酿酒酵母菌葡萄糖信号传导途径研究进展

简要概述了酿酒酵母细胞的葡萄糖信号传导途径的研究进展,总结了葡萄糖的抑制途径和诱导途径.     

Glucose Metabolism in gcr Mutants of Saccharomyces cerevisiae

A gcr2 null mutant of Saccharomyces cerevisiae grows well on glucose in spite of its lower level of glycolytic enzymes between triose phosphates and pyruvate. A quantitative analysis shows that these levels are adequate to the flux but glycerate phosphates are elevated.     

Induction of homologous recombination in Saccharomyces cerevisiae

Summary We have investigated the effects of UV irradiation of Saccharomyces cerevisiae in order to distinguish whether UV-induced recombination results from the induction of enzymes required for homologous recombination, of the production of substrate sites for recombination containing regions of DNA damage. We utilized split-dose experiments to investigate the induction of proteins required for survival, gene conversion, and mutation in a diploid strain of S. cerevisiae. We demonstrate that inducing doses of UV irradiation followed by a 6 h period of incubation render the cells resistant to challenge doses of UV irradiation. The effects of inducing and challenge doses of UV irradiation upon interchromosomal gene conversion and mutation are strictly additive. Using the yeast URA3 gene cloned in non-replicating single- and double-stranded plasmid vectors that integrate into chromosomal genes upon transformation, we show that UV irradiation of haploid yeast cells and homologous plasmid DNA sequences each stimulate homologous recombination approximately two-fold, and that these effects are additive. Non-specific DNA damage has little effect on the stimulation of, homologous recombination, as shown by studies in which UV-irradiated heterologous DNA was included in transformation/recombination experiments. We further demonstrate that the effect of competing single- and double-stranded heterologous DNA sequences differs in UV-irradiated and unirradiated cells, suggesting an induction of recombinational machinery in UV-irradiated S. cerevisiae cells.     

Kinetics and pH-dependence of glycine-proton symport in Saccharomyces cerevisiae

Interactions between intracellular pH (pHi) and H+-coupled transmembrane transport of glycine have been studied by means of 31P-NMR, using both aerobic and 'energy starved' cells of the yeast Saccharomyces cerevisiae. The general features of glycine transport in the yeast strain used (NCYC 239) are similar to those already reported for Saccharomyces carlsbergensis and S. cerevisiae, there being two kinetically distinct glycine uptake systems, with pH-independent K1/2 values near 14 and 0.4mM, respectively, but pH-dependent maximal velocities. Glycine transport itself has no measurable effect on pHi in aerobic cells, and only a marginal effect in energy-starved cells, but changes of pHi, imposed by extracellular addition of butyric acid, strongly influence glycine transport. Indeed, the dependence of glycine influx (in energy-starved cells) upon cytoplasmic H+ concentration appears to be third order, showing Hill slopes of 2.7-3.0. A crucial kinetic role for cytoplasmic pH in glycine transport is further indicated by a proportionality between the decline of flux and the decline of pHi produced by various metabolic inhibitors and uncouplers. Extracellular pH (pHo), by contrast, has only a weak effect on glycine influx, showing a Hill slope of 0.5. The major observations can be accommodated by a simple cyclic carrier scheme, in which 2 or more protons are transported along with glycine, but only one extracellular proton binding site dissociates in the testing range, with a pK near 5.5. The model requires a finite membrane potential, which must be somewhat sensitive to both pHi and pHo, and accommodates the discrepancy between measured net proton flux (one per glycine) and the kinetically required proton flux (two or more per glycine) by shunting through other proton-conducting pathways in the yeast membrane.     

Kinetics of the mating-specific aggregation in Saccharomyces cerevisiae

Procedures are described for obtaining efficient mating (zygote formation) in the heterothallic yeast Saccharomyces cerevisiae. The methods separate operationally the initial, mating-specific aggregation from subsequent steps in conjugation. This cell-cell interaction has been characterized. The data support the conclusions that (i) aggregation in liquid suspension is a random collision process, and (ii) only non-budded cells participate in aggregation. A mathematical model for the kinetics of aggregation in liquid suspension has been developed which is in good agreement with the experimental data.     

Kinetics of active alpha-glucoside transport in Saccharomyces cerevisiae

alpha-Glucosides are the most abundant fermentable sugars in the industrial applications of Saccharomyces cerevisiae, and the active transport across the plasma membrane is the rate-limiting step for their metabolism. In this report we performed a detailed kinetic analysis of the active alpha-glucoside transport system(s) present in a wild-type strain, and in strains with defined alpha-glucoside permeases. Our results indicate that the wild-type strain harbors active transporters with high and low affinity for maltose and trehalose, and low-affinity transport systems for maltotriose and alpha-methylglucoside. The maltose permease encoded by the MAL21 gene showed a high affinity (K(m) approximately 5 mM) for maltose, and a low affinity (K(m) approximately 90 mM) for trehalose. On the other hand, the alpha-glucoside permease encoded by the AGT1 gene had a high affinity (K(m) approximately 7 mM) for trehalose, a low affinity (K(m) approximately 18 mM) for maltose and maltotriose, and a very low affinity (K(m) approximately 35 mM) for alpha-methylglucoside.     

Glucose uptake in the cell cycle of Saccharomyces cerevisiae

Glucose uptake was determined in the cell cycle of the yeast Saccharomyces cerevisiae. It was observed that there are two periods per cell cycle at which cells utilize glucose. This finding could give an explanation for the known fact that yeast cells in the stationary phase of growth are of two size classes.     

Glucose regulation of Saccharomyces cerevisiae cell cycle genes

Nutrient-limited Saccharomyces cerevisiae cells rapidly resume proliferative growth when transferred into glucose medium. This is preceded by a rapid increase in CLN3, BCK2, and CDC28 mRNAs encoding cell cycle regulatory proteins that promote progress through Start. We have tested the ability of mutations in known glucose signaling pathways to block glucose induction of CLN3, BCK2, and CDC28. We find that loss of the Snf3 and Rgt2 glucose sensors does not block glucose induction, nor does deletion of HXK2, encoding the hexokinase isoenzyme involved in glucose repression signaling. Rapamycin blockade of the Tor nutrient sensing pathway does not block the glucose response. Addition of 2-deoxy glucose to the medium will not substitute for glucose. These results indicate that glucose metabolism generates the signal required for induction of CLN3, BCK2, and CDC28. In support of this conclusion, we find that addition of iodoacetate, an inhibitor of the glyceraldehyde-3-phosphate dehydrogenase step in yeast glycolysis, strongly downregulates the levels CLN3, BCK2, and CDC28 mRNAs. Furthermore, mutations in PFK1 and PFK2, which encode phosphofructokinase isoforms, inhibit glucose induction of CLN3, BCK2, and CDC28. These results indicate a link between the rate of glycolysis and the expression of genes that are critical for passage through G₁.     

Glucose transport in a kinaseless Saccharomyces cerevisiae mutant.

Wild-type Saccharomyces cerevisiae organisms contain three kinases which catalyze the phosphorylation of glucose: two hexokinase isozymes (PI and PII) and one glucokinase. Glucose transport measurements for triple-kinaseless mutants, which lack all three of these kinases, confirm that the kinases are involved in the low apparent Km transport process observed in metabolizing cells. Thus kinase-positive cells containing one or more of the three kinases exhibit biphasic transport kinetics with a low apparent Km (1 to 2 mM) and high apparent Km (40 to 50 mM) component. Triple-kinaseless cells, however, exhibit only the high apparent Km component of kinase-positive cells (60 mM). Kinetic analysis of glucose transport in the triple-kinaseless cells shows that glucose is transported by a facilitated diffusion process which exhibits trans-stimulated equilibrium exchange and influx counterflow.     

Glucose and Fructose Metabolism in a Phosphoglucoisomeraseless Mutant of Saccharomyces cerevisiae

A mutant of Saccharomyces cerevisiae deficient in phosphoglucoisomerase (EC 5.3.1.9) is described. It does not grow on glucose or sucrose but does grow on galactose or maltose. Addition of glucose to cultures growing on fructose, mannose, or acetate arrests further growth without altering viability; removal of glucose permits resumption of growth. Glucose causes accumulation of nearly 30 mumoles of glucose-6-phosphate per g (wet weight) of cells and suppresses synthesis of ribonucleic acid. Inhibition of growth by glucose does not appear to be due to a loss of adenosine triphosphate or inorganic orthophosphate. The mutant, however, utilizes glucose-6-phosphate produced intracellularly. Release of carbon dioxide from specifically labeled glucose suggests a C-l preferential cleavage. The kinetics of glucose-6-phosphate accumulation during glucose utilization in the mutant is not consistent with the notion that the utilization of glucose is controlled by glucose-6-phosphate.     

Glucose metabolism and cell size in continuous cultures of Saccharomyces cerevisiae

A detailed analysis of the cell size, monitored as protein content, has been performed in glucose-limited continuous cultures, so as to obtain the values of the average protein content for various subpopulations at different cell cycle stages, as a function of the growth rate. Glucose metabolism appears to affect cell size, since there is an increase of the average protein content of the population when cells produce ethanol above the critical dilution rate. If the production of ethanol is forced at low growth rates by the addition of formate, the average protein content increases. These results indicate a link between glucose metabolism and cell size in budding yeast, as observed for mammalian cells.     

Glucose and fructose consumption in a phosphoglucoseisomeraseless mutant in Saccharomyces cerevisiae

Saccharomyces cerevisiae with a practically complete absence of phosphoglucoseisomerase activity when grown in fructose or glucose minimal medium showed different consumption of fructose and glucose during different periods of the culture. At the beginning of growth, cells had a great quantity of glucose available relative to their requirements and a large quantity of trehalose accumulated from ¹⁴C-glucose in comparison with the wild type strain. A second phase arises when the concentration of glucose in the medium was practically absent and the cells obtain glucose by mobilisation of stored glucose containing compounds. It is very likely that at this moment a balance rate between glucose 6-phosphate formation and consumption occurs. Finally cells reach conditions of glucose starvation and fructose consumption increases in this last stage. The different consumption of fructose throughout different periods of cell growth most probably indicates a strict regulation at the level of sugar uptake.Non Standard Abbreviation pgi phosphoglucoseisomerase     

Induction of respiration-deficient mutants in Saccharomyces cerevisiae by chelerythrine

Abstract Chelerythrine and sanguinarine, two structurally related benzo/c/phenanthridine alkaloids, prevented growth of yeast cells in medium containing either glucose or non-fermentable carbon sources. At concentrations permitting growth of the yeast Saccharomyces cerevisiae , chelerythrine, but not sanquinarine, induced cytoplasmic respiration-deficient mutants. The petite clones that were analysed exhibited suppressiveness and contained different fragments of the wild-type mitochondrial genome.     

Induction of Stress Genes in Saccharomyces cerevisiae during Starvation

In order to analyze the response of Saccharomyces cerevisiae to starvation on a gene expression level, microarray experiments were performed using a yeast whole genome array. It is well known that under stress conditions like heat, high salt concentrations, pressure or the presence of toxins, special stress response genes are induced in Saccharomyces cerevisiae. This includes the genes encoding the typical heat shock proteins as well as numerous genes concerning cell membrane composition, central carbon metabolism or cell cycle. In this contribution, the Saccharomyces cerevisiae starvation‐stress response is analyzed. Starvation is a living condition often experienced by yeast in natural surroundings. As Saccharomyces cerevisiae is an eukaryote, many results from the gene expression analysis are valid for mammalians as well. The understanding of response of the yeast to the absence of a nutrient is also important for the development of feeding strategies in cultivations. Therefore, knowledge about the gene expression during starvation is important for both research and industrial applications. The regulation of 233 genes, which are involved in the stress response according to the literature, was examined via microarray experiments. In addition, a screening was carried out identifying 115 genes, which are hitherto not known to be comprised in the stress response, but which were significantly up‐regulated during starvation.     

Induction of polypeptides in Saccharomyces cerevisiae after ultraviolet irradiation

Alterations in the synthesis of proteins following exposure of Saccharomyces cerevisiae to UV light were investigated using radioactive labelling and two dimensional electrophoresis. UV-irradiation induced the synthesis of various proteins. Among them the analogue of the RecA protein of Escherichia coli (Angulo et al. 1985) and two other polypeptides (34 Kd and 35 Kd, pI 5.8) were observed in all four strains analyzed namely two DNA-repair deficient (rad-) strains: (rad6-1 and pso2-1) and their isogenic wild type RAD+ strains.