Juana M. Gancedo

Fructose-1,6-diphosphatase, phosphofructokinase and glucose-6-phosphate dehydrogenase from fermenting and non fermenting yeasts

Summary1.Levels of phosphofructokinase, glucose-6-phosphate dehydrogenase and fructose-1,6-diphosphatase activities have been compared in different yeasts belonging to glucose fermenting and non-fermenting groups grown in different conditions.2.Phosphofructokinase was present in all the fermentative species tested. On the contrary its level was not measurable in any of the aerobic yeasts tested with the exception of Pichia species.3.No significant variations were observed in the values of glucose-6-phosphate dehydrogenase from the two groups of yeasts.4.The synthesis of fructose-1,6-diphosphatase was repressed in both groups, by growth in sugar carbon sources. However, a remarkable difference in the sensitivity of the fructose-1,6-diphosphatase from both groups towards inhibition by AMP was observed. The enzyme from all fermentative yeasts tested showed a strong inhibition by AMP (1 mM producing about 80% inhibition) while the enzyme from aerobic yeasts showed different responses, inhibition ranging from 10% in Rhodotorula and Sporobolomyces, to 90% in Pichia.

Trehalose-6-phosphate, a new regulator of yeast glycolysis that inhibits hexokinases

Trehalose‐6‐phosphate (P) competitively inhibited the hexokinases from Saccharomyces cerevisiae. The strongest inhibition was observed upon hexokinase II, with a K i, of 40 μM, while in the case of hexokinase I the K i was 200 μM. Glucokinase was not inhibited by trehalose‐6‐P up to 5 mM. This inhibition appears to have physiological significance, since the intracellular levels of trehalose‐6‐P were about 0.2 mM. Hexokinases from other organisms were also inhibited, while glucokinases were unaffected. The hexokinase from the yeast, Yarrowia lipolytica, was particularly sensitive to the inhibition by trehalose‐6‐P: when assayed with 2 mM fructose an apparent K i, of 5 μM was calculated. Two S. cerevisiae mutants with abnormal levels of trehalose‐6‐P exhibited defects in glucose metabolism. It is concluded that trehalose‐6‐P plays an important role in the regulation of the first steps of yeast glycolysis, mainly through the inhibition of hexokinase II.

The early steps of glucose signalling in yeast

In the presence of glucose, yeast undergoes an important remodelling of its metabolism. There are changes in the concentration of intracellular metabolites and in the stability of proteins and mRNAs; modifications occur in the activity of enzymes as well as in the rate of transcription of a large number of genes, some of the genes being induced while others are repressed. Diverse combinations of input signals are required for glucose regulation of gene expression and of other cellular processes. This review focuses on the early elements in glucose signalling and discusses their relevance for the regulation of specific processes. Glucose sensing involves the plasma membrane proteins Snf3, Rgt2 and Gpr1 and the glucose-phosphorylating enzyme Hxk2, as well as other regulatory elements whose functions are still incompletely understood. The similarities and differences in the way in which yeasts and mammalian cells respond to glucose are also examined. It is shown that in Saccharomyces cerevisiae, sensing systems for other nutrients share some of the characteristics of the glucose-sensing pathways.

Concentrations of intermediary metabolites in yeast

Summary The concentrations of some intermediary metabolites in yeast in different metabolic situations have been tabulated from data available in the literature. A critical examination of the extraction procedures and their influence on the values obtained is performed.

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.

Biological roles of cAMP: variations on a theme in the different kingdoms of life

Cyclic AMP (cAMP) plays a key regulatory role in most types of cells; however, the pathways controlled by cAMP may present important differences between organisms and between tissues within a specific organism. Changes in cAMP levels are caused by multiple triggers, most affecting adenylyl cyclases, the enzymes that synthesize cAMP. Adenylyl cyclases form a large and diverse family including soluble forms and others with one or more transmembrane domains. Regulatory mechanisms for the soluble adenylyl cyclases involve either interaction with diverse proteins, as happens in Escherichia coli or yeasts, or with calcium or bicarbonate ions, as occurs in mammalian cells. The transmembrane cyclases can be regulated by a variety of proteins, among which the α subunit and the βγ complex from G proteins coupled to membrane receptors are prominent. cAMP levels also are controlled by the activity of phosphodiesterases, enzymes that hydrolyze cAMP. Phosphodiesterases can be regulated by cAMP, cGMP or calcium‐calmodulin or by phosphorylation by different protein kinases. Regulation through cAMP depends on its binding to diverse proteins, its proximal targets, this in turn causing changes in a variety of distal targets. Specifically, binding of cAMP to regulatory subunits of cAMP‐dependent protein kinases (PKAs) affects the activity of substrates of PKA, binding to exchange proteins directly activated by cAMP (Epac) regulates small GTPases, binding to transcription factors such as the cAMP receptor protein (CRP) or the virulence factor regulator (Vfr) modifies the rate of transcription of certain genes, while cAMP binding to ion channels modulates their activity directly. Further studies on cAMP signalling will have important implications, not only for advancing fundamental knowledge but also for identifying targets for the development of new therapeutic agents.

Contribution of the pentose-phosphate pathway to glucose metabolism in Saccharomyces cerevisiae: A critical analysis on the use of labelled glucose

Abstract The validity of the procedure of Katz and Wood for the determination of the contribution of the pentose-phosphate pathway (PPP) to glucose metabolism in yeast is established, and it is shown that a number of methods are liable to yield erroneous results. In yeast growing on glucose with NH4+ as nitrogen source, the pentose-phosphate pathway was found to account for 2.5% of the total metabolism of glucose, while in yeast growing on an enriched medium where NH4+ had been replaced by Difco yeast extract the contribution was lowered to 0.9%. These results confirm that a major role for this pathway is to supply NADPH for biosynthetic reactions.

Pseudohyphal growth is induced in Saccharomyces cerevisiae by a combination of stress and cAMP signalling.

In Saccharomyces cerevisiae pseudohyphae formation may be triggered by nitrogen deprivation and is stimulated by cAMP. It was observed that even in a medium with an adequate nitrogen supply, cAMP can induce pseudohyphal growth when S. cerevisiae uses ethanol as carbon source. This led us to investigate the effects of the carbon source and of a variety of stresses on yeast morphology. Pseudohyphae formation and invasive growth were observed in a rich medium (YP) with poor carbon sources such as lactate or ethanol. External cAMP was required for the morphogenetic transition in one genetic background, but was dispensable in strain Σ1278b which has been shown to have an overactive Ras2/cAMP pathway. Pseudohyphal growth and invasiveness also took place in YPD plates when the yeast was subjected to different stresses: a mild heat-stress (37 °C), an osmotic stress (1 m NACl), or addition of compounds which affect the lipid bilayer organization of the cell membrane (aliphatic alcohols at 2%) or alter the glucan structure of the cell wall (Congo red). We conclude that pseudohyphal growth is a physiological response not only to starvation but also to a stressful environment; it appears to require the coordinate action of a MAP kinase cascade and a cAMP-dependent pathway.

Regulatory regions in the yeast FBP1 and PCK1 genes

By deletion analysis of the fusion genes FBP1‐lacZ and PCK1‐lacZ we have identified a number of strong regulatory regions in the genes FBP1 and PCK1 which encode fructose‐1,6‐bisphosphatase and phosphoenolpyruvate carboxykinase. Lack of expression of β‐galactosidase in fusions lacking sequences from the coding regions suggests the existence of downstream activating elements. Both promoters have several UAS and URS regions as well as sites implicated in catabolite repression. We have found in both genes consensus sequences for the binding of the same regulatory proteins, such as yAP1, MIG1 or the complex HAP2/HAP3/HAP4. Neither deletion nor overexpression of the MIG1 gene affected the regulated expression of the FBP1 or PCK1 genes.

Regions in the promoter of the yeast FBP1 gene implicated in catabolite repression may bind the product of the regulatory gene MIG1

We have identified in the promoter of the yeast FBP1 gene two sites able to bind nuclear proteins. These sites have a nucleotide sequence strongly similar to that of sites which bind the regulatory protein MIG1 in the promoters of GAL4 and SUC2. Deletions performed in the FBP1 promoter showed that one of the sites contributes to catabolite repression of this gene. In the same promoter, another region was identified with a strong effect on the catabolite repression of FBP1. In this region a sequence similar to the consensus for the binding site of the MIG1 protein was also present.