Expression Of HXT1 Research Articles

Role of Calcium/Calcineurin Signalling in Regulating Intracellular Reactive Oxygen Species Homeostasis in Saccharomyces cerevisiae.

The calcium/calcineurin signalling pathway is required for cell survival under various environmental stresses. Using Saccharomyces cerevisiae, we explored the mechanism underlying calcium-regulated homeostasis of intracellular reactive oxygen species (ROS). We found that deletion of acyltransferase Akr1 and C-5 sterol desaturase Erg3 increased the intracellular ROS levels and cell death, and this could be inhibited by the addition of calcium. The hexose transporter Hxt1 and the amino acid permease Agp1 play crucial roles in maintaining intracellular ROS levels, and calcium induced the expression of the HXT1 and AGP1 genes. The cytosolic calcium concentration was decreased in both the akr1Δ and erg3Δ mutants relative to wild-type cells, potentially lowering basal expression of HXT1 and AGP1. Moreover, the calcium/calcineurin signalling pathway also induced the expression of AKR1 and ERG3, indicating that Akr1 and Erg3 might perform functions that help yeast cells to survive under high calcium concentrations. Our results provided mechanistic insight into how calcium regulated intracellular ROS levels in yeast.

Glucose regulation of the paralogous glucose sensing receptors Rgt2 and Snf3 of the yeast Saccharomyces cerevisiae

BackgroundThe yeast Saccharomyces cerevisiae senses extracellular glucose levels through the two paralogous glucose sensing receptors Rgt2 and Snf3, which appear to sense high and low levels of glucose, respectively. MethodsWestern blotting and qRT-PCR were used to determine expression levels of the glucose sensing receptors. ResultsRgt2 and Snf3 are expressed at different levels in response to different glucose concentrations. SNF3 expression is repressed by high glucose, whereas Rgt2 is turned over in response to glucose starvation. As a result, Rgt2 is predominant in cells grown on high glucose, whereas Snf3 is more abundant of the two paralogs in cells grown on low glucose. When expressed from a constitutive promoter, however, Snf3 behaves like Rgt2, being able to transduce the high glucose signal that induces HXT1 expression. Of note, constitutively active Rgt2 does not undergo glucose starvation-induced endocytic downregulation, whereas signaling defective Rgt2 is constitutively targeted for vacuolar degradation. These results suggest that glucose protects Rgt2 from endocytic degradation and reveal a previously unknown function of glucose as a signaling molecule that regulates the stability of its receptor. ConclusionExpression of Rgt2 and Snf3 is regulated by different mechanisms: Rgt2 expression is highly regulated at the level of protein stability; Snf3 expression is mainly regulated at the level of transcription. General significanceThe difference in the roles of Rgt2 and Snf3 in glucose sensing is a consequence of their cell surface abundance rather than a result of the two paralogous proteins having different functions.

Simulating Extracellular Glucose Signals Enhances Xylose Metabolism in Recombinant Saccharomyces cerevisiae.

Efficient utilization of both glucose and xylose from lignocellulosic biomass would be economically beneficial for biofuel production. Recombinant Saccharomyces cerevisiae strains with essential genes and metabolic networks for xylose metabolism can ferment xylose; however, the efficiency of xylose fermentation is much lower than that of glucose, the preferred carbon source of yeast. Implications from our previous work suggest that activation of the glucose sensing system may benefit xylose metabolism. Here, we show that deleting cAMP phosphodiesterase genes PDE1 and PDE2 increased PKA activity of strains, and consequently, increased xylose utilization. Compared to the wild type strain, the specific xylose consumption rate (rxylose) of the pde1Δ pde2Δ mutant strains increased by 50%; the specific ethanol-producing rate (rethanol) of the strain increased by 70%. We also show that HXT1 and HXT2 transcription levels slightly increased when xylose was present. We also show that HXT1 and HXT2 transcription levels slightly increased when xylose was present. Deletion of either RGT2 or SNF3 reduced expression of HXT1 in strains cultured in 1 g L−1 xylose, which suggests that xylose can bind both Snf3 and Rgt2 and slightly alter their conformations. Deletion of SNF3 significantly weakened the expression of HXT2 in the yeast cultured in 40 g L−1 xylose, while deletion of RGT2 did not weaken expression of HXT2, suggesting that S. cerevisiae mainly depends on Snf3 to sense a high concentration of xylose (40 g L−1). Finally, we show that deletion of Rgt1, increased rxylose by 24% from that of the control. Our findings indicate how S. cerevisiae may respond to xylose and this study provides novel targets for further engineering of xylose-fermenting strains.

Dynamic response of the expression of hxt1, hxt5 and hxt7 transport proteins in Saccharomyces cerevisiae to perturbations in the extracellular glucose concentration

Glucose transport in Saccharomyces cerevisiae relies on a multi-factorial uptake system. The modulation of its efficiency depends on the differential expression of various sets of hexose transport-related proteins whose glucose affinity differs considerably. The expression of three different glucose transport proteins (HXT1, HXT5 and HXT6/7 with low-, intermediate- and high-affinity, respectively) was monitored as a result of modified extracellular glucose concentrations. Cultivation at glucose-limited (continuous) conditions was instantly replaced by a batch (and thus, non-limited) mode. Further, to mimic concentration gradients in large-scale production bioreactors, multiple and rapid transient glucose pulses were applied to chemostat cultivation. Antibodies against the HXT-proteins were used to monitor the proteins’ expression levels prior to and after perturbing the external glucose concentrations. HXT5 and HXT6/7 were either expressed during the starvation-like steady-state phases in the chemostat cultivations, whereas HXT1 could not be detected at all. HXT1, however, is subsequently expressed during the excess of glucose in the batch mode, while the HXT5 and HXT6/7 transporters were at least found to decline. These findings coincide well with the transporters’ affinity profiles. As a result of repeated and rapid transient glucose pulses during continuous fermentation, especially HXT6/7 pointed out to alter the protein expression pattern.

Reduced TOR Signaling Extends Chronological Life Span via Increased Respiration and Upregulation of Mitochondrial Gene Expression

The relationships between mitochondrial respiration, reactive oxygen species (ROS), and life span are complex and remain controversial. Inhibition of the target of rapamycin (TOR) signaling pathway extends life span in several model organisms. We show here that deletion of the TOR1 gene extends chronological life span in Saccharomyces cerevisiae, primarily by increasing mitochondrial respiration via enhanced translation of mtDNA-encoded oxidative phosphorylation complex subunits. Unlike previously reported pathways regulating chronological life span, we demonstrate that deletion of TOR1 delays aging independently of the antioxidant gene SOD2. Furthermore, wild-type and tor1 null strains differ in life span only when respiration competent and grown in normoxia in the presence of glucose. We propose that inhibition of TOR signaling causes derepression of respiration during growth in glucose and that the subsequent increase in mitochondrial oxygen consumption limits intracellular oxygen and ROS-mediated damage during glycolytic growth, leading to lower cellular ROS and extension of chronological life span.

Two Glucose-sensing Pathways Converge on Rgt1 to Regulate Expression of Glucose Transporter Genes in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae deploys two different types of glucose sensors on its cell surface that operate in distinct glucose signaling pathways: the glucose transporter-like Snf3 and Rgt2 proteins and the Gpr1 receptor that is coupled to Gpa2, a G-protein alpha subunit. The ultimate target of the Snf3/Rgt2 pathway is Rgt1, a transcription factor that regulates expression of HXT genes encoding glucose transporters. We have found that the cAMP-dependent protein kinase A (PKA), which is activated by the Gpr1/Gpa2 glucose-sensing pathway and by a glucose-sensing pathway that works through Ras1 and Ras2, catalyzes phosphorylation of Rgt1 and regulates its function. Rgt1 is phosphorylated in vitro by all three isoforms of PKA, and this requires several serine residues located in PKA consensus sequences within Rgt1. PKA and the consensus serine residues of Rgt1 are required for glucose-induced removal of Rgt1 from the HXT promoters and for induction of HXT expression. Conversely, overexpression of the TPK genes led to constitutive expression of the HXT genes. The PKA consensus phosphorylation sites of Rgt1 are required for an intramolecular interaction that is thought to regulate its DNA binding activity. Thus, two different glucose signal transduction pathways converge on Rgt1 to regulate expression of glucose transporters.

Different signalling pathways mediate glucose induction of SUC2, HXT1 and pyruvate decarboxylase in yeast

The glucose sensors Gpr1, Snf3 and Rgt2 generate the earliest signals produced by glucose in yeast. We showed that a lack of Gpr1 or Snf3/Rgt2 decreased by twofold the glucose induction of SUC2, but had no effect on the induction of pyruvate decarboxylase (Pdc). The induction of HXT1 was not affected by the absence of Gpr1. In an hxk1 hxk2 glk1 strain, high glucose fully induced SUC2, caused partial induction of HXT1 and had no effect on Pdc. In this strain, SUC2 induction was dependent on Gpr1, but HXT1 induction was not. Hxk2, required for the high expression of HXT1, was dispensable for the full induction of SUC2 or Pdc. These results indicate that glucose does not induce transcription through a single signalling pathway, but that several pathways may, in different combinations, regulate the transcription of different genes.

Glc7–Reg1 Phosphatase Signals to Yck1,2 Casein Kinase 1 to Regulate Transport Activity and Glucose-Induced Inactivation of Saccharomyces Maltose Permease

The Saccharomyces casein kinase 1 isoforms encoded by the essential gene pair YCK1 and YCK2 control cell growth and morphogenesis and are linked to the endocytosis of several membrane proteins. Here we define roles for the Yck1,2 kinases in Mal61p maltose permease activation and trafficking, using a yck1delta yck2-2(ts) (yck(ts)) strain with conditional Yck activity. Moreover, we provide evidence that Glc7-Reg1 phosphatase acts as an upstream activator of Yck1,2 kinases in a novel signaling pathway that modulates kinase activity in response to carbon source availability. The yck(ts) strain exhibits significantly reduced maltose transport activity despite apparently normal levels and cell surface localization of maltose permease protein. Glucose-induced internalization and rapid loss of maltose transport activity of Mal61/HAp-GFP are not observed in the yck(ts) strain and maltose permease proteolysis is blocked. We show that a reg1delta mutant exhibits a phenotype remarkably similar to that conferred by yck(ts). The reg1delta phenotype is not enhanced in the yck(ts) reg1delta double mutant and is suppressed by increased Yck1,2p dosage. Further, although Yck2p localization and abundance do not change in the reg1delta mutant, Yck1,2 kinase activity, as assayed by glucose-induced HXT1 expression and Mth1 repressor stability, is substantially reduced in the reg1delta strain.

TOR kinase pathway and 14‐3‐3 proteins regulate glucose‐induced expression of HXT1, a yeast low‐affinity glucose transporter

Expression of HXT1, a gene encoding a Saccharomyces cerevisiae low-affinity glucose transporter, is regulated by glucose availability, being activated in the presence of glucose and inhibited when the levels of the sugar are scarce. In this study we show that 14-3-3 proteins are involved in the regulation of the expression of HXT1 by glucose. We also demonstrate that 14-3-3 proteins, in complex with Reg1, a regulatory subunit of Glc7 protein phosphatase, interact physically with Grr1 (a component of the SCF-Grr1 ubiquitination complex), a key player in the process of HXT1 induction by glucose. In addition, we show that the TOR kinase pathway participates actively in the induction of HXT1 expression by glucose. Inhibition of the TOR kinase pathway by rapamycin treatment abolishes HXT1 glucose induction. A possible involvement of PP2A protein phosphatase complex, through the Cdc55 B-subunit, in the glucose induction of HXT1 is also discussed.

How the Rgt1 transcription factor of Saccharomyces cerevisiae is regulated by glucose.

Rgt1 is a transcription factor that regulates expression of HXT genes encoding glucose transporters in the yeast Saccharomyces cerevisiae. Rgt1 represses HXT gene expression in the absence of glucose; high levels of glucose cause Rgt1 to activate expression of HXT1. We identified four functional domains of Rgt1. A domain required for transcriptional repression (amino acids 210-250) is required for interaction of Rgt1 with the Ssn6 corepressor. Another region of Rgt1 (320-380) is required for normal transcriptional activation, and sequences flanking this region (310-320 and 400-410) regulate this function. A central region (520-830) and a short sequence adjacent to the zinc cluster DNA-binding domain (80-90) inhibit transcriptional repression when glucose is present. We found that this middle region of Rgt1 physically interacts with the N-terminal portion of the protein that includes the DNA-binding domain. This interaction is inhibited by the Rgt1 regulator Mth1, which binds to Rgt1. Our results suggest that Mth1 promotes transcriptional repression by Rgt1 by binding to it and preventing the intramolecular interaction, probably by preventing phosphorylation of Rgt1, thereby enabling Rgt1 to bind to DNA. Glucose induces HXT1 gene expression by causing Mth1 degradation, allowing Rgt1 phosphorylation, and leading to the intramolecular interaction that inhibits DNA binding of Rgt1.

Methylglyoxal, a Metabolite Derived from Glycolysis, Functions as a Signal Initiator of the High Osmolarity Glycerol-Mitogen-activated Protein Kinase Cascade and Calcineurin/Crz1-mediated Pathway in Saccharomyces cerevisiae

Methylglyoxal (MG) is a typical 2-oxoaldehyde derived from glycolysis, although it inhibits the growth of cells in all types of organism. Hence, it has been questioned why such a toxic metabolite is synthesized via the ubiquitous energy-generating pathway. We have previously reported that expression of GLO1, coding for the major enzyme detoxifying MG, was induced by osmotic stress in a high osmolarity glycerol (HOG)-mitogen-activated protein (MAP) kinase-dependent manner in Saccharomyces cerevisiae. Here we show that MG activates the HOG-MAP kinase cascade. Two osmosensors, Sln1 and Sho1, have been identified to function upstream of the HOG-MAP kinase cascade, and we reveal that MG initiates the signal transduction to this MAP kinase cascade through the Sln1 branch. We also demonstrate that MG activates the Msn2 transcription factor. Moreover, MG activated the uptake of Ca(2+) in yeast cells, thereby stimulating the calcineurin/Crz1-mediated Ca(2+) signaling pathway. We propose that MG functions as a signal initiator in yeast.

Expression of the HXT1 Low Affinity Glucose Transporter Requires the Coordinated Activities of the HOG and Glucose Signalling Pathways

Expression of the HXT1 gene, which encodes a low affinity glucose transporter in Saccharomyces cerevisiae, is regulated positively in response to glucose by the general glucose induction pathway, involving the Snf3/Rgt2 membrane glucose sensors, the SCF-Grr1 ubiquitination complex and the Rgt1 transcription factor. In this study we show that, in addition to the glucose signaling pathway, regulation of HXT1 expression also requires the HOG pathway. Deletion of components in the glucose signaling pathway or in the HOG pathway results in impaired HXT1 expression. Genetic analyses showed that, whereas the glucose signaling pathway regulates HXT1 through modulation of the Rgt1 transcription factor, the HOG pathway modulates HXT1 through regulation of the Sko1-Tup1-Ssn6 complex. Coordinated regulation of the two signaling pathways is required for expression of HXT1 by glucose and in response to osmostress.

Glucose sensing and signaling in Saccharomyces cerevisiae through the Rgt2 glucose sensor and casein kinase I.

The yeast Saccharomyces cerevisiae senses glucose through two transmembrane glucose sensors, Snf3 and Rgt2. Extracellular glucose causes these sensors to generate an intracellular signal that induces expression of HXT genes encoding glucose transporters by inhibiting the function of Rgt1, a transcriptional repressor of HXT genes. We present the following evidence that suggests that the glucose sensors are coupled to the membrane-associated protein kinase casein kinase I (Yck1). (i) Overexpression of Yck1 leads to constitutive HXT1 expression; (ii) Yck1 (or its paralogue Yck2) is required for glucose induction of HXT1 expression; (iii) Yck1 interacts with the Rgt2 glucose sensor; and (iv) attaching the C-terminal cytoplasmic tail of Rgt2 to Yck1 results in a constitutive glucose signal. The likely targets of Yck1 in this signal transduction pathway are Mth1 and Std1, which bind to and regulate function of the Rgt1 transcription factor and bind to the C-terminal cytoplasmic domain of glucose sensors. Potential casein kinase I phosphorylation sites in Mth1 and Std1 are required for normal glucose regulation of HXT1 expression, and Yck1 catalyzes phosphorylation of Mth1 and Std1 in vitro. These results support a model of glucose signaling in which glucose binding to the glucose sensors causes them to activate Yck1 in the cell membrane, which then phosphorylates Mth1 and Std1 bound to the cytoplasmic face of the glucose sensors, triggering their degradation and leading to the derepression of HXT gene expression. Our results add nutrient sensing to the growing list of processes in which casein kinase I is involved.

Active Snf1 protein kinase inhibits expression of the Saccharomyces cerevisiae HXT1 glucose transporter gene.

Expression of HXT1, a gene encoding a Saccharomyces cerevisiae low-affinity glucose transporter, is regulated by glucose availability, being activated in the presence of glucose and inhibited when the levels of the sugar are scarce. In this study we show that Snf1 protein kinase participates actively in the inhibition of HXT1 expression. Activation of Snf1, either by physiological conditions (growth in low-glucose conditions) or by eliminating any of its negative regulators, such as Hxk2 or Reg1, leads to an inhibition of HXT1 expression. We also show that Std1, another known negative regulator of HXT1 expression, interacts physically with active Snf1 protein kinase. Std1 also interacts physically with Rgt1, a transcription factor involved in HXT1 expression, suggesting that the transcriptional properties of Rgt1 could be modulated either directly or indirectly by Std1 and Snf1 protein kinase. Finally, we show that Rgt1 interacts physically with Ssn6, a major transcriptional repressor, to regulate negatively HXT1 expression when glucose is depleted.

Mth1 receives the signal given by the glucose sensors Snf3 and Rgt2 in Saccharomyces cerevisiae‡

We have determined that the mutant genes DGT1-1 and BPC1-1, which impair glucose transport and catabolite repression in Saccharomyces cerevisiae, are allelic forms of MTH1. Deletion of MTH1 had only slight effects on the expression of HXT1 or SNF3, but increased expression of HXT2 in the absence of glucose. A two-hybrid screen revealed that the Mth1 protein interacts with the cytoplasmic tails of the glucose sensors Snf3 and Rgt2. This interaction was affected by mutations in Mth1 and by the concentration of glucose in the medium. A double mutant, snf3 rgt2, recovered sensitivity to glucose when MTH1 was deleted, thus showing that glucose signalling may occur independently of Snf3 and Rgt2. A model for the possible mode of action of Snf3 and Rgt2 is presented.

A novel signal transduction pathway in Saccharomyces cerevisiae defined by Snf3-regulated expression of HXT6.

We show that cells deleted for SNF3, HXT1, HXT2, HXT3, HXT4, HXT6, and HXT7 do not take up glucose and cannot grow on media containing glucose as a sole carbon source. The expression of Hxt1, Hxt2, Hxt3, Hxt6, or Gal2 in these cells resulted in glucose transport and allowed growth on glucose media. In contrast, the expression of Snf3 failed to confer glucose uptake or growth on glucose. HXT6 is highly expressed on raffinose, low glucose, or nonfermentable carbon sources but is repressed in the presence of high concentrations of glucose. The maintenance of HXT6 glucose repression is strictly dependent on Snf3 and not on intracellular glucose. In snf3 delta cells expression of HXT6 is constitutive even when the entire repertoire of HXT genes is present and glucose uptake is abundant. In addition, glucose repression of HXT6 does not require glucose uptake by HXT1, HXT2, HXT3 or HXT4. We show that a signal transduction pathway defined by the Snf3-dependent hexose regulation of HXT6 is distinct from but also overlaps with general glucose regulation pathways in Saccharomyces cerevisiae. Finally, glucose repression of ADH2 and SUC2 is intact in snf3 delta hxt1 delta hxt2 delta hxt3 delta hxt4 delta hxt6 delta hxt7 delta gal2 cells, suggesting that the sensing and signaling mechanism for general glucose repression is independent from glucose uptake.