Rosario Lagunas

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.

Catabolite inactivation of the glucose transport system in Saccharomyces cerevisiae

The sugar transport systems of Saccharomyces cerevisiae are irreversibly inactivated when protein synthesis is inhibited. This inactivation is responsible for the drastic decrease in fermentation observed in ammonium-starved yeast and is related to the occurrence of the Pasteur effect in these cells. Our study of the inactivation of the glucose transport system indicates that both the high-affinity and the low-affinity components of this system are inactivated. Inactivation of the high-affinity component evidently requires the utilization of a fermentable substrate by the cells, since inactivation did not occur during carbon starvation, when a fermentable sugar was added to starved cells, inactivation began, when the fermentation inhibitors iodoacetate or arsenate were added in addition to sugars, the inactivation was prevented, when a non-fermentable substrate was added instead of sugars, inactivation was also prevented. The inactivation of the low-affinity component appeared to show similar requirements. It is concluded that the glucose transport system in S. cerevisiae is regulated by a catabolite-inactivation process.

Interferences by sulfhydryl, disulfide reagents and potassium ions on protein determination by Lowry's method

Abstract Sulfhydryl, disulfide reagents, and also potassium ions interfere with the protein determination by Lowrys method. The concentrations of these substances which can be used without serious interference are given. Sulfhydryl and disulfide reagents yield a color with the same absorption spectrum as that caused by proteins. At moderate concentrations this interference can be overcome by running appropriate blanks. Potassium ions produce a white precipitate that can be removed without change of color intensity by centrifuging the reaction mixtures after color development.

Half-life of the plasma membrane ATPase and its activating system in resting yeast cells

The stability of the yeast plasma membrane ATPase and its activating system has been investigated in resting Saccharomyces cerevisiae. The half-life of ATPase in the presence of glucose is about 11 h whereas in the presence of ethanol it is greater than 30 h. In the case of the ATPase activating system half-life values of about 5 and 14 h have been observed, respectively, in the presence of these substrates. These results indicate that, similarly to sugar transport systems, plasma membrane ATPase as well as its activating system are less stable than the bulk of proteins in this organism. The fact that all plasma membrane proteins so far examined show low half-life values suggests that a low stability could be a general characteristic of these proteins.

Internal Trehalose Protects Endocytosis from Inhibition by Ethanol in Saccharomyces cerevisiae

ABSTRACT Endocytosis in Saccharomyces cerevisiae is inhibited by concentrations of ethanol of 2 to 6% (vol/vol), which are lower than concentrations commonly present in its natural habitats. In spite of this inhibition, endocytosis takes place under enological conditions when high concentrations of ethanol are present. Therefore, it seems that yeast has developed some means to circumvent the inhibition. In this work we have investigated this possibility. We identified two stress conditions under which endocytosis was resistant to inhibition by ethanol: fermentation during nitrogen starvation and growth on nonfermentable substrates. Under these conditions, yeast accumulates stress protectors, primarily trehalose and Hsp104, a protein required for yeast to survive ethanol stress. We found the following. (i) The appearance of ethanol resistance was accompanied by trehalose accumulation. (ii) Mutant cells unable to synthesize trehalose also were unable to develop resistance. (iii) Mutant cells that accumulated trehalose during growth on sugars were resistant to ethanol even under this nonstressing condition. (iv) Mutant cells unable to synthesize Hsp104 were able to develop resistance. We conclude that trehalose is the major factor in the protection of endocytosis from ethanol. Our results suggest another important physiological role for trehalose in yeast.

Apparent unbalance between the activities of 6-phosphogluconate and glucose-6-phosphate dehydrogenases in rat liver.

Abstract The ratio of activities of 6-phosphogluconate dehydrogenase/glucose-6-phosphate dehydrogenase measured in liver extracts of rats in lipogenic nutritional conditions is only 0.2, suggesting an apparent physiological unbalance between the two dehydrogenases of the hexosemonophosphate shunt. This potential unbalance is enhanced by the fact that TPNH is a more powerful competitive inhibitor of 6-phosphogluconate dehydrogenase than of glucose-6-phosphate dehydrogenase. Accordingly, a strong activation of 6-phosphogluconate dehydrogenase would be required for efficient functioning of this pathway, unless there is an alternative outlet for 6-phosphogluconate so far unrecognized in animal tissues.

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.

Catabolite inactivation of the yeast maltose transporter is due to proteolysis

The maltose transport capacity of fermenting Saccharomyces cerevisiae rapidly decreases when protein synthesis is impaired. Using polyclonal antibodies against a recombinant maltose transporter‐protein we measured the cellular content of the transporter along this inactivation process. Loss of transport capacity was paralleled by a decrease of cross‐reacting material which suggests degradation of the transporter. We also show that in ammonium‐starved cells the half‐life of the maltose transporter is 1.3 h during catabolism of glucose and > 15 h during catabolism of ethanol.

Identification of two forms of the maltose transport system in Saccharomyces cerevisiae and their regulation by catabolite inactivation

Abstract The maltose transport system of Saccharomyces cerevisiae exists in two forms with K m values of approx. 4 mM and 70 mM, respectively. The V max of the high -K m form is about 4-fold greater than the V max of the low one. A rapid and irreversible inactivation of both forms is detected on protein synthesis impairment. This inactivation is stimulated by the catabolism of fermentable sugars and prevented during ethanol catabolism. It is concluded that both forms of the maltose transport system are regulated by catabolite inactivation.

In vivo activation of the yeast plasma membrane ATPase during nitrogen starvation. Identification of the regulatory domain that controls activation.

Yeast plasma membrane ATPase is activated during nitrogen starvation when a fermentable substrate is present. This activation is due to changes in the V max and it is irreversible, independent of protein synthesis and apparently triggered by a decrease in the intracellular pH. It is shown that the ATPase regulatory domain implicated in the activation by fermentable carbon sources is also implicated in activation by nitrogen starvation and by external acidification.