Helmut Holzer

MOLECULAR BIOLOGY OF TREHALOSE AND THE TREHALASES IN THE YEAST SACCHAROMYCES CEREVISIAE

The present state of knowledge of the role of trehalose and trehalose hydrolysis catalyzed by trehalase (EC 3.2.1.28) in the yeast Saccharomyces cerevisiae is reviewed. Trehalose is believed to function as a storage carbohydrate because its concentration is high during nutrient limitations and in resting cells. It is also believed to function as a stress metabolite because its concentration increases during certain adverse environmental conditions, such as heat and toxic chemicals. The exact way trehalose may perform the stress function is not understood, and conditions exist under which trehalose accumulation and tolerance to certain stress situations cannot be correlated. Three trehalases have been described in S. cerevisiae: 1) the cytosolic neutral trehalase encoded by the NTH1 gene, and regulated by cAMP-dependent phosphorylation process, nutrients, and temperature; 2) the vacuolar acid trehalase encoded by the ATH1 gene, and regulated by nutrients; and 3) a putative trehalase Nth1p encoded by the NTH2 gene (homolog of the NTH1 gene) and regulated by nutrients and temperature. The neutral trehalase is responsible for intracellular hydrolysis of trehalose, in contrast to the acid trehalase, which is responsible for utilization of extracellular trehalose. The role of the putative trehalase Nth2p in trehalose metabolism is not known. The NTH1 and NTH2 genes are required for recovery of cells after heat shock at 50 degrees C, consistent with their heat inducibility and sequence similarity. Other stressors, such as toxic chemicals, also induce the expression of these genes. We therefore propose that the NTH1 and NTH2 genes have stress-related function and the gene products may be called stress proteins. Whether the stress function of the trehalase genes is linked to trehalose is not clear, and possible mechanisms of stress protective function of the trehalases are discussed.

Regulation of fructose-1,6-bisphosphatase in yeast by phosphorylation/dephosphorylation.

Abstract Fructose-1,6-bisphosphatase was precipitated with purified rabbit antiserum from extracts of 32 P-orthophosphate labelled yeast cells, submitted to SDS polyacrylamide gel electrophoresis, extracted from the gels and counted for radioactivity due to 32 P incorporation. Fructose-1,6-bisphosphatase from glucose starved yeast cells contained a very low 32 P label. During 3 min treatment of the glucose starved cells with glucose the 32 P-label increased drastically. Subsequent incubation of the cells in an acetate containing, glucose-free medium led to a label which was again low. Analysis for phosphorylated amino acids in the immunpprecipitated fructose-1,6-bisphosphatase protein from the 3 min glucose-inactivated cells exhibited phospho-serine as the only labelled phosphoamino acid. These data demonstrate a phosphorylation of a serine residue of fructose-1,6-bisphosphatase during this 3 min glucose treatment of glucose starved cells. A concomitant about 60 % inactivation of the enzyme had been shown to occur. The data in addition show a release of the esterified phosphate from the enzyme upon incubation of cells in a glucose-free medium, a treatment which leads to peactivation of enzyme activity. A protein kinase and a protein phosphatase catalysing this metabolic interconversion of fructose-1,6-bisphosphatase are postulated. It is assumed that metabolites accumulating after the addition of glucose exert a positive effect on the kinase activity and/or have a negative effect on the phosphatase activity. A role of the enzymic phosphorylation of fructose-1,6-bisphosphatase in the initiation of complete proteolysis of the enzyme during “catabolite inactivation” is discussed.

Phenotypic features of trehalase mutants in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, some studies have shown that trehalose and its hydrolysis may play an important physiological role during the life cycle of the cell. Recently, other studies demonstrated a close correlation between trehalose levels and tolerance to heat stress, suggesting that trehalose may be a protectant which contributes to thermotolerance. We had reported lack of correlation between trehalose accumulation and increase in thermotolerance under certain conditions, suggesting that trehalose may not mediate thermotolerance [Nwaka, S., et al. (1994) FEBS Lett. 344, 225–228]. Using mutants of the trehalase genes, NTH1 and YBR0106, we have demonstrated the necessity of these genes in recovery of yeast cells after heat shock, suggesting a role of these genes in thermotolerance (Nwaka, S., Kopp, M., and Holzer, H., submitted for publication). In the present paper, we have analysed the expression of the trehalase genes under heat stress conditions and present genetic evidence for the ‘poor‐heat‐shock‐recovery’ phenotype associated with NTH1 and YBR0106 mutants. Furthermore, we show a growth defect of neutral and acid trehalase‐deficient mutants during transition from glucose to glycerol, which is probably related to the ‘poor‐heat‐shock‐recovery’ phenomenon.

Mechanism of the enzymatic inactivation of glutamine synthetase from E. coli.

Abstract The previously described “glutamine synthetatse inactivating enzyme” from E. coli catalyzes the incorporation of 14C into glutamine synthetase in presence of 14C-labelled ATP, Mg2+ and glutamine. A comparison of glutamine with other stimulating effectors (methionine, asparagine) in the inactivating system and in the 14C incorporating system shows parallel effects in both reactions. Furthermore the ratio of 14C-incorporation and glutamine synthetase inactivation is constant during the course of the reaction. It is concluded that adenylylation is the mechanism of the inactivation of glutamine synthethase.

Isoenzyme der malatdehydrogenase und ihre regulation in Saccharomyces cerevisiae

Abstract 1. 1. From Saccharomyces cerevisiae , incubated on a glucose-free medium with acetate as the only carbon source, two different malate dehydrogenases ( l -malate: NAD + oxidoreductase, EC 1.1.1.37) have been isolated by DEAE-cellulose ion-exchange chromatography. One of these enzymes was only found in the mitochondria and is called enzyme A or m-malate dehydrogenase; the other enzyme was found in the extramitochondrial c-space and is called enzyme B or c-malate dehydrogenase. At present it cannot be decided whether m-malate dehydrogenase also exists in the c-space or leaks when the mitochondria are injured. 2. 2. The reaction velocity plotted against the concentration of oxaloacetic acid showed a characteristic substrate inhibition in the case of m-malate dehydrogenase In contrast, c-malate dehydrogenase showed no substrate inhibition. This difference corresponds to the behaviour of m-malate dehydrogenase and c-malate dehydrogenase from liver. 3. 3. In yeast grown on glucose only m-malate dehydrogenase could be found, but after incubating the cells on acetate as the sole carbon source, both m-malate dehydrogenase and c-malate dehydrogenase were found. In reference to earlier experiments concerning the regulation of malate dehydrogenase activity in yeast, it is concluded that a repression of c-malate dehydrogenase synthesis by glucose occurs. This regulating mechanism is useful for the cell, because in the glycoxylate cycle c-malate dehydrogenase participates in the gluconeogenesis from acetate or ethanol. This enzyme is not necessary when glucose is in the medium.

Cyclic AMP-dependent phosphorylation of fructose-1,6-bisphosphatase in yeast

Summary Previous in vivo experiments have shown that simultaneously with the glucose-induced inactivation of yeast fructose-1,6-bisphosphatase a phosphorylation of serine residues of the enzyme occurs. The inactivation of fructose-1,6-bisphosphatase dependent on ATP, Mg ++ and cyclic AMP is now demonstrated in a cell-free yeast extract suggesting the existence of a cyclic AMP-dependent fructose-1,6-bisphosphatase kinase. When glucose is added to intact yeast cells within 30 sec the cyclic AMP concentration increases from 0.7 to 3 nmol per g wet weight. This observation suggests that upon addition of glucose to yeast cells cyclic AMP functions as the mediating signal for the protein kinase catalyzed phosphorylation of fructose-1,6-bisphosphatase. The levels of glucose-6-phosphate and fructose-6-phosphate also show a transient rise with a maximum 15 to 30 sec after the addition of glucose to yeast cells, i.e. shortly before the observed increase of the cyclic AMP concentration. Thus, the sugar phosphates may function as allosteric effectors which stimulate adenylate cyclase and/or inhibit cyclic AMP phosphodiesterase thereby leading to a transient rise of the cyclic AMP levels, which in turn may be the signal for the phosphorylation of fructose-1,6-bisphosphatase.

Neutral trehalase Nth1p of Saccharomyces cerevisiae encoded by the NTH1 gene is a multiple stress responsive protein.

We have shown previously that expression of the NTH1 gene is increased at heat stress (40°C) both at the mRNA and enzymatic activity levels. This increased expression was correlated to the requirement of the NTH1 gene for recovery after heat shock at 50°C and the presence of stress responsive elements STRE (CCCCT) 3 times in its promoter region [S. Nwaka et al., FEBS Lett. 360 (1995) 286–290; S. Nwaka et al., J. Biol. Chem. 270 (1995) 10193–10198]. We show here that expression of the NTH1 gene and its product, neutral trehalase (Nth1p), are also induced by other stressors such as H2O2, CuSO4, NaAsO2, and cycloheximide (CHX). Heat‐induced expression of the NTH1 gene is shown to be accompanied by accumulation of trehalose. In contrast, the chemical stressors which also induce the expression of NTH1 did not lead to accumulation of trehalose under similar conditions. Our data suggest that: (1) heat‐ and chemical stress‐induced expression of neutral trehalase is largely due to de novo protein synthesis, and (2) different mechanisms may control the heat‐ and chemical stress‐induced expression of NTH1 at the transcriptional level. Participation of neutral trehalase (Nth1p) in multiple stress response dependent and independent on trehalose is discussed.

Rapid reversible inactivation of fructose-1,6-bisphosphatase in Saccharomyces cerivisiae by glucose

Inactivation of -90% of the activity of fructose1,6_bisphosphatase in 1 h after addition of glucose or fructose to acetate-grown yeast cells has been mentioned [l] and described with experimental data [2]. The inactivation can be reversed by transfer of the sugar-treated inactivated cells to an acetate or ethanol containing growth medium [2]. This reappearance of fructose-l ,6-bisphosphatase activity is prevented by addition of cycloheximide, a potent inhibitor of protein synthesis in yeast, and is therefore dependent on de novo protein synthesis [2]. In the course of our studies on catabolite inactivation of gluconeogenic enzymes in Saccharomyces cerevisiae [ 3,4] a rapid disappearance of 50-70% of fructose-l ,6-bisphosphatase activity in 3 min after addition of glucose was observed, which was followed by a much slower disappearance of the remaining activity [5]. In contrast to the ‘long term’ inactivation described [2], the rapid inactivation observed after 3 min incubation with glucose is reversible after transfer of the cells to a sugar-free, acetate containing medium also in the presence of cycloheximide. Thus, in contrast to the situation after ‘long term’ inactivation reactivation after ‘short term’ inactivation is independent on the novo protein synthesis. The possibility of a rapid covalent interconversion of fructose-l ,6-bisphosphatase (for definition of ‘interconversion’ see [6]), preceeding the irreversible inactivation which can only be restored by de novo protein synthesis, is considered as an explanation for these observations.

Intracellular proteinases in microorganisms.

Publisher Summary One of the main functions of intracellular proteinases lies in their participation in protein turnover by the hydrolytic degradation of proteins. The rates of synthesis and degradation differ for the various enzymes and groups of enzymes. Moreover, they are influenced by many variables, such as nutritional conditions, growth phase, processes of differentiation. Protein synthesis is regulated at the level of transcription and translation by positive and negative control. The degradation of proteins is generally assumed to occur through the combined action of proteinases and peptidases, yielding amino acids after complete hydrolysis. On the other hand, it is possible that “limited proteolysis” leads to an accumulation of distinct macromolecular products that are either inactive or different in their catalytic properties from their uncleaved precursors. Microbial proteinases are commonly divided into intracellular and extracellular enzymes. Extracellular enzymes usually occur in the active state in the growth medium and are quite stable. Many of them are even available in large quantities allowing their application to medicine and industry.

Deletion of the ATH1 gene in Saccharomyces cerevisiae prevents growth on trehalose

The biological function of the yeast trehalases (EC 3.2.1.28) consists of down‐regulation of the concentration of trehalose via glucose formation by trehalose hydrolysis. While it is generally accepted that the cytosolic neutral trehalase (encoded by the NTH1 gene) is responsible for trehalose hydrolysis in intact cells, very little is known about a role of the vacuolar acid trehalase and the product of the recently described neutral trehalase gene YBR0106 (NTH2). We have analyzed the role of the acid trehalase in trehalose hydrolysis using the ATH1 deletion mutant (Δathl) of Saccharomyces cerevisiae [M. Destruelle et al. (1995) Yeast 11, 1015–1025] deficient in acid trehalase activity under various nutritional conditions. In contrast to wild‐type and a mutant deficient in the neutral trehalase (Δathl ), the Aathl mutant does not grow on trehalose as a carbon source. Experiments with diploid strains heterozygous for Δathl show a gene dosage effect for the ATH1 gene for growth on trehalose. The need for acid trehalase for growth on trehalose is supported by the finding that acid trehalase activity is induced during exponential growth of cells on trehalose while no such induction is measurable during growth on glucose. Our results show that the vacuolar acid trehalase Ath1p is necessary for the phenotype of growth on trehalose, i.e. trehalose utilization, in contrast to cytosolic neutral trehalase Nth1p which is necessary for intracellular degradation of trehalose. For explanation of the need for vacuolar acid trehalase and not cytosolic neutral trehalase for growth on trehalose, the participation of endocytosis for uptake of trehalose from medium to the vacuoles is discussed.