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Reduction of glucose uptake through inhibition of hexose transporters and enhancement of their endocytosis by methylglyoxal in Saccharomyces cerevisiae.

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

SUBMITTER: Yoshida A

PROVIDER: S-EPMC3249125 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Reduction of glucose uptake through inhibition of hexose transporters and enhancement of their endocytosis by methylglyoxal in Saccharomyces cerevisiae.

Yoshida Aya A Wei Dandan D Nomura Wataru W Izawa Shingo S Inoue Yoshiharu Y

The Journal of biological chemistry 20111117 1

Diabetes mellitus is characterized by an impairment of glucose uptake even though blood glucose levels are increased. Methylglyoxal is derived from glycolysis and has been implicated in the development of diabetes mellitus, because methylglyoxal levels in blood and tissues are higher in diabetic patients than in healthy individuals. However, it remains to be elucidated whether such factors are a cause, or consequence, of diabetes. Here, we show that methylglyoxal inhibits the activity of mammali ...[more]

PMID: 22094464

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Project description:BackgroundIn the yeast Saccharomyces cerevisiae, which is widely applied for industrial bioethanol production, uptake of hexoses is mediated by transporters with a facilitated diffusion mechanism. In anaerobic cultures, a higher ethanol yield can be achieved when transport of hexoses is proton-coupled, because of the lower net ATP yield of sugar dissimilation. In this study, the facilitated diffusion transport system for hexose sugars of S. cerevisiae was replaced by hexose-proton symport.ResultsIntroduction of heterologous glucose- or fructose-proton symporters in an hxt0 yeast background strain (derived from CEN.PK2-1C) restored growth on the corresponding sugar under aerobic conditions. After applying an evolutionary engineering strategy to enable anaerobic growth, the hexose-proton symporter-expressing strains were grown in anaerobic, hexose-limited chemostats on synthetic defined medium, which showed that the biomass yield of the resulting strains was decreased by 44.0-47.6%, whereas the ethanol yield had increased by up to 17.2% (from 1.51 to 1.77 mol mol hexose-1) compared to an isogenic strain expressing the hexose uniporter HXT5. To apply this strategy to increase the ethanol yield on sucrose, we constructed a platform strain in which all genes encoding hexose transporters, disaccharide transporters and disaccharide hydrolases were deleted, after which a combination of a glucose-proton symporter, fructose-proton symporter and extracellular invertase (SUC2) were introduced. After evolution, the resulting strain exhibited a 16.6% increased anaerobic ethanol yield (from 1.51 to 1.76 mol mol hexose equivalent-1) and 46.6% decreased biomass yield on sucrose.ConclusionsThis study provides a proof-of-concept for the replacement of the endogenous hexose transporters of S. cerevisiae by hexose-proton symport, and the concomitant decrease in ATP yield, to greatly improve the anaerobic yield of ethanol on sugar. Moreover, the sugar-negative platform strain constructed in this study acts as a valuable starting point for future studies on sugar transport or development of cell factories requiring specific sugar transport mechanisms.

Project description:The mechanisms by which bacteria uptake solutes across the cell membrane broadly impact their cellular energetics. Here, we use functional genomic, genetic, and biophysical approaches to reveal how Clostridium (Lachnoclostridium) phytofermentans, a model bacterium that ferments lignocellulosic biomass, uptakes plant hexoses using highly specific, nonredundant ATP-binding cassette (ABC) transporters. We analyze the transcription patterns of its 173 annotated sugar transporter genes to find those upregulated on specific carbon sources. Inactivation of these genes reveals that individual ABC transporters are required for uptake of hexoses and hexo-oligosaccharides and that distinct ABC transporters are used for oligosaccharides versus their constituent monomers. The thermodynamics of sugar binding shows that substrate specificity of these transporters is encoded by the extracellular solute-binding subunit. As sugars are not phosphorylated during ABC transport, we identify intracellular hexokinases based on in vitro activities. These mechanisms used by Clostridia to uptake plant hexoses are key to understanding soil and intestinal microbiomes and to engineer strains for industrial transformation of lignocellulose.IMPORTANCE Plant-fermenting Clostridia are anaerobic bacteria that recycle plant matter in soil and promote human health by fermenting dietary fiber in the intestine. Clostridia degrade plant biomass using extracellular enzymes and then uptake the liberated sugars for fermentation. The main sugars in plant biomass are hexoses, and here, we identify how hexoses are taken in to the cell by the model organism Clostridium phytofermentans We show that this bacterium uptakes hexoses using a set of highly specific, nonredundant ABC transporters. Once in the cell, the hexoses are phosphorylated by intracellular hexokinases. This study provides insight into the functioning of abundant members of soil and intestinal microbiomes and identifies gene targets to engineer strains for industrial lignocellulosic fermentation.

Project description:In Saccharomyces cerevisiae, TRK1 and TRK2 are required for high- and low-affinity K+ transport. Among suppressors of the K+ transport defect in trk1 delta trk2 delta cells, we have identified members of the sugar transporter gene superfamily. One suppressor encodes the previously identified glucose transporter HXT1, and another encodes a new member of this family, HXT3. The inferred amino acid sequence of HXT3 is 87% identical to that of HXT1, 64% identical to that of HXT2, and 32% identical to that of SNF3. Like HXT1 and HXT2, overexpression of HXT3 in snf3 delta cells confers growth on low-glucose or raffinose media. The function of another new member of the HXT superfamily, HXT4 (previously identified by its ability to suppress the snf3 delta phenotype; L. Bisson, personal communication), was revealed in experiments that deleted all possible combinations of the five members of the glucose transporter gene family. Neither SNF3, HXT1, HXT2, HXT3, nor HXT4 is essential for viability. snf3 delta hxt1 delta hxt2 delta hxt3 delta hxt4 delta cells are unable to grow on media containing high concentrations of glucose (5%) but can grow on low-glucose (0.5%) media, revealing the presence of a sixth transporter that is itself glucose repressible. This transporter may be negatively regulated by SNF3 since expression of SNF3 abolishes growth of hxt1 delta hxt2 delta hxt3 delta hxt4 delta cells on low-glucose medium. HXT1, HXT2, HXT3, and HXT4 can function independently: expression of any one of these genes is sufficient to confer growth on medium containing at least 1% glucose. A synergistic relationship between SNF3 and each of the HXT genes is suggested by the observation that SNF2 hxt1 delta hxt2 delta hxt3 delta hxt4 delta cells and snf3 delta HXT1 HXT2 HXT3 HXT4 cells are unable to grow on raffinose (low fructose) yet SNF3 in combination with any single HXT gene is sufficient for growth on raffinose. HXT1 and HXT3 are differentially regulated. HXT1::lacZ is maximally expressed during exponential growth whereas HXT3::lacZ is maximally expressed after entry into stationary phase.

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Reduction of glucose uptake through inhibition of hexose transporters and enhancement of their endocytosis by methylglyoxal in Saccharomyces cerevisiae.

Publications

Reduction of glucose uptake through inhibition of hexose transporters and enhancement of their endocytosis by methylglyoxal in Saccharomyces cerevisiae.

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