Activity of human Delta5 and Delta6 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 Delta5 desaturase is active on 20:3n-6 and 20:4n-3. Endogenous Delta6 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 Delta6 desaturase is active on 18:2n-6, 18:3n-3, 24:4n-6 and 24:5n-3.

Contribution of Hsc70 to barotolerance in the yeast Saccharomyces cerevisiae.

The contribution of Hsc70 to barotolerance in logarithmic-phase cells of the HSC70 (ssb1 and ssb2) deletion mutant and in strains expressing the HSC70 gene on either a low- or a high-copy-number plasmid was studied. The deletion-mutant strain had higher thermotolerance and a slightly lower barotolerance than the control strain. The strain that expresses the HSC70 gene in high copy number had a higher barotolerance than the strain that expresses the gene in low copy number. These results suggest that Hsc70 contributes to barotolerance during exponentially growing conditions as does Hsp104 during heat-shock treatment.

Evidence for contribution of neutral trehalase in barotolerance of Saccharomyces cerevisiae.

In yeast, trehalose accumulation and its hydrolysis, which is catalyzed by neutral trehalase, are believed to be important for thermotolerance. We have shown that trehalose is one of the important factors for barotolerance (resistance to hydrostatic pressure); however, nothing is known about the role of neutral trehalase in barotolerance. To estimate the contribution of neutral trehalase in resisting high hydrostatic pressure, we measured the barotolerance of neutral trehalase I and/or neutral trehalase II deletion strains. Under 180 MPa of pressure for 2 h, the neutral trehalase I deletion strain showed higher barotolerance in logarithmic-phase cells and lower barotolerance in stationary-phase cells than the wild-type strain. Introduction of the neutral trehalase I gene (NTH1) into the deletion mutant restored barotolerance defects in stationary-phase cells. Furthermore, we assessed the contribution of neutral trehalase during pressure and recovery conditions by varying the expression of NTH1 or neutral trehalase activity with a galactose-inducible GAL1 promoter with either glucose or galactose. The low barotolerance observed with glucose repression of neutral trehalase from the GAL1 promoter was restored during recovery with galactose induction. Our results suggest that neutral trehalase contributes to barotolerance, especially during recovery.

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.

Opposite roles of trehalase activity in heat-shock recovery and heat-shock survival in Saccharomyces cerevisiae.

A variety of results has been obtained consistent with activation of neutral trehalase in Saccharomyces cerevisiae through direct phosphorylation by cAMP-dependent protein kinase (PKA). A series of neutral trehalase mutant alleles, in which all evolutionarily conserved putative phosphorylation sites were changed into alanine, was tested for activation in vitro (by PKA) and in vivo (by glucose addition). None of the mutations alone affected the activation ratio, whereas all mutations combined resulted in an inactive enzyme. All mutant alleles were expressed to similar levels, as shown by Western blotting. Several of the point mutations significantly lowered the specific activity. Using this series of mutants with different activity levels we show an inverse relationship between trehalase activity and heat-shock survival during glucose-induced trehalose mobilization. This is consistent with a stress-protective function of trehalose. On the other hand, reduction of trehalase activity below a certain threshold level impaired recovery from a sublethal heat shock. This suggests that trehalose breakdown is required for efficient recovery from heat shock, and that the presence of trehalase protein alone is not sufficient for efficient heat-stress recovery.

Evidence for the interplay between trehalose metabolism and Hsp104 in yeast.

Disruption of the HSP104 gene in a mutant which cannot accumulate trehalose during heat shock treatment caused trehalose accumulation (H. Iwahashi, K. Obuchi, S. Fujii, and Y. Komatsu, Lett. Appl. Microbiol 25:43-47, 1997). This implies that Hsp104 affects trehalose metabolism. Thus, we measured the activities of enzymes involved in trehalose metabolism. The activities of trehalose-synthesizing and -hydrolyzing enzymes are low in the HSP104 disruption mutant during heat shock. This data is correlated with intracellular trehalose and glucose levels observed in the HSP104 disruption mutant. These results suggest that during heat shock, Hsp104 contributes to the simultaneous increase in both accumulation and degradation of trehalose.

Stability of neutral trehalase during heat stress in Saccharomyces cerevisiae is dependent on the activity of the catalytic subunits of cAMP-dependent protein kinase, Tpk1 and Tpk2.

In Saccharomyces cerevisiae cAMP-dependent protein kinase (cAPK) is involved in nutrient sensing and growth regulation via the Ras/cAMP pathway. Target enzymes, e.g. neutral trehalase, are activated or inactivated rapidly by cAPK-mediated phosphorylation. In addition, stress-induced transcription of genes of the general stress-response, e.g. HSP12, is negatively regulated via cAPK. We have investigated the effect of low cAPK activity on the stress-induced expression of neutral trehalase Nth1p. For this purpose we used mutants (tpk1tpk2TPK3, tpk1TPK2tpk3 and TPK1tpk2tpk3) with double knockouts of the three TPK genes encoding catalytic subunits of cAPK. It is shown that the tpk1tpk2TPK3 mutant, which has very low cAPK activity, exhibits a heat-stress-induced inactivation of neutral trehalase that is not observed in tpk1TPK2tpk3, TPK1tpk2tpk3 mutants and wild-type cells. However, heat stress induces an increase in NTH1 mRNA in the tpk1tpk2TPK3 mutant. Introduction of a plasmid carrying the TPK1 or TPK2 gene into tpk1tpk2TPK3 cells restores the heat-induced increase of neutral trehalase activity. In vitro and in vivo results suggest that the heat induced inactivation of neutral trehalase is due to a reversible inactivation of Nth1p. Our data indicate that a certain level of phosphorylation is essential for maintenance of neutral trehalase activity during heat shock in S. cerevisiae. Two identical putative cAPK phosphorylation sites have been found in the sequence predicted for the Nth1p. Stabilization and activation of neutral trehalase may be regulated by these sites. Furthermore, our data suggest that the heat-stress-induced transcription of the NTH1 gene is not negatively regulated by cAPK, that the TPK genes have no effect on the glucose repression of the NTH1 gene, and that non-detectable neutral trehalase activity in derepressed tpk1tpk2TPK3 cells is correlated with the reduced thermotolerance observed in this strain, similar to the heat-shock-recovery defect reported for the nth1delta mutant.

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.

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 degrees 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 degrees 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 (Nthlp), 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.

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 degrees C) is defective in recovery after heat shock at 50-52 degrees 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.

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 YBRO106 (NTH2). We have analyzed the role of the acid trehalase in trehalose hydrolysis using the ATH1 deletion mutant (delta ath1) of Saccharomyces cerevisiae [M. Destruelle et al. (1995) Yeast 11, 1015-10251 deficient in acid trehalase activity under various nutritional conditions. In contrast to wild-type and a mutant deficient in the neutral trehalase (delta nth1), the delta ath1 mutant does not grow on trehalose as a carbon source. Experiments with diploid strains heterozygous for delta ath1 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.

Expression and function of the trehalase genes NTH1 and YBR0106 in Saccharomyces cerevisiae.

The biological function of the trehalose-degrading yeast enzyme neutral trehalase consists of the control of the concentration of trehalose, which is assumed to play a role in thermotolerance, in germination of spores, and in other life functions of yeast. Resequencing of the neutral trehalase gene NTH1 on chromosome IV resulted in the observation of two possible start codons (Kopp, M., Nwaka, S., and Holzer, H. (1994) Gene (Amst.) 150, 403-404). We show here that only the most upstream start codon which initiates translation of the longest possible ORF is used for expression of NTH1 in vivo. A gene with 77% identity with NTH1, YBR0106, which was discovered during sequencing of chromosome II (Wolfe, K. H., and Lohan, A. J. E. (1994) Yeast 10, S41-S46), is shown here to be expressed into mRNA. Experiments with a mutant disrupted in the YBR0106 ORF showed, in contrast to a NTH1 deletion mutant, no changes in trehalase activity and in trehalose concentration. However, similar to the NTH1 gene a requirement of the intact YBR0106 gene for thermotolerance is demonstrated in experiments with the respective mutants. This indicates that the products of the likely duplicated YBR0106 gene and the NTH1 gene serve a heat shock protein function. In case of the YBR0106 gene, this is the only phenotypic feature found at present.

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.

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.

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 degrees C to 30 degrees 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 trehalose [corrected], mediates thermotolerance.