Functional Dissection of the Glucose Signaling Pathways That Regulate the Yeast Glucose Transporter Gene (\u3ci\u3eHXT\u3c/i\u3e) Repressor Rgt1

The yeast Rgt1 repressor is a bifunctional protein that acts as a transcriptional repressor and activator. Under glucose-limited conditions, Rgt1 induces transcriptional repression by forming a repressive complex with its corepressors Mth1 and Std1. Here, we show that Rgt1 is converted from a transcriptional repressor into an activator under high glucose conditions and this occurs through two independent but consecutive events mediated by two glucose signaling pathways: (1) disruption of the repressive complex by the Rgt2/Snf3 pathway; (2) phosphorylation of Rgt1 by the cAMP-dependent protein kinase (cAMP-PKA) pathway. Rgt1 is phosphorylated by PKA at four serine residues within its amino-terminal region, but this does not occur until the repressive complex is disrupted. While phosphorylation of any one of these sites is sufficient to enable Rgt1 to induce transcriptional activation, phosphorylation of all the sites results in the release of Rgt1 from DNA. We discuss how the bifunctional properties of Rgt1 are regulated through differential phosphorylation. J. Cell. Biochem. 112: 3268-3275, 2011. (C) 2011 Wiley Periodicals, Inc

Understanding the mechanism of glucose-induced relief of Rgt1-mediated repression in yeast

The yeast Rgt1 repressor inhibits transcription of the glucose transporter (HXT) genes in the absence of glucose. It does so by recruiting the general corepressor complex Ssn6-Tup1 and the HXT corepressor Mth1. In the presence of glucose, Rgt1 is phosphorylated by the cAMP-activated protein kinase A (PKA) and dissociates from the HXT promoters, resulting in expression of HXT genes. In this study, using Rgt1 chimeras that bind DNA constitutively, we investigate how glucose regulates Rgt1 function. Our results show that the DNA-bound Rgt1 constructs repress expression of the HXT1 gene in conjunction with Ssn6-Tup1 and Mth1, and that this repression is lifted when they dissociate from Ssn6-Tup1 in high glucose conditions. Mth1 mediates the interaction between the Rgt1 constructs and Ssn6-Tup1, and glucose-induced downregulation of Mth1 enables PKA to phosphorylate the Rgt1 constructs. This phosphorylation induces dissociation of Ssn6-Tup1 from the DNA-bound Rgt1 constructs, resulting in derepression of HXT gene expression. Therefore, Rgt1 removal from DNA occurs in response to glucose but is not necessary for glucose induction of HXT gene expression, suggesting that glucose regulates Rgt1 function by primarily modulating the Rgt1 interaction with Ssn6-Tup1

The Glucose Signaling Network in Yeast

Background Most cells possess a sophisticated mechanism for sensing glucose and responding to it appropriately. Glucose sensing and signaling in the budding yeast Saccharomyces cerevisiae represent an important paradigm for understanding how extracellular signals lead to changes in the gene expression program in eukaryotes. Scope of review This review focuses on the yeast glucose sensing and signaling pathways that operate in a highly regulated and cooperative manner to bring about glucose-induction of HXT gene expression. Major conclusions The yeast cells possess a family of glucose transporters (HXTs), with different kinetic properties. They employ three major glucose signaling pathways—Rgt2/Snf3, AMPK, and cAMP-PKA—to express only those transporters best suited for the amounts of glucose available. We discuss the current understanding of how these pathways are integrated into a regulatory network to ensure efficient uptake and utilization of glucose. General significance Elucidating the role of multiple glucose signals and pathways involved inglucose uptake and metabolism in yeast may reveal the molecular basis of glucose homeostasis in humans, especially under pathological conditions, such as hyperglycemia in diabetics and the elevated rate of glycolysis observed in many solid tumors

The Role of the Pleckstrin Homology Domain-Containing Protein CKIP-1 in Activation of p21-activated Kinase 1 (PAK1)

Upon growth factor stimulation, PAK1 is recruited to the plasma membrane and activated by a mechanism that requires its phosphorylation at S223 by the protein kinase CK2. However, the upstream signaling molecules that regulate this phosphorylation event are not clearly defined. Here, we demonstrate a major role of the CK2α-interacting protein CKIP-1 in activation of PAK1. CK2α, CKIP-1 and PAK1 are translocated to membrane ruffles in response to the epidermal growth factor (EGF), where CKIP-1 mediates the interaction between CK2α, and PAK1 in a PI3K-dependent manner. Consistently, we observe that PAK1 mediates phosphorylation and modulation of the activity of p41-Arc, one of its plasma membrane substrate, in a fashion that requires PI3K and CKIP-1. Moreover, CKIP-1 knockdown or PI3K inhibition suppresses PAK1-mediated cell migration and invasion, demonstrating the physiological significance of the PI3K-CKIP-1-CK2α-PAK1 signaling pathway. Taken together, these findings identify a novel mechanism for the activation of PAK1 at the plasma membrane, which is critical for cell migration and invasion

Mth1 regulates the interaction between the Rgt1 repressor and the Ssn6-Tup1 corepressor complex by modulating PKA-dependent phosphorylation of Rgt1

Glucose uptake, the first, rate-limiting step of its utilization, is facilitated by glucose transporters. Expression of several glucose transporter (HXT) genes in yeast is repressed by the Rgt1 repressor, which recruits the glucose-responsive transcription factor Mth1 and the general corepressor complex Ssn6-Tup1 in the absence of glucose; however, it is derepressed when Mth1 is inactivated by glucose. Here we show that Ssn6-Tup1 interferes with the DNA-binding ability of Rgt1 in the absence of Mth1 and that the Rgt1 function abrogated by Ssn6 overexpression is restored by co-overexpression of Mth1. Thus Mth1 likely regulates Rgt1 function not by modulating its DNA-binding activity directly but by functionally antagonizing Ssn6-Tup1. Mth1 does so by acting as a scaffold-like protein to recruit Ssn6-Tup1 to Rgt1. Supporting evidence shows that Mth1 blocks the protein kinase A–dependent phosphorylation of Rgt1 that impairs the ability of Rgt1 to interact with Ssn6-Tup1. Of note, Rgt1 can bind DNA in the absence of Ssn6-Tup1 but does not inhibit transcription, suggesting that dissociation of Rgt1 from Ssn6-Tup1, but not from DNA, is necessary and sufficient for the expression of its target genes. Taken together, these findings show that Mth1 is a transcriptional corepressor that facilitates the recruitment of Ssn6-Tup1 by Rgt1

Assessing Glucose Uptake through the Yeast Hexose Transporter 1 (Hxt1)

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

The glucose metabolite methylglyoxal inhibits expression of the glucose transport genes by inactivating the cell surface glucose sensors Rgt2 and Snf3 in yeast

Methylglyoxal (MG) is a cytotoxic by-product of glycolysis. MG has inhibitory effect on the growth of cells ranging from microorganisms to higher eukaryotes, but its molecular targets are largely unknown. The yeast cell-surface glucose sensors Rgt2 and Snf3 function as glucose receptors that sense extracellular glucose and generate a signal for induction of expression of genes encoding glucose transporters (HXTs). Here we provide evidence that these glucose sensors are primary targets of MG in yeast. MG inhibits the growth of glucose-fermenting yeast cells by inducing endocytosis and degradation of the glucose sensors. However, the glucose sensors with mutations at their putative ubiquitin-acceptor lysine residues are resistant to MG-induced degradation. These results suggest that the glucose sensors are inactivated through ubiquitin-mediated endocytosis and degraded in the presence of MG. In addition, the inhibitory effect of MG on the glucose sensors is greatly enhanced in cells lacking Glo1, a key component of the MG detoxification system. Thus the stability of these glucose sensors seems to be critically regulated by intracellular MG levels. Taken together, these findings suggest that MG attenuates glycolysis by promoting degradation of the cell-surface glucose sensors and thus identify MG as a potential glycolytic inhibitor

Methylglyoxal Inhibits Expression of the Glucose Transporter Genes by Inactivating Rgt2/Snf3 Glucose Sensors

Methylglyoxal (MG) is a highly reactive, cytotoxic dicarbonyl compound, mainly formed as a by-product of glycolysis. It is one of the most potent glycating agents and readily reacts with proteins, lipids and nucleic acids to form advanced glycation end products (AGEs). However, the molecular targets of MG are largely unknown. Glucose is the preferred carbon source of yeast Saccharomyces cerevisiae and can sense and utilize it efficiently over a broad range of concentrations. It prefers to ferment rather than oxidize glucose, even when oxygen is abundant. The yeast cell-surface glucose sensors Rgt2 and Snf3 function as glucose receptors that sense extracellular glucose and generate a signal for induction of genes encoding glucose transporters (HXTs). Using molecular and cell biology approaches, including Western blotting, qRT-PCR analysis and fluorescence microscopy, we provide evidence that MG inhibits expression of the HXTs by inactivating the yeast glucose sensors Rgt2 and Snf3. MG inhibits the growth of glucose-fermenting yeast cells by inducing endocytosis and degradation of the glucose sensors. However, the glucose sensors with mutations at their putative ubiquitin-acceptor lysine residues are resistant to MG-induced degradation. Our results suggest that the glucose sensors are inactivated through ubiquitin-mediated endocytosis and degraded in the presence of MG. Under physiological conditions, MG is detoxified by the glyoxalase system into D-lactate, with glyoxalase 1 (Glo1) as the key enzyme in the anti-glycation defense. This study further indicates that the inhibitory effect of MG on the glucose sensors is greatly enhanced in the cells lacking Glo1. Thus, the stability of these glucose sensors seems to be critically regulated by intracellular MG levels. Taken together, these findings suggest that MG attenuates glycolysis by promoting degradation of the cell surface glucose sensors and thus identify MG as a potential glycolytic inhibitor

Detailed Computational Modeling of Laminar and Turbulent Sooting Flames

This study reports development and validation of two parallel flame solvers with soot models based on the open-source computation fluid dynamics (CFD) toolbox code OpenF0AM. First, a laminar flame solver is developed and validated against experimental data. A semi-empirical two-equation soot model and a detailed soot model using a method of moments with interpolative closure (MOMIC) are implemented in the laminar flame solver. An optically thin radiation model including gray soot radiation is also implemented. Preliminary results using these models show good agreement with experimental data for the laminar axisymmetric diffusion flame studied. Second, a turbulent flame solver is developed using Reynolds-averaged equations and transported probability density function (tPDF) method. The MOMIC soot model is implemented on this turbulent solver. A sophisticated photon Monte-Carlo (PMC) model with line-by-line spectral radiation database for modeling is also implemented on the turbulent solver. The validation of the turbulent solver is under progress. Both the solvers show good scalability for a moderate-sized chemical mechanism, and can be expected to scale even more strongly when larger chemical mechanisms are used

Detailed computational modeling of laminar and turbulent sooting flames

This study reports development and validation of two parallel flame solvers with soot models based on the open-source computation fluid dynamics (CFD) toolbox code OpenF0AM. First, a laminar flame solver is developed and validated against experimental data. A semi-empirical two-equation soot model and a detailed soot model using a method of moments with interpolative closure (MOMIC) are implemented in the laminar flame solver. An optically thin radiation model including gray soot radiation is also implemented. Preliminary results using these models show good agreement with experimental data for the laminar axisymmetric diffusion flame studied. Second, a turbulent flame solver is developed using Reynolds-averaged equations and transported probability density function (tPDF) method. The MOMIC soot model is implemented on this turbulent solver. A sophisticated photon Monte-Carlo (PMC) model with line-by-line spectral radiation database for modeling is also implemented on the turbulent solver. The validation of the turbulent solver is under progress. Both the solvers show good scalability for a moderate-sized chemical mechanism, and can be expected to scale even more strongly when larger chemical mechanisms are used