Respiratory inhibitors affect incorporation of glucose into Saccharomyces cerevisiae cells,but not the activity of glucose transport

Incubation of starved galactose-grown S. cerevisiae cells with cyanide reduced glucose uptake as measured over a 5-s period. The V_max for glucose uptake was decreased by over a factor of two but the apparent affinity for glucose doubled. When measured in the sub-second time scale, however, there was no significant inhibition of glucose uptake, by cyanide, up to 200-ms, clearly demonstrating that, in cyanide treated cells, glucose uptake was not linear for the first 5-s. After a 200-ms exposure of untreated cells to radio-labelled glucose, less than 10% of the intracellular label resided in soluble uncharged compounds. In cyanide-treated cells up to 43% of the labelled compounds were uncharged, with a concurrent reduction of intracellular label residing in anionic compounds. The results suggest that, in the presence of 10 mM cyanide when respiration is inhibited, a reduction in the cellular ATP concentration causes a reduction in hexose-kinase activity which results in an accumulation of internal free glucose, which in turn causes a reduction in net glucose transport.     

A high-affinity uptake system for branched-chain amino acids in Saccharomyces cerevisiae.

In order to isolate mutants with impaired uptake of branched-chain amino acids, mutants were induced which on complex medium were sensitive to an inhibitor of branched-chain amino acid biosynthesis. Eighteen mutants of independent origin were found. Ten of them were assayed for branched-chain amino acid uptake. Three of these were impaired in the uptake of L-valine, L-isoleucine and L-leucine, while the rest were unaffected in uptake of any of the three amino acids. Kinetics of the uptake by one selected mutant and the parental strain S288C were compared to models for one or two systems obeying Michaelis-Menten kinetics. This analysis suggested that a high-affinity system for all three amino acids is absent in the mutant, whereas low-affinity uptake of L-isoleucine and L-leucine by one or more systems remains unaffected. Moreover, medium-affinity uptake components for L-valine and L-leucine, not originally seen in the wild type, were identified in the mutant. In the wild type, 10 mM of any of the amino acids L-alanine, L-cysteine, L-isoleucine, L-leucine, L-tryptophan and L-valine reduce uptake of any of the three branched-chain amino acids. We propose that a permease responsible for high-affinity uptake of the branched-chain amino acids in strain S288C is partially or completely inactive in the mutant. Tetrad analysis shows that the phenotype can be ascribed to a single Mendelian gene. The wild-type allele is denoted BAP1 for branched-chain amino acid permease. The BAP1-dependent system is different from the general amino acid permease.     

Carnitine/organic cation transporter OCTN2-mediated transport of carnitine in primary-cultured epididymal epithelial cells

Carnitine is essential for the acquisition of motility and maturation of spermatozoa in the epididymis, and is accumulated in epididymal fluid. In this study, carnitine transport into primary-cultured rat epididymal epithelial cells was characterized to clarify the nature of the transporter molecules involved. Uptake of carnitine by primary-cultured epididymal epithelial cells was time, Na(+) and concentration dependent. Kinetic analysis of carnitine uptake by the cells revealed the involvement of high- and low-affinity transport systems with Km values of 21 microM and 2.2 mM respectively. The uptake of carnitine by the cells was significantly reduced by inhibitors of carnitine/organic cation transporter (OCTN2), such as carnitine analogues and cationic compounds. In RT-PCR analysis, OCTN2 expression was detected. These results demonstrated that the high-affinity carnitine transporter OCTN2, which is localized at the basolateral membrane of epididymal epithelial cells, mediates carnitine supply into those cells from the systemic circulation as the first step of permeation from blood to spermatozoa.     

A comparative study on the transport of L(-)malic acid and other short-chain carboxylic acids in the yeast Candida utilis: Evidence for a general organic acid permease

Cells of the yeast Candida utilis grown in medium with short-chain mono-, di- or tricarboxylic acids transported L(-)malic acid by two transport systems at pH 3·0. Results indicate that probably a proton symport for the ionized form of the acid and a facilitated diffusion for the undissociated form were present. Dicarboxylic acids such as succinic, fumaric, oxaloacetic and α-ketoglutaric acids were competitive inhibitors of the malic acid for the high-affinity system, suggesting that these acids used the same transport system. In turn, competitive inhibition uptake studies of labelled carboxylic acid in the low-affinity range indicated that this system was non-specific and able to accept not only carboxylic (mono-, di- or tri-) acids but also some amino acids. Additionally, under the same growth conditions, C. utilis produced two mediated transport systems for lactic acid: a proton symport for the anionic form which appeared to be a common monocarboxylate carrier and a facilitated diffusion system for the undissociated acid displaying a substrate specificity similar to that observed for the low-affinity dicarboxylic acid transport. The mediated carboxylic acid transport systems were inducible and subjected to repression by glucose. In glucose-grown cells the undissociated dicarboxylic acids entered the cells slowly by simple diffusion. Repressed glucose-grown cells were only able to produce both transport systems if an inducer, at low concentration (0·5%, w/v), was present during starvation in buffer. This process was inhibited by the presence of cycloheximide indicating that induction requires de novo protein synthesis. If a higher acid concentration was used, only the low-affinity transport system was detectable, showing that the high-affinity system was also repressed by high concentrations of the inducer.     

Transport of L-leucine in the fission yeast Schizosaccharomyces pombe

Transport of L -leucine into Schizosaccharomyces pombe cells from the stationary phase of growth (after preincubation for 60 min with 1% glucose) proceeds uphill, practically unidirectionally, and is mediated by at least two systems: a high-affinity system with a K_T of 0·045 mmol 1^?1 and J_max of 3·3 nmol min^?1 (mg dry weight)^?1 and a low-affinity system with a K_T of 1·25 mmol 1^?1 and J_max of 16·0 nmol min^?1 (mg dry weight)^?1. The high-affinity system has a pH optimum at 3.2, the accumulation ratio is highest at a cell density of 2–4 mg dry weight per ml and decreases with increasing leucine concentration. Transport of leucine by the high-affinity system is strongly inhibited by proton conductors, ammonium ions and by most amino acids, but only L -phenylalanine, L -isoleucine, L -valine and L -cysteine behave as fully competitive inhibitors. Systems of L -leucine transport in S. pombe are not constitutive. Transport activity appears only after preincubation of cells with a suitable source of energy. If cycloheximide is added during preincubation with glucose, no transport systems for leucine are synthesized. After removal of glucose, the activity of transport systems decays with a half-time of about 20 min. The presence of cyclic AMP increases the initial rate of leucine uptake only in cells preincubated with glucose and in the absence of cycloheximide.     

Disruption of URA7 and GAL6 improves the ethanol tolerance and fermentation capacity of Saccharomyces cerevisiae

Screening of the homozygous diploid yeast deletion pool of 4741 non-essential genes identified two null mutants (Deltaura7 and Deltagal6) that grew faster than the wild-type strain in medium containing 8% v/v ethanol. The survival rate of the gal6 disruptant in 10% ethanol was higher than that of the wild-type strain. On the other hand, the glucose consumption rate of the ura7 disruptant was better than that of the wild-type strain in buffer containing ethanol. Both disruptants were more resistant to zymolyase, a yeast lytic enzyme containing mainly beta-1,3-glucanase, indicating that the integrity of the cell wall became more resistance to ethanol stress. The gal6 disruptant was also more resistant to Calcofluor white, but the ura7 disruptant was more sensitive to Calcofluor white than the wild-type strain. Furthermore, the mutant strains had a higher content of oleic acid (C18 : 1) in the presence of ethanol compared to the wild-type strain, suggesting that the disruptants cope with ethanol stress not only by modifying the cell wall integrity but also the membrane fluidity. When the cells were grown in medium containing 5% ethanol at 15 degrees C, the gal6 and ura7 disruptants showed 40% and 14% increases in the glucose consumption rate, respectively.     

The Branched-Chain Amino Acid Permease Gene of Saccharomyces cerevisiae,BAP2, Encodes the High-Affinity Leucine Permease (S1)

The amino acid leucine has been shown previously to be transported into a yeast cell by at least three permeases: the general amino acid permease, a high-affinity permease (S1) and a low-affinity permease (S2). We isolated the gene BAP2 as a multicopy suppressor of the YPD⁻ phenotype of aat1leu2 yeast. BAP2 has been identified previously as encoding an amino acid permease which transports branched-chain amino acids. In order to align the genetic and biochemical studies of leucine uptake we completed a detailed kinetic analysis of yeast strains in which the BAP2 gene was disrupted and compared this to the kinetics of uptake of the parental strain. We demonstrate that BAP2 encodes the high-affinity leucine permease previously called S1. © 1997 John Wiley & Sons, Ltd.     

Absorption of anthocyanins through intestinal epithelial cells – Putative involvement of GLUT2

Anthocyanins bioavailability is a major issue regarding their biological effects and remains unclear due to few data available on this matter. This work aimed to evaluate the absorption of anthocyanins at the intestine using Caco‐2 cells. Anthocyanin extract, rich in malvidin‐3‐glucoside, was obtained from red grape skins and tested on Caco‐2 cells. The absorption of anthocyanins, in absence or presence of 1% ethanol, was detected by HPLC/DAD/LC‐MS. Our results showed that this transport was significantly increased in the presence of ethanol especially after 60 min of incubation. In addition, cells that were pretreated for 96 h with anthocyanins (200 μg/mL) showed an increase of their own transport (about 50% increase). Expression of glucose transporters sodium‐dependent glucose transporter 1, facilitative glucose transporters 5, and facilitative glucose transporters 2 was assessed by RT‐PCR. It was found that facilitative glucose transporters 2 expression was increased (60%) in Caco‐2 cells pretreated with anthocyanins, by comparison with controls. When the effect of anthocyanin extract on ³H‐2‐deoxy‐D ‐glucose uptake was tested, an inhibitory effect was observed (about 60% decrease). However, the malvidin aglycone was tested and had no effect. In conclusion, anthocyanins could be absorbed through Caco‐2 cells, and can interfere with their own transport and also with glucose intestinal uptake.     

甜玉米芯多糖对胰岛素抵抗HepG2细胞糖代谢功能的影响

目的：探讨甜玉米芯多糖（sweet corncob polysaccharide，SCP）组分SCP-80-I对胰岛素抵抗HepG2（insulin resistant HepG2，IR-HepG2）细胞糖代谢功能的影响。方法：确立IR-HepG2细胞模型建立的最佳条件，并由此建立IR-HepG2细胞模型；分别以50、100、200、400 μg/mL剂量的SCP-80-I孵育细胞24 h，评价其对细胞葡萄糖消耗的影响，同时利用噻唑蓝法评价其细胞毒性；检测氧化应激标志物超氧化物歧化酶（superoxide dismutase，SOD）、丙二醛（malondialdehyde，MDA）及活性氧（reactive oxygen species，ROS）的水平，测定细胞内糖原积累水平及糖酵解关键限速酶己糖激酶（hexokinase，HK）、丙酮酸激酶（pyruvate kinase，PK）的活力。结果：确立以1×10－6 mol/L胰岛素处理24 h作为诱导IR-HepG2细胞形成的最佳条件，并成功建立IR-HepG2细胞模型；在孵育实验中，SCP-80-I能极显著增加IR-HepG2细胞的葡萄糖摄取量（P＜0.01），细胞活力随SCP-80-I质量浓度的增加呈先上升后下降的趋势；200 μg/mL SCP-80-I处理组与模型对照组相比，胞内MDA含量极显著下降，SOD活力极显著提高，ROS水平极显著下降（P＜0.01）；胞内糖原含量极显著增加（P＜0.01），HK与PK活力极显著增加（P＜0.01）。结论：推测SCP-80-I的降血糖功效与其缓解氧化应激所造成的肝脏细胞损伤，改善胰岛素抵抗肝脏细胞的糖代谢功能有关。     

The Emi2 Protein of Saccharomyces cerevisiae is a Hexokinase Expressed under Glucose Limitation

Hexokinases catalyze glucose phosphorylation at the first step in glycolysis in eukaryotes. In the budding yeast Saccharomyces cerevisiae , three enzymes for glucose phosphorylation have long been known: Hxk1, Hxk2, and Glk1. In this study, we focus on Emi2, a previously uncharacterized hexokinase-like protein of S. cerevisiae . Our data show that the recombinant Emi2 protein (rEmi2), expressed in Escherichia coli , possesses glucose-phosphorylating activity in the presence of ATP and Mg ²⁺ . It was also found that rEmi2 phosphorylates not only glucose but also fructose, mannose and glucosamine in vitro . In addition, we examined changes in the level of endogenous Emi2 protein in S. cerevisiae in the presence or absence of glucose and a non-fermentable carbon source. We found that the expression of Emi2 protein is tightly suppressed during proliferation in high glucose, while it is strongly upregulated in response to glucose limitation and the presence of a non-fermentable carbon source. Our data suggest that the expression of the endogenous Emi2 protein in S. cerevisiae is regulated under the control of Hxk2 in response to glucose availability in the environment.     

Kinetics of growth and glucose transport in glucose-limited chemostat cultures of Saccharomyces cerevisiae CBS 8066

The glucose transport capacity of Saccharomyces cerevisiae CBS 8066 was studied in aerobic glucose-limited chemostat cultures. Two different transport systems were encountered with affinity constants of 1 and 20 mM, respectively. The capacity of these carriers (Vmax) was dependent on the dilution rate and the residual glucose concentration in the culture. From the residual glucose concentration in the fermenter and the kinetic constants of glucose transport, their in situ contribution to glucose consumption was determined. The sum of these calculated in situ transport rates correlated well with the observed rate of glucose consumption of the culture. The growth kinetics of S. cerevisiae CBS 8066 in glucose-limited cultures were rather peculiar. At low dilution rates, at which glucose was completely respired, the glucose concentration in the fermenter was constant at 110 microM, independent of the glucose concentration in the reservoir. At higher dilution rates, characterized by the occurrence of both respiration and alcoholic fermentation, the residual substrate concentration followed Monod kinetics. In this case, however, the overall affinity constant was dependent on the reservoir glucose concentration.     

URE3] prion propagation is abolished by a mutation of the primary cytosolic Hsp70 of budding yeast

[URE3] and [PSI(+)] are infectious protein forms of the Saccharomyces cerevisiae Ure2p and Sup35p, respectively. We isolated an allele of SSA2, the primary cytosolic Hsp70, in a screen for mutants unable to maintain [URE3]. Designated ssa2-10, the mutation results in a leucine substitution for proline 395, a conserved residue of the peptide-binding domain. This allele also unexpectedly destabilizes [URE3] in newly formed heterozygotes: [URE3] is either absent in heterozygotes formed by crossing wild-type [URE3] cells with ssa2-10 mutants, or present and fully stable. SSA2 deletion mutants are weakly capable of maintaining [URE3]. The ssa2-10 allele is compatible with propagation of [PSI(+)]. However, in combination with a deletion of SSA1, ssa2-10 eliminates the nonsense-suppression phenotype of [PSI(+)] cells.     

Isolation and characterization of peroxisomal protein import (Pim−) mutants of Hansenula polymorpha

In the course of our studies on the molecular mechanisms involved in peroxisome biogenesis, we have isolated several mutants of the methylotrophic yeast Hansenula polymorpha impaired in the import of peroximal matrix proteins. These mutants are characterized by the presence of small intact peroxisomes, while the bulk of the peroxisomal matrix protein is not imported and resides in the cytosol (Pim^? phenotype). Genetic analysis of back-crossed mutants revealed five different complementation groups, which were designated PERI–PER5. Mapping studies to determine the linkage relationships indicated that the observed Pim^? phenotypes were determined by single recessive nuclear mutations. The different mutants had comparable phenotypes: (i) they were impaired to utilize methanol as the sole source of carbon and energy but grew well on various other compounds, including nitrogen sources, the metabolism of which is known to be mediated by peroxisome-borne enzymes in wild-type cells; (ii) all peroxisomal enzymes tested were induced, assembled and activated as in wild-type cells although their activities varied between the different representative mutants; (iii) all peroxisomal proteins, whether constitutive or inducible, were found both in the cytosol and in the small peroxisomes. These results suggest that a general, major import mechanism is affected in all mutants.     

Assembly of amine oxidase and D-amino acid oxidase in the cytosol of peroxisome-deficient mutants of the yeast Hansenula polymorpha during growth of cells on glucose in the presence of primary amines or D-alanine as the sole nitrogen source

We have studied growth of two peroxisome-deficient mutant strains of Hansenula polymorpha on glucose in the presence of different organic nitrogen sources (methylamine, ethylamine and D -alanine), the metabolism of which is mediated by peroxisome-borne oxidases in wild-type (WT) cells. Both strains grew well on each of these substrates with growth rates comparable to WT cells. Growth on both methylamine and ethylamine was associated with enhanced levels of catalase and amine oxidase in the cells; in D -alanine-grown cells D -amino acid oxidase activity and increased. In WT cells of H-polymorpha the activities of these enzymes were confined to the peroxisomal matrix; however, in both peroxisome-deficient strains their activities were localized in the cytosol. Electron microscopy indicated that, dependent on the stage of growth, the enzymes may form large protein aggregates. The molecular masses of both amine oxidase and D -amino acid oxidase in the mutant strains were identical to their respective counterparts in WT cells, indicating that both proteins were correctly assembled and active in the cytosol.