Solomon Nwaka

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.

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.

Activity of human Δ5 and Δ6 desaturases on multiple n-3 and n-6 polyunsaturated fatty acids

Yeast co‐expressing human elongase and desaturase genes were used to investigate whether the same desaturase gene encodes an enzyme able to desaturate n‐3 and n‐6 fatty acids with the same or different carbon chain length. The results clearly demonstrated that a single human Δ5 desaturase is active on 20:3n‐6 and 20:4n‐3. Endogenous Δ6 desaturase substrates were generated by providing to the yeast radiolabelled 20:4n‐6 or 20:5n‐3 which, through two sequential elongations, produced 24:4n‐6 and 24:5n‐3, respectively. Overall, our data suggest that a single human Δ6 desaturase is active on 18:2n‐6, 18:3n‐3, 24:4n‐6 and 24:5n‐3.

Induction of neutral trehalase Nth1 by heat and osmotic stress is controlled by STRE elements and Msn2/Msn4 transcription factors: variations of PKA effect during stress and growth

Saccharomyces cerevisiae neutral trehalase, encoded by NTH1, controls trehalose hydrolysis in response to multiple stress conditions, including nutrient limitation. The presence of three stress responsive elements (STREs, CCCCT) in the NTH1 promoter suggested that the transcriptional activator proteins Msn2 and Msn4, as well as the cAMP‐dependent protein kinase (PKA), control the stress‐induced expression of Nth1. Here, we give direct evidence that Msn2/Msn4 and the STREs control the heat‐, osmotic stress‐ and diauxic shift‐dependent induction of Nth1. Disruption of MSN2 and MSN4 abolishes or significantly reduces the heat‐ and NaCl‐induced increases in Nth1 activity and transcription. Stress‐induced increases in activity of a lacZ reporter gene put under control of the NTH1 promoter is nearly absent in the double mutant. In all instances, basal expression is also reduced by about 50%. The trehalose concentration in the msn2 msn4 double mutant increases less during heat stress and drops more slowly during recovery than in wild‐type cells. This shows that Msn2/Msn4‐controlled expression of enzymes of trehalose synthesis and hydrolysis help to maintain trehalose concentration during stress. However, the Msn2/Msn4‐independent mechanism exists for heat control of trehalose metabolism. Site‐directed mutagenesis of the three STREs (CCCCT changed to CATCT) in NTH1 promoter fused to a reporter gene indicates that the relative proximity of STREs to each other is important for the function of NTH1. Elimination of the three STREs abolishes the stress‐induced responses and reduces basal expression by 30%. Contrary to most STRE‐regulated genes, the PKA effect on the induction of NTH1 by heat and sodium chloride is variable. During diauxic growth, NTH1 promoter‐controlled reporter activity strongly increases, as opposed to the previously observed decrease in Nth1 activity, suggesting a tight but opposite control of the enzyme at the transcriptional and post‐translational levels. Apparently, inactive trehalase is accumulated concomitant with the accumulation of trehalose. These results might help to elucidate the general connection between control by STREs, Msn2/Msn4 and PKA and, in particular, how these components play a role in control of trehalose metabolism.

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.

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.

Is thermotolerance of yeast dependent on trehalose accumulation

During heat stress, trehalose concentration increases in yeast cells in parallel to thermotolerance. This parallelism suggested that trehalose mediated thermotolerance. We show in this work that, under certain conditions, trehalose accumulation and increase in thermotolerance do not go in parallel. A mutant deficient in the trehalose‐degrading neutral trehalase shows, after shift from 40°C to 30°C, low thermotolerance in spite of a high trehalose concentration. When glucose is added to stationary yeast cells with high trehalose concentration and high thermotolerance, trehalose concentration decreases while thermotolerance remains high. A mutant deficient in ubiquitin‐conjugating genes, ubc4ubc5, shows during exponential growth a low trehalose concentration, but a high thermotolerance, in contrast to wild‐type cells. Because the ubc4ubc5 mutant synthesizes heat‐shock proteins constitutively, it is proposed that, under these conditions, accumulation of heat‐shock proteins, and not trehalase, mediates thermotolerance.

Corrected sequence of the yeast neutral trehalase-encoding gene (NTH1): biological implications.

We have identified a sequencing error in the neutral trehalase-encoding gene NTH1 [Kopp et al., J. Biol. Chem. 268 (1993) 4766-4774]. This error extends the deduced amino acid (aa) sequence at the N terminus by 58 aa. The biological implications of this include the presence of an additional phosphorylation site, which is believed to regulate trehalose hydrolysis.

The heat shock factor and mitochondrial Hsp70 are necessary for survival of heat shock in Saccharomyces cerevisiae

A heat shock recovery assay on solid medium (Nwaka et al. (1995) J. Biol. Chem. 270, 10193–10198) as well as the classical cell counting method were used to investigate the function of some heat shock proteins in thermotolerance. We show that expression of intact heat shock factor protein (HSF), which regulates the stress induced expression of heat shock proteins (HSPs), is necessary for recovery from heat shock. A HSF1 mutant (hsf1‐m3) which does not induce the expression of some heat shock proteins at heat stress (37–40°C) is defective in recovery after heat shock at 50–52 C compared to a corresponding wild‐type strain in both stationary and exponentially growing cells. Using two temperature sensitive mutants of the mitochondrial Hsp70 (ssc1–2 and ssc1–3) encoded by the SSC1 gene, we show that the ssc1–3 mutant, which has a mutation in the ATPase domain, is defective in recovery after heat shock in contrast to the ssc1–2 mutant, which has a mutation in the peptide binding domain. Different binding capacities for unfolded proteins are shown to be the molecular reason for the observed phenotypes. The thermotolerance defect of the hsf1‐m3 and ssc1–3 mutants is demonstrated for both glucose and glycerol media.