compounds were examined for their effect on the transport activity of GLUT1 [47], GLUT4 [47], and GLUT5 [48] expressed in hexose transporter null yeast cells (hxt 0 ). Yeast cell culturing was done at 30°C with shaking (180 rpm). ...
... Synthesized compounds were examined for their effect on the transport activity of GLUT1 [47], GLUT4 [47], and GLUT5 [48] expressed in hexose transporter null yeast cells (hxt 0 ). Yeast cell culturing was done at 30°C with shaking (180 rpm). ...
Epigenetics plays a fundamental role in cancer progression, and developing agents that regulate epigenetics is crucial for cancer management. Among Class I and Class II HDACs, HDAC8 is one of the essential epigenetic players in cancer progression. Therefore, we designed, synthesized, purified, and structurally characterized novel compounds containing N-substituted TZD (P1-P25). Cell viability assay of all compounds on leukemic cell lines (CEM, K562, and KCL22) showed the cytotoxic potential of P8, P9, P10, P12, P19, and P25. In-vitro screening of different HDACs isoforms revealed that P19 was the most potent and selective inhibitor for HDAC8 (IC50 – 9.3 μM). Thermal shift analysis (TSA) confirmed the binding of P19 to HDAC8. In-vitro screening of all compounds on the transport activity of GLUT1, GLUT4, and GLUT5 indicated that P19 inhibited GLUT1 (IC50 – 28.2 μM). P10 and P19 induced apoptotic cell death in CEM cells (55.19% and 60.97% respectively) and P19 was less cytotoxic on normal WBCs (CC50 – 104.2 μM) and human fibroblasts (HS27) (CC50 – 105.0 μM). Thus, among this novel series of TZD derivatives, compound P19 was most promising HDAC8 inhibitor and cytotoxic on leukemic cells. Thus, P19 could serve as a lead for further development of optimized molecules with enhanced selectivity and potency.
... The resulting strain was named EBY.VW4000 and is unable to take up and grow on glucose or related hexoses as sole carbon source. The functional expression of human GLUTs in this hexose transporter deficient (hxt 0 ) yeast strain restores its ability to grow on glucose or fructose enabling compound screening for the particular human GLUT via simple cell growth assays (Wieczorke et al., 2002). Even though cell growth is the simplest parameter to determine the functionality of the transporters or potency of the inhibitors, compound screening is not limited to this method. ...
... However, the functional expression of human GLUTs in yeast cells require additional modifications either within the transporter or in the genome of the yeast strain. Whereas wildtype GLUT1, GLUT4 and GLUT5 Kasahara, 1996, 1997;Wieczorke et al., 2002;Tripp et al., 2017) were not active in the hxt 0 strain, single point mutations in the transmembrane region 2 of GLUT1 and GLUT5 mediated their functional expression (Wieczorke et al., 2002;Tripp et al., 2017). Wild-type GLUT1 was active only in the hxt 0 strain that additionally acquired the fgy1 (for functional expression of GLUT1 in yeast) mutation (Wieczorke et al., 2002). ...
... However, the functional expression of human GLUTs in yeast cells require additional modifications either within the transporter or in the genome of the yeast strain. Whereas wildtype GLUT1, GLUT4 and GLUT5 Kasahara, 1996, 1997;Wieczorke et al., 2002;Tripp et al., 2017) were not active in the hxt 0 strain, single point mutations in the transmembrane region 2 of GLUT1 and GLUT5 mediated their functional expression (Wieczorke et al., 2002;Tripp et al., 2017). Wild-type GLUT1 was active only in the hxt 0 strain that additionally acquired the fgy1 (for functional expression of GLUT1 in yeast) mutation (Wieczorke et al., 2002). ...
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.
... However, limited numbers of studies, only two early works, have shown that 2-NBDG can be incorporated into yeast cells [31,32]. To directly determine whether 2-NBDG is transported into the yeast S. cerevisiae through the glucose transport system, the yeast cells lacking all HXT (glucose transporter) genes [33] were transformed with an empty plasmid or with a plasmid encoding Hxt1-HA. The resulting transformants were first grown in SC-glycerol/ethanol medium till mid log phase and shifted to the same medium containing 60 μM of 2-NBDG. ...
... as compared with those of wild type Hxt1 (Fig. 2C). The hxt null strain is unable to grow on glucose as a sole carbon source and this defect is fully complemented by expression of wild type Hxt1 [33]. To test the mutant Hxt1 proteins for their ability to transport glucose, the hxt null mutant strain was transformed with plasmids encoding the mutant Hxt1-HA transporters and scored for growth on glucose medium. ...
... The Saccharomyces cerevisiae strains used in this study are BY4742 (WT, Mata his3Δ1 leu2Δ0 ura3Δ0 met15Δ), EBY.S7 (MATα hxt1-17Δgal2Δagt1Δstl1Δleu2-3,112 ura3-52 trp1-289 his3-Δ1 MAL2-8c SUC2 hxtΔfgy1-1 [33]) and KFY127 (Matα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 end3:: KanMX [47]). Yeast cells were grown in YP (2% bacto-peptone, 1% yeast extract) and SC (synthetic yeast nitrogen base medium containing 0.17% yeast nitrogen base and 0.5% ammonium sulfate) media supplemented with the appropriate amino acids and carbon sources. ...
The transport of glucose across the plasma membrane is mediated by members of the glucose transporter family. In this study, we investigated glucose uptake through the yeast hexose transporter 1 (Hxt1) by measuring incorporation of 2-NBDG, a non-metabolizable, fluorescent glucose analog, into the yeast Saccharomyces cerevisiae. We find that 2-NBDG is not incorporated into the hxt null strain lacking all glucose transporter genes and that this defect is rescued by expression of wild type Hxt1, but not of Hxt1 with mutations at the putative glucose-binding residues, inferred from the alignment of yeast and human glucose transporter sequences. Similarly, the growth defect of the hxt null strain on glucose is fully complemented by expression of wild type Hxt1, but not of the mutant Hxt1 proteins. Thus, 2-NBDG, like glucose, is likely to be transported into the yeast cells through the glucose transport system. Hxt1 is internalized and targeted to the vacuole for degradation in response to glucose starvation. Among the mutant Hxt1 proteins, Hxt1N370A and HXT1W473A are resistant to such degradation. Hxt1N370A, in particular, is able to neither uptake 2-NBDG nor restore the growth defect of the hxt null strain on glucose. These results demonstrate 2-NBDG as a fluorescent probe for glucose uptake in the yeast cells and identify N370 as a critical residue for the stability and function of Hxt1.
... Constructs were introduced into the hexose uptake-deficient yeast strain EBY.S7 [20]. Transformants were grown on minimal media containing 2% maltose. ...
... A similarly truncated STP1 variant is expected to be expressed in stp1-1 plants; however, the STP1 protein expressed in stp1-1 plants should contain several additional residues from the T-DNA sequence compared with STP1ΔC. The EBY.S7 yeast strain used in this study is not able to grow on media containing glucose as the sole carbon source because it lacks multiple monosaccharide transporters [20]. Consistent with our previous report [4], the introduction of STP1-GFP complemented yeast growth on glucose media ( Fig 2B). ...
Membrane trafficking is highly organized to maintain cellular homeostasis in any organisms. Membrane-embedded transporters are targeted to various organelles to execute appropriate partition and allocation of their substrates, such as ions or sugars. To ensure the fidelity of targeting and sorting, membrane proteins including transporters have sorting signals that specify the subcellular destination and the trafficking pathway by which the destination is to be reached. Here, we have identified a novel sorting signal (called the tri-aromatic motif) which contains three aromatic residues, two tryptophans and one histidine, for the plasma membrane localization of sugar transporters in the STP family in Arabidopsis. We firstly found that a C-terminal deletion disrupted the sugar uptake activity of STP1 in yeast cells. Additional deletion and mutation analyses demonstrated that the three aromatic residues in the C-terminus, conserved among all Arabidopsis STP transporters, were critical for sugar uptake by not only STP1 but also another STP transporter STP13. We observed that, when the tri-aromatic motif was mutated, STP1 was largely localized at the endomembrane compartments in yeast cells, indicating that this improper subcellular localization led to the loss of sugar absorption. Importantly, our further analyses uncovered that mutations of the tri-aromatic motif resulted in the endoplasmic reticulum (ER) retention of STP1 and STP13 in plant cells, suggesting that this motif is involved at the step of ER exit of STP transporters to facilitate their plasma membrane localization. Together, we here identified a novel ER export signal, and showed that appropriate sorting via the tri-aromatic motif is important for sugar absorption by STP transporters.
... In initial trials, the human glucose transporters GLUT1 and GLUT4 did not confer growth of the hxt 0 strain on glucose 30,31 . In a later approach, the complementation of the hxt 0 phenotype by GLUT1 and GLUT4 could be achieved by prolonged incubation on glucose-containing media or UV-mutagenesis of the transformed yeast cells 32 . By genetic analyses, this could be attributed to mutations either in the GLUT transporter sequence or in the genome of the yeast host. ...
... Strikingly, the positions S72 and S76 are directly adjacent to residues that align to positions W65 and V69 of GLUT1 (see alignment in the supplementary information, Supplementary Fig. S5). W65R and V69M mutations were found in GLUT1 variants, which were functional in yeast 32 . All these mutations are located in the second transmembrane helix (TM2) of GLUT5 or GLUT1. ...
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.
... In addition, because various sugars and the aforementioned mutants would affect the expression of numerous genes, we cannot completely exclude the possibility that TC sensitivity and its alleviation by additional sugars are due to the expression of non-HXT genes. To avoid the complicated effects of multiple HXT genes and their regulation, we examined the drug effects in the hxt 0 mutant (EBY.S7) (Wieczorke et al. 2003), in which all HXT genes are deleted but a HXT gene is expressed under the same ADH1 promoter. The ADH1 promoter-driven HXT1 and HXT2 genes were expressed on single-copy vectors (pHXT1s and pHXT2s) or multicopy vectors (pHXT1m and pHXT2m) in the hxt 0 mutant. ...
... The function of Hxts is highly conserved from yeast to animals (Kasahara and Kasahara 1997;Wieczorke et al. 2003). Administration of volatile and local anesthetics is known to increase blood glucose levels in animals and humans (Diltoer and Camu 1988;Nakamura et al. 2001). ...
Action mechanisms of anesthetics remain unclear because of difficulty in explaining how structurally different anesthetics cause similar effects. In Saccharomyces cerevisiae, local anesthetics and antipsychotic phenothiazines induced responses similar to those caused by glucose starvation, and they eventually inhibited cell growth. These drugs inhibited glucose uptake but additional glucose conferred resistance to their effects; hence, the primary action of the drugs is to cause glucose starvation. In hxt(0) strains with all hexose transporter (HXT) genes deleted, a strain harboring a single copy of HXT1 (HXT1s) was more sensitive to tetracaine than a strain harboring multiple copies (HXT1m), which indicates that quantitative reduction of HXT1 increases tetracaine sensitivity. However, additional glucose rather than the overexpression of HXT1/2 conferred tetracaine resistance to wild-type yeast; therefore, Hxts that actively transport hexoses apparently confer tetracaine resistance. Additional glucose alleviated sensitivity to local anesthetics and phenothiazines in the HXT1m strain but not HXT1s strain; thus, the glucose-induced effects required a certain amount of Hxt1. At low concentrations, fluorescent phenothiazines were distributed in various membranes. At higher concentrations, they destroyed the membranes and thereby delocalized Hxt1-GFP from the plasma membrane, similar to local anesthetics. These results suggest that the aforementioned drugs affect various membrane targets via nonspecific interactions with membranes. However, the drugs preferentially inhibit the function of abundant Hxts, resulting in glucose starvation. When Hxts are scarce, this preference is lost, thereby losing the alleviation by additional glucose. These results provide a mechanism that explains how different compounds induce similar effects according to lipid theory.
... Contrary to the leaves, the root monosaccharide contents increased more between the sampling periods in comparison to disaccharides. This was probably because the roots are also able to absorb monosaccharides from the rhizosphere [44]. Additionally, according to Yamada et al. [45], monosaccharides are stored in the roots, but are transferred into other plant organs to be transformed into other sugar forms. ...
The present study examined the effects of different nitrogen (NH4NO3) and potassium (KNO3) fertilization levels in combination with a nitrogen-fixing, plant growth-promoting rhizobacteria (PGPR) inoculation on the carbohydrate (CHO), amino acid content, and nutrient concentrations (N, P, K) in the spears and the root system of asparagus plants. No significant differences were indicated between the different fertilization treatments regarding N, P, and K in the leaves and roots of asparagus. The inoculation of the asparagus fields with PGPR, no matter the type of the inorganic fertilizer, resulted in increased CHO and amino acid content of the foliage and roots of asparagus. The highest CHO content and amino acid content were recorded in the treatment that combined PGPR inoculation along with KNO3 fertilizer, indicating that higher K applications acted synergistically with the added PGPR.
... 97 In this regard, the yeast Saccharomyces cerevisiae is often a useful expression host as its relatively easy to genetically manipulate in the removal of competing transporters. 98 The use of Xenopus oocytes is also a useful expression host due to its limited competition with endogenous transport activity in the oocyte plasma membrane, e.g., glucose (GLUT) transporters, 99,100 but like yeast, transporters may not be functional in Xenopus oocytes as they may require mammalian cells due to the requirement for certain lipids and/or complex N-linked glycosylation for folding. 101 Cell-based assays further have the limitation that it is not possible to control the internal environment, which might be critical for functional characterization of antiporters, for example. ...
The major facilitator superfamily (MFS) is the largest known superfamily of secondary active transporters. MFS transporters are responsible for transporting a broad spectrum of substrates, either down their concentration gradient or uphill using the energy stored in the electrochemical gradients. Over the last 10 years, more than a hundred different MFS transporter structures covering close to 40 members have provided an atomic framework for piecing together the molecular basis of their transport cycles. Here, we summarize the remarkable promiscuity of MFS members in terms of substrate recognition and proton coupling as well as the intricate gating mechanisms undergone in achieving substrate translocation. We outline studies that show how residues far from the substrate binding site can be just as important for fine-tuning substrate recognition and specificity as those residues directly coordinating the substrate, and how a number of MFS transporters have evolved to form unique complexes with chaperone and signaling functions. Through a deeper mechanistic description of glucose (GLUT) transporters and multidrug resistance (MDR) antiporters, we outline novel refinements to the rocker-switch alternating-access model, such as a latch mechanism for proton-coupled monosaccharide transport. We emphasize that a full understanding of transport requires an elucidation of MFS transporter dynamics, energy landscapes, and the determination of how rate transitions are modulated by lipids.
... The yeast strain used was BY4741 as the wild type (MATa his3Δ1 leu2Δ0 ura3Δ0 met15Δ0) and EBY.S7, deleting all 18 hexose transporter genes (MATa hxt1−17Δgal2Δagt1Δstl1Δleu2−3, 112 ura3−52 trp1−289 his3-Δ1 MAL2−8c SUC2 fgy1−1). 53 EBY.S7 harboring Hxt2mnx-pVT, the ADH1 promoter-driven HXT2 gene on multicopy vector pVT102-U (2 μ ori, URA3), was used as the HXT2m strain expressing a high-affinity hexose transporter alone. 35,54 Cellular turbidity (OD 600 ) was monitored by a UV spectrophotometer (Ultrospec 1100 Pro, GE Healthcare), and the cell numbers were counted with a hematocytometer. ...
Quinacrine (QC) and chloroquine (CQ) have antimicrobial and antiviral activities as well as antimalarial activity, although the mechanisms remain unknown. QC increased the antimicrobial activity against yeast exponentially with a pH-dependent increase in the cationic amphiphilic drug (CAD) structure. CAD-QC localized in the yeast membranes and induced glucose starvation by noncompetitively inhibiting glucose uptake as antipsychotic chlorpromazine (CPZ) did. An exponential increase in antimicrobial activity with pH-dependent CAD formation was also observed for CQ, indicating that the CAD structure is crucial for its pharmacological activity. A decrease in CAD structure with a slight decrease in pH from 7.4 greatly reduced their effects; namely, these drugs would inefficiently act on falciparum malaria and COVID-19 pneumonia patients with acidosis, resulting in resistance. The decrease in CAD structure at physiological pH was not observed for quinine, primaquine, or mefloquine. Therefore, restoring the normal blood pH or using pH-insensitive quinoline drugs might be effective for these infectious diseases with acidosis.
... It has been described that HTLV's receptor-binding domain interacts with GLUT-1 -the mammalian glucose transporter -, inhibiting the sugar transport and finally perturbing the glucose metabolism (Manel et al. 2003). The expression of GLUT-1 in a "null" hexose transport S. cerevisiae background has already been described by Wieczorke et al. (2003). We envision that the GPCR/YSD strategy could enable HTLV's sensing through extracellular glucose concentration variation due to the virus interaction with GLUT-1 (acting as the membrane receptor) followed by GPCR-mediated glucose detection coupled, for instance, to the mating pathway. ...
Viral infections pose intense burdens to healthcare systems and global economies. The correct diagnosis of viral diseases represents a crucial step towards effective treatments and control. Biosensors have been successfully implemented as accessible and accurate detection tests for some of the most important viruses. While most biosensors are based on physical or chemical interactions of cell-free components, the complexity of living microorganisms holds a poorly explored potential for viral detection in the face of the advances of synthetic biology. Indeed, cell-based biosensors have been praised for their versatility and economic attractiveness, however, yeast platforms for viral disease diagnostics are still limited to indirect antibody recognition. Here we propose a novel strategy for viral detection in Saccharomyces cerevisiae, which combines the transductive properties of G Protein-Coupled Receptors (GPCRs) with the Yeast Surface Display (YSD) of specific enzymes enrolled in the viral recognition process. The GPCR/YSD complex might allow for active virus detection through a modulated signal activated by a GPCR agonist, whose concentration correlates to the viral titer. Additionally, we explore this methodology in a case study for the detection of highly pathogenic coronaviruses that share the same cell receptor upon infection (i.e. the Angiotensin-Converting Enzyme 2, ACE2), as a conceptual example of the potential of the GPCR/YSD strategy for the diagnosis of COVID-19.
... While for each transporter, mannose was always the best sugar, or among the best, in terms of final biomass yield at the end of the culture period (4 days), this yield was particularly low for several transporters (namely, spB4, spS2, and spS7) on all of the tested concentrations of the different sugars. This may be the result of improper expression of these proteins in yeast (as already reported for sugar transporters by, e.g., Dreyer et al. 1999;Wieczorke et al. 2003) or may suggest that in vivo, in their respective fungal species, these transporters may preferentially transport other sugars. ...
Sugar transporters are essential components of carbon metabolism and have been extensively studied to control sugar uptake by yeasts and filamentous fungi used in fermentation processes. Based on published information on characterized fungal sugar porters, we show that this protein family encompasses phylogenetically distinct clades. While several clades encompass transporters that seemingly specialized on specific “sugar-related” molecules (e.g., myo-inositol, charged sugar analogs), others include mostly either mono- or di/oligosaccharide low-specificity transporters. To address the issue of substrate specificity of sugar transporters, that protein primary sequences do not fully reveal, we screened “multi-species” soil eukaryotic cDNA libraries for mannose transporters, a sugar that had never been used to select transporters. We obtained 19 environmental transporters, mostly from Basidiomycota and Ascomycota. Among them, one belonged to the unusual “Fucose H⁺ Symporter” family, which is only known in Fungi for a rhamnose transporter in Aspergillus niger. Functional analysis of the 19 transporters by expression in yeast and for two of them in Xenopus laevis oocytes for electrophysiological measurements indicated that most of them showed a preference for d-mannose over other tested d-C6 (glucose, fructose, galactose) or d-C5 (xylose) sugars. For the several glucose and fructose-negative transporters, growth of the corresponding recombinant yeast strains was prevented on mannose in the presence of one of these sugars that may act by competition for the binding site. Our results highlight the potential of environmental genomics to figure out the functional diversity of key fungal protein families and that can be explored in a context of biotechnology.
Key points
• Most fungal sugar transporters accept several sugars as substrates.
• Transporters, belonging to 2 protein families, were isolated from soil cDNA libraries.
• Environmental transporters featured novel substrate specificities.
... Increased glucose transport in malignant cells has also been associated with increased expression of glucose transporters, with overexpression of GLUT1 and/or GLUT3. 45 The GLUT family is divided into three classes based on their sequence homology. They can be stimulated by Tumor-suppressor Figure 1 p16 is able to inhibit the action of cyclin D kinase which is responsible for retinoblastoma protein (pRb) phosphorylation leading to pRb inactivation and cell cycle progression stimulating the S phase. ...
Non-small cell lung cancer (NSCLC) is the leading cause of cancer death and in most cases it is often diagnosed at an advanced stage. Many genetic and microenvironmental factors are able to modify the cell cycle inducing carcinogenesis and tumor growth. Among the metabolic and genetic factors that come into play in carcinogenesis and tumor cell differentiation and growth there are two different proteins that should be considered which are glucose transporters (GLUTs) and p16INK4 The first are glucose transporters which are strongly involved in tumor metabolism, notably accelerating cancer cell metabolism both in aerobic and anaerobic conditions. There are different subtypes of GLUT family factors of which GLUT 1 is the most important and widely expressed. By contrast, p16 is mainly a tumor-suppressor protein that acts on cyclin-dependent kinase favoring cell cycle arrest in the G1 phase. Our search focused on the action of the aforementioned factors.
... Human GLUT4 transports D-glucose, D-galactose, 2ODG, and 3OMG but does not accept D-fructose as substrate (Table 1). For trans-zero uptake of D-glucose by human GLUT4, an apparent K m value of 12.6 mM was determined [442], whereas for trans-zero uptake of 2DOG, an apparent K m value of 4.6 mM has been reported [49]. Similar to GLUT1 and GLUT3, GLUT4 accepts dehydroascorbic acid as substrate [350]. ...
Energy demand of neurons in brain that is covered by glucose supply from the blood is ensured by glucose transporters in capillaries and brain cells. In brain, the facilitative diffusion glucose transporters GLUT1-6 and GLUT8, and the Na+-D-glucose cotransporters SGLT1 are expressed. The glucose transporters mediate uptake of D-glucose across the blood-brain barrier and delivery of D-glucose to astrocytes and neurons. They are critically involved in regulatory adaptations to varying energy demands in response to differing neuronal activities and glucose supply. In this review, a comprehensive overview about verified and proposed roles of cerebral glucose transporters during health and diseases is presented. Our current knowledge is mainly based on experiments performed in rodents. First, the functional properties of human glucose transporters expressed in brain and their cerebral locations are described. Thereafter, proposed physiological functions of GLUT1, GLUT2, GLUT3, GLUT4, and SGLT1 for energy supply to neurons, glucose sensing, central regulation of glucohomeostasis, and feeding behavior are compiled, and their roles in learning and memory formation are discussed. In addition, diseases are described in which functional changes of cerebral glucose transporters are relevant. These are GLUT1 deficiency syndrome (GLUT1-SD), diabetes mellitus, Alzheimer's disease (AD), stroke, and traumatic brain injury (TBI). GLUT1-SD is caused by defect mutations in GLUT1. Diabetes and AD are associated with changed expression of glucose transporters in brain, and transporter-related energy deficiency of neurons may contribute to pathogenesis of AD. Stroke and TBI are associated with changes of glucose transporter expression that influence clinical outcome.
... SLC2A9 transcripts were identified in human islets and GLUT9 proteins were allocated to human βcells by immunodetection [31,48]. Expression of GLUT4, an insulin-regulated low affinity glucose transporter (Km1 2 mmol/l) [161] was detected in human and rat islets on mRNA and protein level as well as in the murine β-cellderived β-TC cell line [12,13,64]. SLC2A10, encoding for GLUT10 was reported to be associated with Type 2 diabetes (T2D) and SLC2A10 transcripts were found in human islets [48,83]. ...
The fine-tuning of glucose uptake mechanisms is rendered by various glucose transporters with distinct transport characteristics. In the pancreatic islet, facilitative diffusion glucose transporters (GLUTs), and sodium-glucose cotransporters (SGLTs) contribute to glucose uptake and represent important components in the glucose-stimulated hormone release from endocrine cells, therefore playing a crucial role in blood glucose homeostasis. This review summarizes the current knowledge about cell type-specific expression profiles as well as proven and putative functions of distinct GLUT and SGLT family members in the human and rodent pancreatic islet and further discusses their possible involvement in onset and progression of diabetes mellitus. In context of GLUTs, we focus on GLUT2, characterizing the main glucose transporter in insulin-secreting β-cells in rodents. In addition, we discuss recent data proposing that other GLUT family members, namely GLUT1 and GLUT3, render this task in humans. Finally, we summarize latest information about SGLT1 and SGLT2 as representatives of the SGLT family that have been reported to be expressed predominantly in the α-cell population with a suggested functional role in the regulation of glucagon release.
... q Glc increases. The majority of glucose transporters (GLUTs) in mammalian cells show affinity to both glucose and galactose and, therefore, a regulation of the glucose and galactose flux from the GLUTs is possible(Wieczorke, Dlugai, Krampe, & Boles, 2003;Zhao & Keating, 2007). ...
Exerting control over the glycan moieties of antibody therapeutics is highly desirable from a product safety and batch‐to‐batch consistency perspective. Strategies to improve antibody productivity may compromise quality, while interventions for improving glycoform distribution can adversely affect cell growth and productivity. Process design therefore needs to consider the trade‐off between preserving cellular health and productivity while enhancing antibody quality. In this work, we present a modeling platform that quantifies the impact of glycosylation precursor feeding – specifically that of galactose and uridine – on cellular growth, metabolism as well as antibody productivity and glycoform distribution. The platform has been parameterized using an initial training data set yielding an accuracy of ±5% with respect to glycoform distribution. It was then used to design an optimized feeding strategy that enhances the final concentration of galactosylated antibody in the supernatant by over 90% compared with the control without compromising the integral of viable cell density or final antibody titer. This work supports the implementation of Quality by Design towards higher‐performing bioprocesses.
... This complexity has turned S. cerevisiae into an attractive and powerful model to study the mechanisms connecting extracellular signals to transcriptional responses in eukaryotes (Rolland, Winderickx and Thevelein 2001). While its redundancy in hexose transporter genes initially complicated the use of S. cerevisiae as a testbed for heterologous transporters, the construction of an S. cerevisiae hexose-transport deficient strain (named EBY.VW4000 or Hxt 0 ) (Wieczorke et al. 1999) provided a unique and intensively used platform for functional analysis of native hexose transporters and studies on hexose transport (see for instance Wieczorke et al. 2003;Schussler et al. 2006;Price et al. 2010;Young et al. 2011;Xuan et al. 2013;Boles and Oreb 2018) and of transport of other biotechnologically relevant sugars (Young, Lee and Alper 2010;Thomik et al. 2017). ...
Hexose transporter-deficient yeast strains are valuable testbeds for the study of sugar transport by native and heterologous transporters. In the popular Saccharomyces cerevisiae strain EBY.VW4000, deletion of 21 transporters completely abolished hexose transport. However, repeated use of the LoxP/Cre system in successive deletion rounds also resulted in major chromosomal rearrangements, gene loss and phenotypic changes. In the present study CRISPR/SpCas9 was used to delete the 21 hexose transporters in a S. cerevisiae strain from the CEN.PK family in only three deletion rounds, using 11 unique guide RNAs. Even upon prolonged cultivation, the resulting strain IMX1812 (CRISPR-Hxt0) was unable to consume glucose, while its growth rate on maltose was the same as that of a strain equipped with a full set of hexose transporters. Karyotyping and whole-genome sequencing of the CRISPR-Hxt0 strain with Illumina and Oxford Nanopore technologies did not reveal chromosomal rearrangements or other unintended mutations besides a few SNPs. This study provides a new, 'genetically unaltered' hexose transporter-deficient strain and supplies a CRISPR toolkit for removing all hexose transporter genes from most S. cerevisiae laboratory strains in only three transformation rounds.
... Instead, growth on glucose was only completely abolished after subsequent deletion of the remaining HXT3, HXT6 and HXT7 genes (Wieczorke et al. 1999). The resulting 'HXT null' strain has become an invaluable platform for functional analysis of native and heterologous hexose and pentose transporter genes (for examples see Buziol et al. 2002;Wieczorke et al. 2003;Subtil and Boles 2011; for a complete overview see Solis-Escalante et al. 2015). At the time of its construction, deletion of so many genes represented a herculean task. ...
CRISPR/Cas9-based genome editing allows rapid, simultaneous modification of multiple genetic loci in Saccharomyces cerevisiae. Here, this technique was used in a functional analysis study aimed at identifying the hitherto unknown mechanism of lactate export in this yeast. First, an S. cerevisiae strain was constructed with deletions in 25 genes encoding transport proteins, including the complete aqua(glycero)porin family and all known carboxylic-acid transporters. The 25-deletion strain was then transformed with an expression cassette for Lactobacillus casei lactate dehydrogenase (LcLDH). In anaerobic, glucose-grown batch cultures, this strain exhibited a lower specific growth rate (0.15 vs. 0.25 h-1) and biomass-specific lactate production rate (0.7 vs. 2.4 mmol (g biomass)-1 h-1) than an LcLDH-expressing reference strain. However, a comparison of the two strains in anaerobic glucose-limited chemostat cultures (dilution rate 0.10 h-1) showed identical lactate production rates. These results indicate that, although deletion of the 25 transporter genes affected the maximum specific growth rate, it did not impact lactate export rates when analysed at a fixed specific growth rate. The 25-deletion strain provides a first step towards a 'minimal transportome' yeast platform, which can be applied for functional analysis of specific (heterologous) transport proteins as well as for evaluation of metabolic engineering strategies.
... The K M for GLUT3 (11.9 ± 1.6 mM) and GLUT4 (33.3 ± 5.1 mM) reconstituted in liposomes was higher than reported when expressed in cells, which is consistent with previously published studies comparing transporter kinetics in cells versus reconstituted into liposomes (35)(36)(37)(38)(39). The K M (30.5 ± 2.1 mM) and k cat (19.5 ± 0.57 s -1 ) measured for GLUT4 in liposomes containing PC, PE, PI, PS and PA (68.3, 16.7, 1.6, 10.6, and 2.8 mole % respectively) were similar to those shown in Fig. 8. Measuring transporter kinetics of GLUT4 expressed in HEK293 cells prior to purification and reconstitution, we observed a lower K M of 7.1 ± 1.1 mM (Fig. 9), similar to previously published studies (34,40). These results indicate that additional cellular components, not present in the purified four-component liposome system, might play a role in altering the transporter's K M . ...
The regulated movement of glucose across mammalian cell membranes is mediated by facilitative glucose transporters (GLUTs)
embedded in lipid bilayers. Despite the known importance of phospholipids in regulating protein structure and activity, lipid-induced
effects on the GLUTs remain poorly understood. We systematically examined the effects of physiologically relevant phospholipids
on glucose transport in liposomes containing purified GLUT4 and GLUT3. The anionic phospholipids, phosphatidic acid, phosphatidylserine,
phosphatidylglycerol, and phosphatidylinositol were found to be essential for transporter function by activating it and stabilizing
its structure. Conical lipids,
phosphatidylethanolamine and diacylglycerol enhanced transporter activity up to threefold in the presence of anionic phospholipids
but did not stabilize protein structure. Kinetic analyses revealed that both lipids increase the kcat of transport without
changing the Km. These results allowed us to elucidate the activation of GLUT by plasma membrane phospholipids and extend
the field of membrane protein-lipid interactions to the family of structurally and functionally related human solute carriers.
... Since its construction, EBY.VW4000 has become an essential tool to elucidate the function and kinetic characteristics of individual S. cerevisiae hexose transporters (Wieczorke et al., 1999;Buziol et al., 2002). Moreover, it has served as a splendid screening platform for transporters from organisms ranging from fungi to plants and mammals (Wieczorke et al., 2003;Liu et al., 2006). Expression of putative membrane-proteins encoding genes in EBY.VW4000 included the characterization of large libraries of transporters (Price et al., 2010;Young et al., 2011;Coelho et al., 2013;Xuan et al., 2013) and the discovery of previously unknown hexose transporters (Schneidereit, Scholz-Starke and Buttner 2003;Vignault et al., 2005;Schussler et al., 2006;Stasyk et al., 2008;Dae-Hee, Soo-Jung and Jin-Ho 2014). ...
Saccharomyces cerevisiae harbours a large group of tightly controlled hexose transporters with different characteristics. Construction and characterization of S. cerevisiae EBY.VW4000, a strain devoid of glucose import, was a milestone in hexose-transporter research. This strain has become a widely used platform for discovery and characterization of transporters from a wide range of organisms. To abolish glucose uptake, 21 genes were knocked out, involving 16 successive deletion rounds with the LoxP/Cre system. Although such intensive modifications are known to increase the risk of genome alterations, the genome of EBY.VW4000 has hitherto not been characterized. Based on a combination of whole genome sequencing, karyotyping and molecular confirmation, the present study reveals that construction of EBY.VW4000 resulted in gene losses and chromosomal rearrangements. Recombinations between the LoxP scars have led to the assembly of four neo-chromosomes, truncation of two chromosomes and loss of two subtelomeric regions. Furthermore, sporulation and spore germination are severely impaired in EBY.VW4000. Karyotyping of the EBY.VW4000 lineage retraced its current chromosomal architecture to four translocations events occurred between the 6th and the 12th rounds of deletion. The presented data facilitate further studies on EBY.VW4000 and highlight the risks of genome alterations associated with repeated use of the LoxP/Cre system.
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... könnte es UPR auslösen, auf diesem Weg in die Vakuole gelangen und so ein PEP4-abhängiges Expressionsniveau zeigen.Wege der Qualitätskontrolle des Golgi-Apparates und der Plasmamembran sowie der Qualitätskontrolle des ER (Autophagie) konvergieren somit in der Vakuole. Aus der PEP4-Abhängigkeit der Steady-State-Expression von GlpT kann daher nicht darauf geschlossen werden, welche Qualitätskontrollinstanzen wirksam werden.4.2.8 Weitere mögliche Strategien für die Verbesserung der Expression undLokalisierung von GlpT in S. cerevisiaeEinzelne Mutationen haben sich für die heterologe Expression eines Transmembranproteins in Hefe als förderlich erwiesen, beispielsweise die Deletion von CNE1) oder fgy1-1 im uncharakterisierten Gen YMR212(Wieczorke et al. 2003). Der Erfolg war jedoch proteinspezifisch, da die Mutationen die Expression eines anderen Transmembranproteins nicht beeinflussten.Auch der Glykosylierung eines Proteins kommt Bedeutung für seine Passage durch den sekretorischen Weg zu. ...
... The failure of the cells, also of those transformed with SUT1, to grow on sucrose, ultimately indicated that the transporters were not functionally expressed in both EBY yeast strains. A similar problem had also been described for the mammalian sugar transporters GLUT1 and GLUT4 which only became functionally expressed at the plasma membrane of EBY-VW4000 cells after additional spontaneous mutations were introduced [40]. Unfortunately, mutagenesis was not successful in our case (results not shown). ...
... 이러한 [6]. 이러한 연유로 당 수송체들을 E. coli [17,22], Xenopus oocytes [5,9], mammalian cells [1], transgenic mice [14], yeast [8,24] T. ni cells were infected with 50 μl of 10-fold dilutions of virus prepared from the eight individual plaques, ranging from 10 -1 to 10 -8 . The assay was performed as described in Summers and Smith [20]. ...
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.
... A strategy to overcome this problem has focused on the isolation of heterologous sugar transporters with better xylose-transporting properties for functional expression in xylose fermenting S. cerevisiae cells [22,[34][35][36][37][38][39][40][41]. However, up to now very few heterologous xylose transporters have been characterized in S. cerevisiae, probably due to structural or functional barriers for the expression of heterologous membrane permeases in yeasts [34,42,43]. For example, a recent survey with over 23 heterologous known and putative sugar transporters from seven different organisms revealed only five permeases that allowed utilization of xylose by S. cerevisiae cells [44]. ...
... Numbers at nodes are bootstrap values based on 1,000 samplings antibody (Fig. 2a). Incorrect localization of plasma membrane proteins overproduced in a heterologous context in the host cell has often been described (Wieczorke et al. 2003). In order to test this possibility, GFP was used as a reporter for the subcellular localization of heterologously expressed CmFSY1 and CmFFZ1 genes. ...
Sugar transport is very critical in developing an efficient and rapid conversion process of a mixture of sugars by engineered microorganisms. By using expressed sequence tag data generated for the fructophilic yeast Candida magnoliae JH110, we identified two fructose-specific transporters, CmFSY1 and CmFFZ1, which show high homology with known fructose transporters of other yeasts. The CmFSY1 and CmFFZ1 genes harbor no introns and encode proteins of 574 and 582 amino acids, respectively. Heterologous expression of the two fructose-specific transporter genes in a Saccharomyces cerevisiae, which is unable to utilize hexoses, revealed that both transporters are functionally expressed and specifically transport fructose. These results were further corroborated by kinetic analysis of the fructose transport that showed that CmFsy1p is a high-affinity fructose–proton symporter with low capacity (K
M = 0.13 ± 0.01 mM, V
max = 2.1 ± 0.3 mmol h−1 [gdw]−1) and that CmFfz1p is a low-affinity fructose-specific facilitator with high capacity (K
M = 105 ± 12 mM, V
max = 8.6 ± 0.7 mmol h−1 [gdw]−1). These fructose-specific transporters can be used for improving fructose transport in engineered microorganisms for the production of biofuels and chemicals from fructose-containing feedstock.
In adipose tissue, insulin stimulates glucose uptake by mediating the translocation of GLUT4 from intracellular vesicles to the plasma membrane. In 2010, insulin was revealed to also have a fundamental impact on the spatial distribution of GLUT4 within the plasma membrane, with the existence of two GLUT4 populations at the plasma membrane being defined: 1) as stationary clusters and 2) as diffusible monomers. In this model, in the absence of insulin, plasma membrane-fused GLUT4 are found to behave as clusters. These clusters are thought to arise from an exocytic event that retains GLUT4 at the fusion site; this has been proposed to function as an intermediate hub between GLUT4 exocytosis and re-internalisation. By contrast, insulin stimulation induces the dispersal of GLUT4 clusters into monomers and favors a distinct type of GLUT4-vesicle fusion event, known as fusion-with-release exocytosis. Here, we review how super-resolution microscopy approaches have allowed investigation of the characteristics of plasma membrane-fused GLUT4 and further discuss regulatory step(s) involved in the GLUT4 dispersal machinery, introducing the scaffold protein EFR3 which facilitates localisation of phosphatidylinositol 4-kinase type IIIα (PI4KIIIα) to the cell surface. We consider how dispersal may be linked to the control of transporter activity, consider whether macro-organisation may be a widely used phenomenon to control proteins within the plasma membrane, and speculate on the origin of different forms of GLUT4-vesicle exocytosis.
Glucose is an essential energy source for cells. In humans, its passive diffusion through the cell membrane is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT2 transports both glucose and fructose with low affinity and plays a critical role in glucose sensing mechanisms. Alterations in the function or expression of GLUT2 are involved in the Fanconi-Bickel syndrome, diabetes, and cancer. Distinguishing GLUT2 transport in tissues where other GLUTs coexist is challenging due to the low affinity of GLUT2 for glucose and fructose and the scarcity of GLUT-specific modulators. By combining in silico ligand screening of an inward-facing conformation model of GLUT2 and glucose uptake assays in a hexose transporter-deficient yeast strain, in which the GLUT1-5 can be expressed individually, we identified eleven new GLUT2 inhibitors (IC 50 ranging from 0.61 to 19.3 µM). Among them, nine were GLUT2-selective, one inhibited GLUT1-4 (pan-Class I GLUT inhibitor), and another inhibited GLUT5 only. All these inhibitors dock to the substrate cavity periphery, close to the large cytosolic loop connecting the two transporter halves, outside the substrate-binding site. The GLUT2 inhibitors described here have various applications; GLUT2-specific inhibitors can serve as tools to examine the pathophysiological role of GLUT2 relative to other GLUTs, the pan-Class I GLUT inhibitor can block glucose entry in cancer cells, and the GLUT2/GLUT5 inhibitor can reduce the intestinal absorption of fructose to combat the harmful effects of a high-fructose diet.
Microalgae are in the focus of research and industry because of their potential as high-value substance producers. Especially diatoms and within this group Phaeodactylum tricornutum are of high interest as biofactory, for e.g. synthesizing high amounts of fucoxanthin or as important source for ω-3 fatty acids. As model organism P. tricornutum can be used for genetic engineering. Here we showed the potential of P. tricornutum as efficient biofactory under mixotrophic conditions, combining the advantages of hetero- and phototrophic growth. We expressed the hexose uptake protein (HUP1) from the Chlorophyte Chlorella kessleri under the control of the light-inducible promoter fcpA or the nitrate reductase promoter. Only in case of fcpA significant overexpression could be demonstrated. With an in situ localization study, we demonstrated that the N-terminal sequence of the heterologous HUP1 protein is sufficient for insertion in the ER, whereas only the full-length protein is correctly targeted to the plasma membrane. The heterologous expression of HUP1 enables the cells to take up glucose, leading to increased biomass, especially when grown under low light intensities. The results demonstrate the capability of P. tricornutum to grow more efficiently using glucose and shed light on the targeting pathway of plasma membrane transporters.
Insulin-regulated trafficking of the facilitative glucose transporter GLUT4 has been studied in many cell types. The translocation of GLUT4 from intracellular membranes to the cell surface is often described as a highly specialised form of membrane traffic restricted to certain cell types such as fat and muscle, which are the major storage depots for insulin-stimulated glucose uptake. Here, we discuss evidence that favours the argument that rather than being restricted to specialised cell types, the machinery through which insulin regulates GLUT4 traffic is present in all cell types. This is an important point as it provides confidence in the use of experimentally tractable model systems to interrogate the trafficking itinerary of GLUT4.
Transporters play an important role in the absorption, distribution, metabolism, and excretion (ADME) of drugs. In recent years, various in vitro, in situ/ex vivo, and in vivo methods have been established for studying transporter function and drug-transporter interaction. In this chapter, the major types of in vitro models for drug transport studies comprise membrane-based assays, cell-based assays (such as primary cell cultures, immortalized cell lines), and transporter-transfected cell lines with single transporters or multiple transporters. In situ/ex vivo models comprise isolated and perfused organs or tissues. In vivo models comprise transporter gene knockout models, natural mutant animal models, and humanized animal models. This chapter would be focused on the methods for the study of drug transporters in vitro, in situ/ex vivo, and in vivo. The applications, advantages, or limitations of each model and emerging technologies are also mentioned in this chapter.
The HpGcr1, a hexose transporter homologue from the methylotrophic yeast Hansenula (Ogataea) polymorpha, was previously identified as being involved in glucose repression. Intriguingly, potential HpGcr1 orthologues are found only in the genomes of a few yeasts phylogenetically closely related to H. polymorpha, but are absent in all other yeasts. The other closest HpGcr1 homologues are fungal high-affinity glucose symporters or putative transceptors suggesting a possible HpGcr1 origin due to a specific archaic gene retention or via horizontal gene transfer from Eurotiales fungi. Herein we report that, similarly to other yeast non-transporting glucose sensors, the substitution of the conserved arginine residue converts HpGcr1R165K into a constitutively signaling form. Synthesis of HpGcr1R165K in gcr1Δ did not restore glucose transport or repression but instead profoundly impaired growth independent of carbon source used. Simultaneously, gcr1Δ was impaired in transcriptional induction of repressible peroxisomal alcohol oxidase and in growth on methanol. Overexpression of the functional transporter HpHxt1 in gcr1Δ partially restored growth on glucose and glucose repression but did not rescue impaired growth on methanol. Heterologous expression of HpGcr1 in a Saccharomyces cerevisiae hxt-null strain did not restore glucose uptake due to protein mislocalization. However, HpGcr1 overexpression in H. polymorpha led to increased sensitivity to extracellular 2-deoxyglucose, suggesting HpGcr1 is a functional glucose carrier. The combined data suggest that HpGcr1 represents a novel type of yeast glucose transceptor functioning also in the absence of glucose.
The biotinylated photolabeling assay enables quantification of cell-surface glucose transporters (GLUTs). This technique has been successfully applied to quantify the cell-surface GLUT protein content in striated muscles and adipose tissue, as a means to evaluate GLUT trafficking. Here, we describe the detailed method of quantifying the cell-surface content of several GLUT isoforms (1, 4, 8, and 12) in isolated cardiac myocytes, as well as in the intact perfused atria and ventricle.
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 is metabolized through anaerobic glycolysis and aerobic oxidative phosphorylation (OXPHOS). Perturbing glucose uptake and its subsequent metabolism can alter both glycolytic and OXPHOS pathways and consequently lactate and/or oxygen consumption. Production and secretion of lactate, as a consequence of glycolysis, leads to acidification of the extracellular medium. Molecular oxygen is the final electron acceptor in the electron transport chain, facilitating oxidative phosphorylation of ADP to ATP. The alterations in extracellular acidification and/or oxygen consumption can thus be used as indirect readouts of glucose metabolism and assessing the impact of inhibiting glucose transport through specific glucose transporters (GLUTs). The Seahorse bioenergetics analyzer can measure both the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). The proposed methodology affords a robust, high-throughput method to screen for GLUT inhibition in cells engineered to express specific GLUTs, providing live cell read-outs upon GLUT inhibition.
Efficient substrate utilization is the first and most important prerequisite for economically viable production of biofuels and chemicals by microbial cell factories. However, production rates and yields are often compromised by low transport rates of substrates across biological membranes and their diversion to competing pathways. This is especially true when common chassis organisms are engineered to utilize nonphysiological feedstocks. Here, we addressed this problem by constructing an artificial complex between an endogenous sugar transporter and a heterologous xylose isomerase in Saccharomyces cerevisiae. Direct feeding of the enzyme through the transporter resulted in acceleration of xylose consumption and substantially diminished production of xylitol as an undesired side product, with a concomitant increase in the production of ethanol. This underlying principle could also likely be implemented in other biotechnological applications.
Dueling for sugars
Bacteria thrive on sugar. So do plant cells. Yamada et al. now show how the fight for sugar plays out in the extracellular spaces around plant cells when pathogenic bacteria are invading the plant (see the Perspective by Dodds and Lagudah). In the model plant Arabidopsis , part of the defense response incited by the presence of pathogenic bacteria includes transcriptional and posttranscriptional regulation of sugar transporters. The resulting uptake of monosaccharides from the extracellular space makes life a little bit more difficult for the invading bacteria.
Science , this issue p. 1427 ; see also p. 1377
The biochemical study of sugar uptake in yeasts started five decades ago and led to the early production of abundant kinetic and mechanistic data. However, the first accurate overview of the underlying sugar transporter genes was obtained relatively late, due mainly to the genetic complexity of hexose uptake in the model yeast, Saccharomyces cerevisiae. The genomic era generated in turn a massive amount of information, allowing the identification of a multitude of putative sugar transporter and sensor-encoding genes in yeast genomes, many of which are phylogenetically related. This review aims to briefly summarize our current knowledges on the biochemical and molecular features of the transporters of pentoses in yeasts, when possible establishing links between previous kinetic studies and genomic data currently available. Emphasis is given to recent developments concerning the identification of D-xylose transporter genes, which are thought to be key players in the optimization of S. cerevisiae for bioethanol production from lignocellulose hydrolysates.
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.
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.
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We are investigating the transport and turnover of the multispanning membrane protein Ste6. The Ste6 protein is a member of the ABC-transporter family and is required for the secretion of the yeast mating pheromone a-factor. In contrast to the prevailing view that Ste6 is a plasma membrane protein, we found that Ste6 is mainly associated with internal membranes and not with the cell surface. Fractionation and immunofluorescence data are compatible with a Golgi localization of Ste6. Despite its mostly intracellular localization, the Ste6 protein is in contact with the cell surface, as demonstrated by the finding that Ste6 accumulates in the plasma membrane in endocytosis mutants. The Ste6 protein which accumulates in the plasma membrane in endocytosis mutants is ubiquitinated. Ste6 is thus the second protein in yeast besides MAT alpha 2 for which ubiquitination has been demonstrated. Ste6 is a very unstable protein (half-life 13 min) which is stabilized approximately 3-fold in a ubc4 ubc5 mutant, implicating the ubiquitin system in the degradation of Ste6. The strongest stabilizing effect on Ste6 is, however, observed in the vacuolar pep4 mutant (half-life > 2 h), suggesting that most of Ste6 is degraded in the vacuole. Secretory functions are required for efficient degradation of Ste6, indicating that Ste6 enters the secretory pathway and is transported to the vacuole by vesicular carriers.
AB~T~S affects ~,5% of people in the United States of America and the incidence is much higher in sporadic populations such as Pima Indians in North America and Australian Aborigines. There are two major forms of diabetes, referred to as insulin-dependent diabetes (IDD) ~ and non-insulin-dependent diabetes (NIDD). The etiology of these diseases is quite different despite similari- ties in their pathophysiologies. IDD often arises in early life and is due to autoimmune destruction of pancreatic/3 cells resulting in partial or complete loss of/3 cell function. NIDD is more common in later life (>40 yr) and ,x,85 % of all dia- betics have this form. The increased prevalence of NIDD in developed countries together with its association with heart disease and stroke makes this one of the most devastating dis- eases in the western world. NIDD appears to be due to a defect in glucose disposal (DeFronzo et al., 1992). Both glucose per se and insulin, play important roles in facilitating glucose uptake into target organs (for review see DeFronzo et ai., 1992). Following in- gestion of a carbohydrate meal, glucose uptake into many or- gans is increased simply due to the elevated blood glucose. A more pronounced increase which is evoked by insulin, is observed specifically in muscle and adipose tissue. The net result is that in the postprandial state, glucose is rapidly and efficiently cleared from the blood and stored primarily in muscle and adipose. Each of these processes is defective in NIDD making it extremely difficult to identify the primary lesion. Studies in first degree relatives of NIDDs indicate that one of the earliest detectable defects is impaired insulin action in muscle and adipose tissue or insulin resistance (Martin et al., 1992). Thus, identifying the physiological and molecular biological processes that control insulin-stimu- lated glucose utilization in these organs is of major signifi- cance.
Turnover numbers for 3-O-methylglucose transport by the homologous glucose transporters GLUT1 and GLUT4 were compared to those for truncated and chimeric
transporters expressed in Xenopus oocytes to assess potential regulatory properties of the C-terminal domain. The ability of high intracellular sugar concentrations
to increase the turnover number for sugar entry (“accelerated exchange”) by GLUT1 and not by GLUT4 was maintained in oocytes.
Replacing the GLUT1 C terminus with that of GLUT4 stimulated turnover 1.6-fold, but abolished accelerated exchange. Thus,
the GLUT1 C terminus permits accelerated exchange by GLUT1, but in doing so must interact with other GLUT1 specific sequences
since the GLUT4ctrm1 chimera did not exhibit this kinetic property. Removal of 38 C-terminal amino acids from GLUT4 reduced
its turnover number by 40%, whereas removing only 20 residues or replacing its C terminus with that of GLUT1 increased its
turnover number 3.5-3.9 fold. Therefore, using mechanisms independent of those which alter transporter targeting to the plasma
membrane, C-terminal mutations in either GLUT1 or GLUT4 can activate transport normally restricted by the native C-terminal
domain. These results implicate the C termini as targets of physiological factors, which through covalent modification or
direct binding might alter C-terminal interactions to regulate intrinsic GLUT1 and GLUT4 transporter activity.
Transcription factor AP-1 transduces environmental signals to the transcriptional machinery. To ensure a quick response yet maintain tight control over AP-1 target genes, AP-1 activity is likely to be negatively regulated in nonstimulated cells. To identify proteins that interact with the Jun subunits of AP-1 and repress its activity, we developed a novel screen for detecting protein-protein interactions that is not based on a transcriptional readout. In this system, the mammalian guanyl nucleotide exchange factor (GEF) Sos is recruited to the Saccharomyces cerevisiae plasma membrane harboring a temperature-sensitive Ras GEF, Cdc25-2, allowing growth at the nonpermissive temperature. Using the Sos recruitment system, we identified new c-Jun-interacting proteins. One of these, JDP2, heterodimerizes with c-Jun in nonstimulated cells and represses AP-1-mediated activation.
The GLUT4 system in muscle and fat cells plays an important role in whole-body glucose homeostasis. Insulin stimulates the translocation of GLUT4 from an intracellular storage compartment to the cell surface. The nature of this compartment remains largely unknown. We review recent studies describing the biogenesis and molecular constituents of the GLUT4 storage compartment and conclude that it is segregated from the endosomal and biosynthetic pathways. Further, we present evidence to suggest that the GLUT4 storage compartment moves directly to the plasma membrane in response to insulin and, hence, is analogous to small synaptic vesicles in neurons. We propose that the GLUT4 storage compartment be referred to as GLUT4 storage vesicles or GSVs.
Fanconi-Bickel syndrome (FBS) is a rare autosomal recessive disorder of carbohydrate metabolism recently demonstrated to be caused by mutations in Glut2, the gene for the glucose transporter protein 2 expressed in liver, pancreas, intestine and kidney. The disease was first described in a 3-year-old Swiss boy in 1949. Here we report a follow up of this original patient over more than 50 years and show that the typical clinical and laboratory findings of FBS (hepatomegaly secondary to glycogen accumulation, glucose and galactose intolerance, fasting hypoglycaemia, a characteristic proximal tubular nephropathy and severe short stature) persist into adulthood. We further summarize the historical observations that eventually led to the identification of the basic defect of FBS and give an overview of the 82 cases from 70 families in the published literature and from personal communications.
Conclusion Although with the first description of a congenital defect of facilitative glucose transport the main steps in the pathophysiology of Fanconi-Bickel syndrome have been elucidated, numerous pathophysiological mechanisms are far from clear and thus encourage the ongoing study of patients with this disorder.
In 1998 we updated earlier descriptions of the largest family of secondary transport carriers found in living organisms, the major facilitator superfamily (MFS). Seventeen families of transport proteins were shown to comprise this superfamily. We here report expansion of the MFS to include 29 established families as well as five probable families. Structural, functional, and mechanistic features of the constituent permeases are described, and each newly identified family is shown to exhibit specificity for a single class of substrates. Phylogenetic analyses define the evolutionary relationships of the members of each family to each other, and multiple alignments allow definition of family-specific signature sequences as well as all well-conserved sequence motifs. The work described serves to update previous publications and allows extrapolation of structural, functional and mechanistic information obtained with any one member of the superfamily to other members with limitations determined by the degrees of sequence divergence.
The human facilitative transporter Glut1 is the major glucose transporter present in all human cells, has a central role in metabolism, and is an archetype of the superfamily of major protein facilitators. Here we describe a three-dimensional structure of Glut1 based on helical packing schemes proposed for lactose permease and Glut1 and predictions of secondary structure, and refined using energy minimization, molecular dynamics simulations, and quality and environmental scores. The Ramachandran scores and the stereochemical quality of the structure obtained were as good as those for the known structures of the KcsA K(+) channel and aquaporin 1. We found two channels in Glut1. One of them traverses the structure completely, and is lined by many residues known to be solvent-accessible. Since it is delimited by the QLS motif and by several well conserved residues, it may serve as the substrate transport pathway. To validate our structure, we determined the distance between these channels and all the residues for which mutations are known. From the locations of sugar transporter signatures, motifs, and residues important to the transport function, we find that this Glut1 structure is consistent with mutagenesis and biochemical studies. It also accounts for functional deficits in seven pathogenic mutants.
In muscle and fat cells, insulin stimulates the delivery of the glucose transporter GLUT4 from an intracellular location to the cell surface, where it facilitates the reduction of plasma glucose levels. Understanding the molecular mechanisms that mediate this translocation event involves integrating our knowledge of two fundamental processes--the signal transduction pathways that are triggered when insulin binds to its receptor and the membrane transport events that need to be modified to divert GLUT4 from intracellular storage to an active plasma membrane shuttle service.
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-K
m isoform expressed in hepatocytes, pancreatic β 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-K
m 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.
The trafficking of the insulin-sensitive glucose transporter, GLUT4, is the paradigm of how cells control the movement of membrane proteins through intricate pathways of transport in response to external stimuli, and how, by doing so, regulate their function. The GLUT4 intracellularly sequestered in resting adipocytes and muscle cells becomes exposed on their surface in response to an increase in insulin levels and muscle contraction, where it facilitates glucose uptake. Ceasing of the stimuli is followed by endocytosis of the GLUT4 molecules exposed on the plasma membrane and their recycling to the original stores, where they are retained. This review discusses current understanding of the organelles that host GLUT4 and the motifs that mediate its trafficking.
The structure of the human erythrocyte facilitative glucose transporter (GLUT1) has been intensively investigated using a wide array of chemical and biophysical approaches. Despite the lack of a crystal structure for any of the facilitative monosaccharide transport proteins, detailed information regarding primary and secondary structure, membrane topology, transport kinetics, and functionally important residues has allowed the construction of a sophisticated working model for GLUT1 tertiary structure. The existing data support the formation of a central aqueous channel formed by the juxtaposition of several amphipathic transmembrane-spanning alpha -helices. The results of extensive mutational analysis of GLUT1 have elucidated many of the structural determinants of the glucose permeation pathway. Continued application of currently available technologies will allow further refinement of this working model. In addition to providing insights into the molecular basis of both normal and disordered glucose homeostasis, this detailed understanding of structure/function relationships within GLUT1 can provide a basis for understanding transport carried out by other members of the major facilitator superfamily.
The GLUT4 facilitative glucose transporter protein is primarily expressed in muscle and adipose tissue and accounts for the majority of post-prandial glucose uptake. In the basal or nonstimulated state, GLUT4 is localized to intracellular membrane compartments sequestered away from circulating glucose. However, in response to agonist stimulation, there is a marked redistribution of the GLUT4 protein to the cell surface membrane providing a transport route for the uptake of glucose. This GLUT4 translocation can be divided into four general steps: (i) GLUT4 vesicle trafficking out of the storage pool, (ii) docking just below the cell surface, (iii) priming via the interactions of the SNARE proteins present on the vesicular and plasma membranes, and (iv) fusion of the GLUT4 vesicle with the plasma membrane. This review focuses on recent advances made in identification and characterization of the molecular events and protein interactions involved in these steps of insulin-stimulated GLUT4 translocation.
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.
The major facilitator superfamily (MFS) is one of the two largest families of membrane transporters found on Earth. It is present ubiquitously in bacteria, archaea, and eukarya and includes members that can function by solute uniport, solute/cation symport, solute/cation antiport and/or solute/solute antiport with inwardly and/or outwardly directed polarity. All homologous MFS protein sequences in the public databases as of January 1997 were identified on the basis of sequence similarity and shown to be homologous. Phylogenetic analyses revealed the occurrence of 17 distinct families within the MFS, each of which generally transports a single class of compounds. Compounds transported by MFS permeases include simple sugars, oligosaccharides, inositols, drugs, amino acids, nucleosides, organophosphate esters, Krebs cycle metabolites, and a large variety of organic and inorganic anions and cations. Protein members of some MFS families are found exclusively in bacteria or in eukaryotes, but others are found in bacteria, archaea, and eukaryotes. All permeases of the MFS possess either 12 or 14 putative or established transmembrane alpha-helical spanners, and evidence is presented substantiating the proposal that an internal tandem gene duplication event gave rise to a primordial MFS protein prior to divergence of the family members. All 17 families are shown to exhibit the common feature of a well-conserved motif present between transmembrane spanners 2 and 3. The analyses reported serve to characterize one of the largest and most diverse families of transport proteins found in living organisms.
A major mechanism by which insulin stimulates glucose transport in muscle and fat is the translocation of glucose transporters from an intracellular membrane pool to the cell surface. The existence of a distinct insulin-regulatable glucose transporter was suggested by the poor cross-reactivity between antibodies specific for either the HepG2 or rat brain glucose transporters and the rat adipocyte glucose transporter. More direct evidence was provided by the production of a monoclonal antibody (mAb 1F8) specific for the rat adipocyte glucose transporter that immunolabels a species of relative molecular mass 43,000 (43K) present only in tissues that exhibit insulin-dependent glucose transport, suggesting that this protein may be encoded by a different gene from the previously described mammalian glucose transporters. This antibody has been used to immunoprecipitate a 43K protein that was photoaffinity-labelled with cytochalasin B in a glucose displaceable way, and to immunolabel a protein in the plasma membrane of rat adipocytes, whose concentration was increased at least fivefold after cellular insulin exposure. Here we describe the cloning and sequencing of cDNAs isolated from both rat adipocyte and heart libraries that encode a protein recognized by mAb 1F8, and which has 65% sequence identity to the human HepG2 glucose transporter. This cDNA hybridizes to an mRNA present only in skeletal muscle, heart and adipose tissue. Our data indicate that this cDNA encodes a membrane protein with the characteristics of the translocatable glucose transporter expressed in insulin-responsive tissues.
Insulin, rapidly and independently of new protein synthesis, stimulates glucose transport in sensitive target tissues. A cDNA has been cloned from a skeletal muscle library that encodes a novel glucose transporter protein exhibiting the following properties of an insulin-regulated hexose carrier protein: it is expressed exclusively in adipose tissue, skeletal muscle and heart, the principal organs with insulin-responsive glucose transport; RNA transcribed from the muscle cDNA, when expressed in Xenopus oocytes, encodes a protein capable of cytochalasin B inhibitable 2-deoxyglucose transport; and treatment of isolated rat adipocytes with insulin effects a redistribution of "muscle" transports from low density microsomes to the plasma membrane to an extent comparable to the activation of glucose transport.
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.
A quenched-flow apparatus is described and applied to measurements of the hydrolysis of 2,4-dinitrophenyl acetate by sodium hydroxide and the entry of D-[U-14C]glucose into human red blood cells at 37 degrees C. Glucose influx into red cells was a saturable process obeying Michaelis-Menten kinetics with a Km for glucose of 6.6 +/- 0.61 mM and a maximum rate for glucose entry under "zero trans" conditions of 20.7 +/- 0.76 mmol (L cell water)-1 s-1. The technique used requires only readily available laboratory equipment and should be easily adaptable to the study of other rapid transport processes.
Activation of growth factor receptors results in tyrosine autophosphorylation and recruitment of SH2 domain-containing effectors, including Grb2. Grb2 recruitment mediates activation of the Ras nucleotide exchanger Sos by an unknown mechanism. To examine the role of membrane recruitment, we prepared Sos derivatives containing either myristoylation or farnesylation signals. This resulted in plasma membrane targeting of Sos and stimulation of the Ras signaling pathway, including ERK and AP-1 activities leading to oncogenic transformation. Sos derivatives with nonfunctional myristoylation or farnesylation sequences were inactive. Farnesylation of Sos also activated Ras signaling in yeast. In both mammalian cells and yeast, membrane-targeted Sos derivatives lacking the C-terminal region were considerably more active. Therefore, targeting of Sos to the plasma membrane in the vicinity of Ras appears to be the primary mechanism leading to activation of the Ras pathway. A secondary mechanism could involve relief of the inhibitory effect of the Sos C-terminal region.
The rate-limiting step in the uptake and metabolism of D-glucose by insulin target cells is thought to be glucose transport mediated by glucose transporters (primarily the GLUT4 isoform) localized to the plasma membrane. However, subcellular fractionation, photolabelling and immunocytochemical studies have shown that the pool of GLUT4 present in the plasma membrane is only one of many subcellular pools of this protein. GLUT4 has been found in occluded vesicles at the plasma membrane, clathrin-coated pits and vesicles, early endosomes, and tubulo-vesicular structures; the latter are analogous to known specialized secretory compartments. Tracking the movement of GLUT4 through these compartments, and defining the mechanism and site of action of insulin in stimulating this subcellular trafficking, are major topics of current investigation. Recent evidence focuses attention on the exocytosis of GLUT4 as the major site of insulin action. Increased exocytosis may be due to decreased retention of glucose transporters in an intracellular pool, or possibly to increased assembly of a vesicle docking and fusion complex. Although details are unknown, the presence in GLUT4 vesicles of a synaptobrevin homologue leads us to propose that a process analogous to that occurring in synaptic vesicle trafficking is involved in the assembly of GLUT4 vesicles into a form suitable for fusion with the plasma membrane. Evidence that the pathways of signalling from the insulin receptor and of GLUT4 vesicle exocytosis may converge at the level of the key signalling enzyme, phosphatidylinositol 3-kinase, is discussed.
By using a modified technique to measure glucose uptake in Saccharomyces cerevisiae, potential uncertainties have been identified in previous determinations. These previous determinations had led to the proposal that S. cerevisiae contained a constitutive low-affinity glucose transporter and a glucose-repressible high-affinity transporter. We show that, upon transition from glucose-repressed to -derepressed conditions, the maximum rate of glucose transport is constant and only the affinity for glucose changes. We conclude that the transporter or group of transporters is constitutive and that regulation of glucose transport occurs via a factor that modifies the affinity of the transporters and not via the synthesis of different kinetically independent transporters. Such a mechanism could, for instance, be accommodated by the binding of kinases causing a change in affinity for glucose.
Growth and carbon metabolism in triosephosphate isomerase (delta tpi1) mutants of Saccharomyces cerevisiae are severely inhibited by glucose. By using this feature, we selected for secondary site revertants on glucose. We defined five complementation groups, some of which have previously been identified as glucose repression mutants. The predominant mutant type, HTR1 (hexose transport regulation), is dominant and causes various glucose-specific metabolic and regulatory defects in TPI1 wild-type cells. HTR1 mutants are deficient in high-affinity glucose uptake and have reduced low-affinity transport. Transcription of various known glucose transporter genes (HXT1, HXT3, and HXT4) was defective in HTR1 mutants, leading us to suggest that HTR mutations affect a negative factor of HXT gene expression. By contrast, transcript levels for SNF3, which encodes a component of high-affinity glucose uptake, were unaffected. We presume that HTR1 mutations affect a negative factor of HXT gene expression. Multicopy expression of HXT genes or parts of their regulatory sequences suppresses the metabolic defects of HTR1 mutants but not their derepressed phenotype at high glucose concentrations. This suggests that the glucose repression defect is not a direct result of the metabolically relevant defect in glucose transport. Alternatively, some unidentified regulatory components of the glucose transport system may be involved in the generation or transmission of signals for glucose repression.
Two glycolytic enzymes, phosphoglucose isomerase and fructose-1,6-bisphosphate aldolase, of Saccharomyces cerevisiae could be replaced by their heterologous counterparts from Escherichia coli and Drosophila melanogaster. Both heterologous enzymes, which show respectively little and no sequence homology to the corresponding yeast enzymes, fully restored wild-type properties when their genes were expressed in yeast deletion mutants. This result does not support notions of an obligatory formation of glycolytic multi-enzyme aggregates in yeast; nor does it support possible regulatory functions of yeast phosphoglucose isomerase.
We present a rapid, cheap and highly efficient method for site-directed mutagenesis using the polymerase chain reaction (PCR). This method is applicable to every DNA fragment which has to be cloned into the multiple cloning site of any vector, or vector pair, in two different orientations. It requires only two primers, one new and specific mutagenic primer and one of the usual sequencing primers. In the first PCR, a mutagenic DNA fragment is synthesized which is amplified exponentially in the second PCR. In contrast, wild-type sequences are only linearly amplified resulting in an efficiency of mutagenesis of nearly 100%.
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.
Saccharomyces cerevisiae accomplishes high rates of hexose transport. The kinetics of hexose transport are complex. The capacity and kinetic complexity of hexose transport in yeast are reflected in the large number of sugar transporter genes in the genome. Twenty hexose transporter genes exist in S. cerevisiae. Some of these have been found by genetic means; many have been discovered by the comprehensive sequencing of the yeast genome. This review codifies the nomenclature of the hexose transporter genes and describes the sequence homology and structural similarity of the proteins they encode. Information about the expression and function of the transporters is presented. Access to the sequences of the genes and proteins at three sequence databases is provided via the World Wide Web.
Five functional mammalian facilitated hexose carriers (GLUTs) have been characterized by molecular cloning. By functional expression in heterologous systems, their specificity and affinity for different hexoses have been defined. There are three high-affinity transporters (GLUT-1, GLUT-3 and GLUT-4) and one low-affinity transporter (GLUT-2), and GLUT-5 is primarily a fructose carrier. Because their Michaelis constants (Km) are below the normal blood glucose concentration, the high-affinity transporters function at rates close to maximal velocity. Thus their level of cell surface expression greatly influences the rate of glucose uptake into the cells. In contrast, the rate of glucose uptake by GLUT-2 (Km = 17 mM) increases in parallel with the rise in blood glucose over the physiological concentration range. High-affinity transporters are found in almost every tissue, but their expression is higher in cells with high glycolytic activity. Glut-2, however, is found in tissues carrying large glucose fluxes, such as intestine, kidney, and liver. As an adaptive response to variations in metabolic conditions, the expression of these transporters is regulated by glucose and different hormones. Thus, because of their specific characteristics and regulated expression, the facilitated glucose transporters control fundamental aspects of glucose homeostasis. I review data pertaining to the structure and regulated expression of the glucose carriers present in intestine, kidney, and liver and discuss their role in the control of glucose flux into or out of these different tissues.
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 K(m) 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 microM). Comparing these properties to those of rat Glut1 we found two notable differences; (1) in Glut1, K(m) 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 K(m) values; and (2) Glut1 was inhibited with pCMB, pCMBS and low concentrations of HgCl2 (IC50, 3.5 microM), 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 microM, whereas K(m), 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.
Transport across the plasma membrane is the first, obligatory step of hexose utilization. In yeast cells the uptake of hexoses is mediated by a large family of related transporter proteins. In baker's yeast Saccharomyces cerevisiae the genes of 20 different hexose transporter-related proteins have been identified. Six of these transmembrane proteins mediate the metabolically relevant uptake of glucose, fructose and mannose for growth, two others catalyze the transport of only small amounts of these sugars, one protein is a galactose transporter but also able to transport glucose, two transporters act as glucose sensors, two others are involved in the pleiotropic drug resistance process, and the functions of the remaining hexose transporter-related proteins are not yet known. The catabolic hexose transporters exhibit different affinities for their substrates, and expression of their corresponding genes is controlled by the glucose sensors according to the availability of carbon sources. In contrast, milk yeast Kluyveromyces lactis contains only a few different hexose transporters. Genes of other monosaccharide transporter-related proteins have been found in fission yeast Schizosaccharomyces pombe and in the xylose-fermenting yeast Pichia stipitis. However, the molecular genetics of hexose transport in many other yeasts remains to be established. The further characterization of this multigene family of hexose transporters should help to elucidate the role of transport in yeast sugar metabolism.
Sugar transport across the plasma membrane is one of the most important transport processes. The cloning and expression of cDNAs from a superfamily of related sugar transporters that all adopt a 12-membrane-spanning-domain structure has opened new avenues of investigation, including presteady-state kinetic analysis. Structure-function analyses of mammalian and bacterial sugar transporters, and comparisons of these transporters with those of parasitic trypanosomatids, indicate that different environmental pressures have tailored the evolution of the various members of the sugar-transporter superfamily. Subtle distinctions in the function of these proteins can be related to particular amino acid residue substitutions.
After addition of high concentrations of glucose, rates of high-affinity glucose uptake in Saccharomyces cerevisiae decrease rapidly. We found that the high-affinity hexose transporters Hxt6 and Hxt7 are subject to glucose-induced proteolytic degradation (catabolite inactivation). Degradation occurs in the vacuole, as Hxt6/7 were stabilized in proteinase A-deficient mutant cells. Degradation was independent of the proteasome. The half-life of Hxt6 and Hxt7 strongly increased in end4, ren1 and act1 mutant strains, indicating that the proteins are delivered to the vacuole by endocytosis. Moreover, both proteins were also stabilized in mutants defective in ubiquitination. However, the initial signal that triggers catabolite inactivation is not relayed via the glucose sensors Snf3 and Rgt2.
The hexose transporter family of Saccharomyces cerevisiae comprises 18 proteins (Hxt1-17, Gal2). Here, we demonstrate that all these proteins, except Hxt12, and additionally three members of the maltose transporter family (Agt1, Ydl247, Yjr160) are able to transport hexoses. In a yeast strain deleted for HXT1-17, GAL2, AGT1, YDL247w and YJR160c, glucose consumption and transport activity were completely abolished. However, as additional deletion of the glucose sensor gene SNF3 partially restored growth on hexoses, our data indicate the existence of even more proteins able to transport hexoses in yeast.
We describe here 20 families of secondary (pmf-driven) carriers which, in addition to nine families within the ATP-dependent ABC superfamily, and seven families of Gram-negative bacterial outer membrane porins, largely account for the stereospecific transport of sugars and their derivatives into and out of all living cells on earth. Family characteristics as well as struc-tural and functional properties of the family constituents are described. By reference to our website (http://www-biology.ucsd.edu/ approximately msaier/transport/), phylogenetic relationships, detailed substrate specificity information and both primary and secondary references are also available. This review provides a comprehensive guide to the diversity of carriers that mediate the transport of sugar-containing molecules across cell and organellar membranes.
During the last 2 years, several novel genes that encode glucose transporter-like proteins have been identified and characterized. Because of their sequence similarity with GLUT1, these genes appear to belong to the family of solute carriers 2A (SLC2A, protein symbol GLUT). Sequence comparisons of all 13 family members allow the definition of characteristic sugar/polyol transporter signatures: (1) the presence of 12 membrane-spanning helices, (2) seven conserved glycine residues in the helices, (3) several basic and acidic residues at the intracellular surface of the proteins, (4) two conserved tryptophan residues, and (5) two conserved tyrosine residues. On the basis of sequence similarities and characteristic elements, the extended GLUT family can be divided into three subfamilies, namely class I (the previously known glucose transporters GLUT1-4), class II (the previously known fructose transporter GLUT5, the GLUT7, GLUT9 and GLUT11), and class III (GLUT6, 8, 10, 12, and the myo-inositol transporter HMIT1). Functional characteristics have been reported for some of the novel GLUTs. Like GLUT1-4, they exhibit a tissue/cell-specific expression (GLUT6, leukocytes, brain; GLUT8, testis, blastocysts, brain, muscle, adipocytes; GLUT9, liver, kidney; GLUT10, liver, pancreas; GLUT11, heart, skeletal muscle). GLUT6 and GLUT8 appear to be regulated by sub-cellular redistribution, because they are targeted to intra-cellular compartments by dileucine motifs in a dynamin dependent manner. Sugar transport has been reported for GLUT6, 8, and 11; HMIT1 has been shown to be a H+/myo-inositol co-transporter. Thus, the members of the extended GLUT family exhibit a surprisingly diverse substrate specificity, and the definition of sequence elements determining this substrate specificity will require a full functional characterization of all members.
We have investigated the role and the kinetic properties of the Hxt5 glucose transporter of Saccharomyces cerevisiae. The HXT5 gene was not expressed during growth of the yeast cells in rich medium with glucose or raffinose. However, it became strongly induced during nitrogen or carbon starvation. We have constructed yeast strains constitutively expressing only Hxt5, Hxt1 (low affinity) or Hxt7 (high affinity), but no other glucose transporters. Aerobic fed-batch cultures at quasi steady-state conditions, and aerobic and anaerobic chemostat cultures at steady-state conditions of these strains were used for estimation of the kinetic properties of the individual transporters under in vivo conditions, by investigating the dynamic responses of the strains to changes in extracellular glucose concentration. The K(m) value and the growth properties of the HXT5 single expression strain indicate that Hxt5 is a transporter with intermediate affinity.
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