Nitrogen-source-induced activation of neutral trehalase in Schizosaccharomyces pombe and Pachysolen tannophilus: role of cAMP as second messenger

Abstract Resting cells of the fission yeast Schizosaccharomyces pombe , suspended in buffer with glucose, responded to the addition of asparagine by increasing trehalase activity. This response was preceded by a peak in cAMP concentration. The addition of the nitrogen source to resting cells, devoid of the catalytic subunit of cAMP-dependent protein kinase, produced the transient increase in cAMP but did not promote any change in trehalase activity. In the budding yeast Pachysolen iannophilus , the activation of trehalase by nitrogen source was also accompanied by a sharp peak in cAMP. These results suggest that in the two yeasts cAMP acts as a second messenger in the transduction of the nitrogen-source-induced signal causing the activation of trehalase.     

Characterization of different forms of yeast acid trehalase in the secretory pathway

The biosynthesis and processing of the vacuolar (lysosomal) acid trehalase (molecular mass about 220 kDa) was followed in vivo using mutants conditionally defective in the secretory pathway. A precursor of 41 kDa was found in sec61 mutant cells deficient in translocation of secretory protein precursors into the lumen of the endoplasmic reticulum. Endoglycosidase H and N-glycosidase F treatment of purified acid trehalase in vitro resulted in a 41 kDa band, indicating that the precursor form found in sec61 mutant cells corresponds to the carbohydrate-free form of the enzyme. sec 18 mutant cells, blocked in the delivery of secretory proteins from the endoplasmic reticulum to the Golgi body accumulate a form with a molecular mass of 76 kDa which probably corresponds to a partially glycosylated precursor of the mature acid trehalase. This precursor partially disappears in favour of the appearance of a higher molecular weight component of 180 kDa in sec7 mutants which are blocked in the delivery step of secretory proteins from the Golgi body to the vacuole. In wild-type cells the fully glycosylated mature form of acid trehalase of about 220 kDa was observed accompanied by some 180 kDa and 76 kDa material.     

Heat-shock response in a yeast tps1 mutant deficient in trehalose synthesis

Exponential cells of the Saccharomyces cerevisiae tps1 mutant underwent a rapid loss of viability upon a non-lethal heat exposure (from 28 to 42°C). However, a further more severe heat stress (52.5°C 5 min) induced an increase in the fraction of viable cells. This mutant can not synthesize trehalose either at 28° C or at 42°C due to the lack of a functional trehalose-6P synthase complex. In control experiments carried out with the wild-type W303-1 B, heat-stressed exponential phase cultures grown on YPgal at 28°C acquired thermotolerance to a higher extent than identical cultures grown on YPD, although in both cultures the level of stored trehalose was negligible. These data suggest that the bulk of trehalose accumulated in yeast upon mild heat treaments is not sufficient to account for the acquisition of thermotolerance.     

Fate and effects of the trehalase inhibitor trehazolin in the migratory locust (Locusta migratoria)

Trehalose is the main haemolymph sugar in many insect species. To be utilized trehalose must be hydrolysed into its glucose units by trehalase (EC 3.2.1.28). Inhibitors of trehalase have attracted interest as possible pesticides and tools for studying the regulation of trehalose metabolism in insects. To make full use of these inhibitors requires knowledge of their fate and effects in vivo. To this end we have measured trehazolin in locusts using a method based on the specific inhibition of a trehalase preparation. After injection of 20 μg, trehazolin decreased in haemolymph with a half-life of 2.6 days and after 10 days almost 95% had disappeared. Trehazolin did not reach the intracellular water space of locust tissues, but appeared with full inhibitory potency in locust faeces, suggesting that it was not metabolized, but quantitatively eliminated via the gut. Haemolymph trehalose increased transiently upon trehazolin injection, it was maximal after 3 days, then decreased and reached control level after 10 days. Inhibition of flight muscle trehalase by trehazolin was prolonged and still conspicuous 21 days post injection, suggesting that trehazolin inhibits trehalase activity irreversibly in vivo and that recovery requires de novo enzyme synthesis.     

Saccharomyces cerevisiae strains from traditional fermentations of Brazilian cachaça: trehalose metabolism, heat and ethanol resistance

Nine indigenous cachaça Saccharomyces cerevisiae strains and one wine strain were compared for their trehalose metabolism characteristics under non-lethal (40°C) and lethal (52°C) heat shock, ethanol shock and combined heat and ethanol stresses. The yeast protection mechanism was studied through trehalose concentration, neutral trehalase activity and expression of heat shock proteins Hsp70 and Hsp104. All isolates were able to accumulate trehalose and activate neutral trehalase under stress conditions. No correlation was found between trehalose levels and neutral trehalase activity under heat or ethanol shock. However, when these stresses were combined, a positive relationship was found. After pre-treatment at 40°C for 60 min, and heat shock at 52°C for 8 min, eight strains maintained their trehalose levels and nine strains improved their resistance against lethal heat shock. Among the investigated stresses, heat treatment induced the highest level of trehalose and combined heat and ethanol stresses activated the neutral trehalase most effectively. Hsp70 and Hsp104 were expressed by all strains at 40°C and all of them survived this temperature although a decrease in cell viability was observed at 52°C. The stress imposed by more than 5% ethanol (v/v) represented the best condition to differentiate strains based on trehalose levels and neutral trehalase activity. The investigated S. cerevisiae strains exhibited different characteristics of trehalose metabolism, which could be an important tool to select strains for the cachaça fermentation process.     

Purification and properties of trehalase in Frankia ArI3

Trehalase was purified from cultures of Frankia strain ArI3 grown on media with or without NH₄Cl. The purified enzyme was specific for trehalose, exhibited a broad pH optimum of pH 4.5 to 5.3 and had a K _m for trehalose of 4.2 mM. The trehalase was inhibited in vitro completely by sucrose, glucose and mannose and partially by mannitol and sorbitol. In addition to the specific trehalase, a mixture of non-specific - and -glucosidases which exhibited some activity with ,-trehalose as a substrate were also partially purified in Frankia extracts made from nitrogen-fixing cells. These enzymes were not detected in the purifications of crude extracts made from non-nitrogen-fixing cells (grown on media supplemented with NH₄Cl). Trehalase activity in crude extracts increased over time when cells were induced to fix nitrogen, and the maximum specific activity of trehalase from nitrogen-fixing cultures was 4 times the maximum activity from non-fixing cultures. Trehalase activity was also examined in crude extracts made from Frankia vesicle clusters isolated from Alnus rubra nitrogen-fixing nodules infected with ArI3. The maximum activity of trehalase in these clusters was 6–7 times greater than in the nitrogenfixing pure cultures of ArI3 and 26–33 times greater than the non-fixing pure cultures.Abbreviations pcv packed cell volume - DTE dithioerythritol - PMSF phenylmethylsulphonylfluoride - EDTA sodium ethylenediaminetetraacetate     

Absence of glucose-induced cAMP signaling in the Saccharomyces cerevisiae mutants cat1 and cat3 which are deficient in derepression of glucose-repressible proteins

Addition of glucose to derepressed cells of the yeast Saccharomyces cerevisiae induces a transient, specific cAMP signal. Intracellular acidification in these cells, as caused by addition of protonophores like 2,4-dinitrophenol (DNP) causes a large, lasting increase in the cAMP level. The effect of glucose and DNP was investigated in glucose-repressed wild type cells and in cells of two mutants which are deficient in derepression of glucose-repressible proteins, cat1 and cat3. Addition of glucose to cells of the cat3 mutant caused a transient increase in the cAMP level whereas cells of the cat1 mutant and in most cases also repressed wild type cells did not respond to glucose addition with a cAMP increase. The glucose-induced cAMP increase in cat3 cells and the cAMP increase occasionally present in repressed wild type cells however could be prevented completely by addition of a very low level of glucose in advance. In derepressed wild type cells this does not prevent the specific glucose-induced cAMP signal at all. These results indicate that repressed cells do not show a true glucose-induced cAMP signal. When DNP was added to glucose-repressed wild type cells or to cells of the cat1 and cat3 mutants no cAMP increase was observed. Addition of a very low level of glucose before the DNP restored the cAMP increase which points to lack of ATP as the cause for the absence of the DNP effect. These data show that intracellular acidification is able to enhance the cAMP level in repressed cells. The glucose-induced artefactual increase occasionally observed in repressed cells is probably caused by the fact that their low intracellular pH is only restored after the ATP level has increased to such an extent that it is no longer limiting for cAMP synthesis. It is unclear why the artefactual increases are not always observed. Measurement of glucose- and DNP-induced activation of trehalase confirmed the physiological validity of the changes observed in the cAMP level. Our results are consistent with the idea that the glucose-induced signaling pathway contains a glucose-repressible protein and that the protein is located before the point where intracellular acidification triggers activation of the pathway.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DNP 2,4-dinitrophenol - Mes 4-morpholineethanesulfonic acid     

Properties of a germination mutant of Phycomyces blakesleeanus

Spores of the Phycomyces blakesleeanus strain S440 germinated only for some 4 to 7% when activated with a heat treatment or with ammonium acetate. Contrary to wild type spores, they showed no increase in trehalase activity during or after the activating treatment. This was not due to a variant trehalase or a defective protein kinase but rather to the absence of an increase in cellular cyclic AMP which normally occurs in the wild type. Phosphodiesterase activity in the mutant was comparable to wild type activity and in both strains phosphodiesterase was inactivated by a heat treatment. The phosphodiesterase inhibitor 1-isobutyl, 3-methyl xanthine caused germination and trehalase activation in the wild type but not in the mutant. The results corroborate the importance of cyclic AMP in the breaking of dormancy and the activation of trehalase in this fungus.     

Purification and properties of trehalase from monkey small intestine

Brush border membrane trehalase was purified from monkey small intestine by a procedure which includes solubilisation by Triton X-100, ammonium sulphate fractionation, and chromatography on DE-52 and hydroxyapatite. The purified enzyme had a specific activity of 11 units/mg protein and was purified 140-fold. The enzyme showed a single protein band on Polyacrylamide gel electrophoresis. It had aK ^m value of 17.4 mM for trehalose and a V_max of 1.33 units. Sucrose and Tris acted as competitive inhibitors of the enzyme.