Dihydroxyacetone Kinases in Saccharomyces cerevisiae Are Involved in Detoxification of Dihydroxyacetone*

Mikael Molin, Joakim NorbeckDagger , and Anders Blomberg§

From the Department of Cell and Molecular Biology-Microbiology, Göteborg University, Lundberg Laboratory, Medicinaregatan 9c, 413 90 Göteborg, Sweden and the Dagger  Swegene Proteomics Core Facility in Göteborg, Göteborg University, Medicinaregatan 7B, 413 90 Göteborg, Sweden

Received for publication, March 28, 2002, and in revised form, September 13, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The genes YML070W/DAK1 and YFL053W/DAK2 in the yeast Saccharomyces cerevisiae were characterized by a combined genetic and biochemical approach that firmly functionally classified their encoded proteins as dihydroxyacetone kinases (DAKs), an enzyme present in most organisms. The kinetic properties of the two isoforms were similar, exhibiting Km(DHA) of 22 and 5 µM and Km(ATP) of 0.5 and 0.1 mM for Dak1p and Dak2p, respectively. We furthermore show that their substrate, dihydroxyacetone (DHA), is toxic to yeast cells and that the detoxification is dependent on functional DAK. The importance of DAK was clearly apparent for cells where both isogenes were deleted (dak1Delta dak2Delta ), since this strain was highly sensitive to DHA. In the opposite case, overexpression of either DAK1 or DAK2 made the dak1Delta dak2Delta highly resistant to DHA. In fact, overexpression of either DAK provided cells with the capacity to grow efficiently on DHA as the only carbon and energy source, with a generation time of about 5 h. The DHA toxicity was shown to be strongly dependent on the carbon and energy source utilized, since glucose efficiently suppresses the lethality, whereas galactose or ethanol did so to a much lesser extent. However, this suppression was found not to be explained by differences in DHA uptake, since uptake kinetics revealed a simple diffusion mechanism with similar capacity independent of carbon source. Salt addition strongly aggravated the DHA toxicity, independent of carbon source. Furthermore, the DHA toxicity was not linked to the presence of oxygen or to the known harmful agents methylglyoxal and formaldehyde. It is proposed that detoxification of DHA may be a vital part of the physiological response during diverse stress conditions in many species.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Stress-induced mechanisms are fundamental to cellular physiology. This is especially true for microbes, which frequently encounter drastic environmental changes. An understanding of these stress mechanisms will provide information about both the metabolic features activated under nonoptimal growth conditions as well as novel insights into mechanisms relevant to basal physiology. Cellular effects from environmental stress currently represent a vividly studied field, and our understanding of quite a number of stress adaptation mechanisms has lately been substantially deepened (1-4). Many of the gene responders characterized have an easily understandable functional role that is more or less specific for the type of stress applied, which can be exemplified by the glutaredoxin gene GLR1 for adaptation to oxidative stress conditions (2) or GPD1, which has been shown to be instrumental for survival and growth in saline media (5). However, for many of these stress genes, the cellular importance appears to be of a more general nature, since their expression is reported regulated under many different stresses (6, 7). This indicates that the processes in which the encoded proteins participate apparently will be of general importance during diverse cellular disturbances.

One example of a gene that appears to be involved in a metabolic pathway central to stress adaptation in general is the putative dihydroxyacetone kinase (DAK)1 gene YML070W/DAK1, since this gene is up-regulated under a large number of environmental conditions. This protein was first identified as a stress responder in the yeast Saccharomyces cerevisiae while exponentially growing in saline media (8). However, it has more recently been shown to be regulated under a wide variety of stress conditions (e.g. heat stress (9), osmotic stress by the addition of external sorbitol or NaCl (6, 10), hydrogen peroxide-administered oxidative stress (11), cadmium stress (12), starvation (6, 7), and recovery from treatment by the mutagenic compound methyl methanesulfonate (13)). However, the cellular role of this protein under these conditions is less clear. Its induction during saline conditions was surprising, considering the prominent production and accumulation of glycerol under these conditions (14). The identification of a similarly salt-regulated putative glycerol dehydrogenase led us to propose salt-induced glycerol dissimilation via the DHA path (8). Under conditions of salt stress, an activation of this pathway would counteract the stress-induced accumulation of the osmolyte glycerol, which appears absolutely essential under these conditions. Thus, different rationales for the increased expression of DAK were proposed (8): (i) the protein is involved in fine tuning of glycerol accumulation and thus constitutes an alternative route for pool regulation besides production and controlled excretion via the glycerol facilitator Fps1p, and (ii) the protein is involved in a stress-induced transhydrogenase cycle. This was based on the fact that the putative glycerol dehydrogenase has sequence similarities to the fungal NADPH-dependent enzyme, whereas Gpd1p is NADH-dependent. An alternative rationale was later proposed, which placed DAK as a component in an ATP futile cycle activated under stress conditions, which was involved in balancing the ATP pool and being mainly important under cellular adaptation to stress (15).

In this work, we characterize the putative DAK genes YML070W/DAK1 and YFL053W/DAK2 in the yeast S. cerevisiae, with a combined genetic and biochemical approach. In doing so, we have shown that theses genes indeed encode dihydroxyacetone kinases with high affinity for the substrate DHA and that these enzymes appear to play an important role in the detoxification mechanisms involved in maintaining a low intracellular concentration of DHA. We propose that detoxification of DHA is a vital part of mechanisms of general importance in stress physiology in many species.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Yeast Strains and Genetic Methods-- Genotypes of all yeast strains used in this study can be found in Table I. Standard molecular genetic and yeast genetic methods were used throughout. The DAK1 gene was deleted in a diploid wild type of W303 (16) (kindly provided by Stefan Hohmann) using the long flanking homology region method to replace the major part of the ORF with the KanMX4 cassette (17), and the DAK2 gene was similarly deleted in a W303 diploid using the HIS3MX6 cassette (18). The following primer oligonucleotides were used: DAK1, L1 (5'-GCATGGCTGAAATGGCAATA-3'), L2 (5'-GGGGATCCGTCGACCTGCAGCGTACCAAACGATTTAGCGGACATTT-3'), L3 (5'-AACGAGCTCGAATTCATCGATGATAGTAAGTACTTGGCTCACGAA-3'), and L4 (5'-CCTTCACTTGTTAACGGTATT-3'); DAK2, L1 (5'-GGCCTTCTGATGGAACTTAA-3'), L2 (5'-GGGGATCCGTCGACCTGCAGCGTACGAATTGTTTGTGAGACATGATT-3'), L3 (5'-AACGAGCTCGAATTCATCGATGATAGGGTACTAGAATTGCTCGTA-3'), L4 (5'-CTGTTTGTTGACTTGCGAGA-3').

                              
View this table:
[in this window]
[in a new window]
 
Table I
Strains used in this study

The two heterozygous diploids obtained were sporulated, and tetrads were dissected. After selection for G418 resistance (200 µg/ml) or histidine prototrophy, the strain yJN001 (W303, MATa, DAK1Delta ::KanMX4) was crossed with yJN008 (W303, MATalpha , DAK2Delta ::HIS3MX6) to produce the diploid strain yJN009-2n. This diploid was sporulated, several tetrads were dissected, and one tetrad was selected, which contained yJN009-1A, yJN009-1B, yJN009-1C, and yJN009-1D (Table I). These strains were used in subsequent experiments.

DAK1 and DAK2 ORFs were amplified from genomic DNA derived from strain yJN009-1A using the Expand long template PCR kit (Roche Molecular Biochemicals) according to recommendations from the manufacturer.

PCR primer sequences were as follows: DAK1 forward (5'-CATGCATGCCATGGCCGCTAAATCGTTTGAAGTCACAGAT-3'), DAK1 reverse (5'-CATGCATGGAGCTCGTACTTACAAGGCGCTTTGAACCCCCTTCAA-3'), DAK2 forward (5'-GTCAGAATTCATCATGTCTCACAAACAATTC-3'), and DAK2 reverse (5'-CTGATGCTAGCTGTTATGTTTGGCTTCTAGT-3').

In sequences shown above, added restriction sites are marked in boldface type, and translation start/stop sites are underlined. To the DAK1 forward primer, a NcoI site was added 5' of the open reading frame. In creating this, the second codon was changed from TCC to GCC, as indicated in italics, resulting in a shift from a serine to an alanine in the second amino acid residue of the expressed protein. The reverse primer contained a SacI site 3' of the ORF.

DAK2 was amplified from 3 base pairs upstream of the ATG translation to 37 base pairs downstream of the translation stop with EcoRI and NheI sites added to the upstream and downstream primers, respectively. The PCR-amplified, digested, and agarose gel-purified DAK1 ORF was cloned into the multicopy plasmid pYX212 (R&D Systems Inc.) polylinker following the TPI1 promoter. This produced the plasmid pYX212-DAK1. The DAK2 ORF was digested and cloned into pYX212 to make the plasmid pYX212-DAK2. Clones containing the correct inserts, as evidenced by multiple restriction enzyme digestion patterns and sequencing, were amplified and used to transform S. cerevisiae strain yJN009-1D using the lithium acetate/polyethylene glycol method. The obtained strains yMM006 and yMM007 (Table I) were saved for further analyses. Strains yMM004 and yMM005 were made by transforming strains yJN009-1A and yJN009-1D, respectively, with pYX212.

Sequences obtained for DAK1 and DAK2, when using strain W303 as a template, were identical to the published sequences, with the exception of a one-nucleotide conflict in DAK2, resulting in a proline instead of a serine at position 350. The DAK2 gene was later on cloned by PCR using the BY strain as a template, and the sequence obtained was in this case identical to the published sequence. Most importantly, the two Dak2 proteins appear functionally similar, since either a proline or a serine at position 350 produced transformants that grew identically on DHA as the sole carbon and energy source and the produced proteins were indistinguishable as far as specific activity and kinetics (Km and Vmax) are concerned (data not shown). It should also be noted that position 350 in Dak2p corresponds to a nonconserved region in the protein as revealed by its multiple alignment to DAKs from other species, further giving support to this position's low functional importance.

The construction and use of the GLO1 overexpression and disruption plasmids (pE24-GLO1 and pIGLK0a, respectively) have been reported earlier (19) and were kindly provided by Dr. Yoshiharu Inoue. Strain yJN201 was constructed by transforming strain yJN009-1A with the XbaI-digested plasmid pIGLKOa and selecting for uracil prototrophy. Strain yJN202 was similarly made by transforming strain yJN009-1A with the undigested plasmid pE24-GLO1. To obtain strain yJN203, strains yJN201 and yJN009-1D were mated, and obtained diploids were sporulated (Table I). A total of 24 tetrads were dissected and screened for the cosegregation of G418 resistance and uracil/histidine prototrophy. Strains used for testing DHA tolerance of pentose pathway mutants were all derivatives of strain BY4742 (20) originating from the collection of systematic deletions of all yeast gene ORFs (21).

Media and Growth Conditions-- Cultures were grown in defined minimal medium (0.17% (w/v) yeast nitrogen base without amino acids and ammonium sulfate) supplemented with 2% (w/v) glucose, 0.5% (w/v) (NH4)2SO4, and the appropriate amino acids or nucleotides (120 mg/ml). Where indicated, the appropriate amounts of dihydroxyacetone or NaCl were added to the medium. In some cases YPD complex medium (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose) was used. Solid medium was made by adding 2% (w/v) agar. Precultures of 5 ml were grown in 14-ml plastic tubes (Falcon) overnight at 30 °C on a rotator. Larger precultures and most ordinary cultures were grown in E-flasks with rotary agitation, 110 rpm, at 30 °C. Cultures were inoculated to an OD610 of 0.07 (roughly 1 × 106 cells/ml), and growth was followed over time. Larger culture volumes were grown in Fernbach flasks.

DAK Enzyme Assays-- The assay for dihydroxyacetone kinase activity was essentially performed as described earlier (8, 22). Extracts for the assays were made as previously described (8) using cells harvested from 100 ml of culture grown to midexponential phase (OD610 of 0.35) (or from 200 ml of culture incubated for 55 h in the case of activity measurements of yJN009-1A and yJN009-1D in YNB 50 mM DHA). Cells were washed in ice-cold 20 mM MES buffer, pH 6.5, and disrupted by vortexing with acid-washed glass beads, with the exception of cells grown on 50 mM DHA that were washed three times in sterile MilliQ water prior to the normal washing procedure. DAK activity was recorded as amount of NADH oxidized/unit of time in a coupled reaction with excess of glycerol-3-phosphate dehydrogenase. During DAK activity measurements, the addition of 4 mM DHA started the reaction. Protein content of extracts was determined using a slightly modified Lowry assay (product number P5656; Sigma) with bovine serum albumin as a standard.

During measurement of kinetic parameters with respect to DHA, measurements were performed at six different concentrations of DHA between 40 and 800 µM and at a constant concentration of 10 mM ATP. Measurements with respect to ATP were performed at six different ATP concentrations in the range 0.1-5 mM and at a constant DHA concentration of 4 mM. Reactions were started with the addition of ATP, when the kinetic parameters of DAK with respect to ATP were determined, and with DHA, when parameters with respect to DHA were determined.

Gel Filtration-- Gel filtration experiments were performed using a fast protein liquid chromatography system (Amersham Biosciences) at 4 °C. Extracts were kept on ice when possible. Cells of strain yMM006 from 500 ml of exponential phase culture (OD610 0.35) grown in defined minimal medium supplemented with 2% glucose were harvested and resuspended in 1 ml of MES/(NH4)2SO4 buffer (0.1 M (NH4)2SO4, 20 mM MES, pH 6.5) supplied with Complete Mini protease inhibitors (Roche Molecular Biochemicals) according to the manufacturer's recommendations. Cells were broken through vortexing in the presence of glass beads (1.5 g; 0.5-mm diameter) four times for 30 s each with intermittent placing on ice for at least 30 s. Extracts were then cleared through centrifugation at 18,000 × g using a SS-34 rotor (Sorvall), and 500 µl of the supernatant (about 5 mg of protein) was loaded onto a 120-ml Superdex 200 column equilibrated with 2 column volumes of MES/(NH4)2SO4 buffer. The column was eluted with the same buffer at a flow rate of 1 ml/min, and 1-ml fractions were collected. Protein concentration in the eluate was continuously monitored as absorbance at 280 nm, and DAK activity of the fractions was measured throughout the whole range. A size standard curve was generated using the high and low molecular weight gel filtration calibration kit (product numbers 17-0441-01 and 17-0442-01; Amersham Biosciences) containing the following proteins: catalase (232 kDa), aldolase (158 kDa), albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and ribonuclease A (13.7 kDa) in addition to blue dextran 2000 reconstituted in elution buffer. The elution of blue dextran 2000 was set to equal the void volume (V0). The elution volumes were all converted to partition coefficient (Kav) values ((elution volume - void volume)/(total bed volume - void volume)). The Kav obtained for Dak1p was fit to the standard curve made by plotting Mr against Kav.

Anaerobiosis-- Strictly anoxic growth conditions were imposed by incubating cells in defined minimal medium, as described above, with the addition of 1 ml/liter of a stock solution of ergosterol/Tween 80 (10 g/liter ergosterol and 420 g/liter Tween 80 in 99.5% ethanol) and under constant nitrogen flushing using a system described elsewhere (23). In short, 100-ml shake flasks were connected to air-tight rubber stoppers and hoses and put under constant flushing with humidified nitrogen gas. To verify anaerobicity, a culture of the gpd1Delta gpd2Delta double deletion mutant was incubated along with the other cultures.

Growth Assays in Microplates Using the Bioscreen C-- Tests for methylglyoxal sensitivity, DHA sensitivity in salt containing medium, formaldehyde sensitivity, and DHA sensitivity of mutants were all performed in a 100-well microcultivation system (24). Cultures were inoculated with overnight cultures grown in 5 ml of medium in plastic tubes (Falcon), and experiments were performed in glucose minimal medium, supplemented with methylglyoxal (Sigma), DHA, NaCl, or formaldehyde (Merck) to the specified amounts (see above), in a Bioscreen C, which is a combined incubator/shaker/spectrophotometer (Labsystems Oy). In short, cells were grown at 30 °C in a culture volume of 350 µl and a shaking regime of 60 s of high intensity shaking followed by 60 s without shaking. For methylglyoxal sensitivity tests, the growth of the different strains was recorded every 20 min during a 24-h period. When DHA sensitivity of mutants was tested, cultures were monitored for growth every 30 min for 72 h. During combined DHA and salt, as well as formaldehyde, sensitivity tests, culture growth was recorded every 40 min for 96 h. Recorded values were subsequently exported to Excel, where OD data were corrected for the nonlinearity of the OD measurements at higher cell densities, and from these corrected OD values the length of the lag phase, the generation time, and the stationary OD increment were calculated.

Glycerol Analysis-- Samples from exponential phase cultures (OD610 = 0.35) were analyzed for glycerol using a commercial kit (Roche Molecular Biochemicals) as described before (5). Intracellular glycerol was calculated by subtracting extracellular glycerol, measured on filtered samples (0.2 µm), from total glycerol values.

DHA Uptake-- DHA uptake measurements were performed essentially as described earlier for glycerol uptake measurements (25). 500 ml of exponential phase cultures (OD610 of 0.35), with either glucose, galactose, or ethanol as carbon and energy source, were harvested and resuspended in 10 mM MES, pH 6.0, to a density of ~70 mg dry weight/ml. Dry weight was determined by filtering and drying (3 days at 80 °C) of 100 µl of cell suspension (duplicate samples). 14C-Labeled DHA (55 mCi/mmol; American Radiolabelled Chemicals) was added to a concentration of 85 µM and mixed with unlabelled DHA solutions to yield DHA concentrations in the range of 0.18-200 mM. Cell suspensions (10 µl) were exposed to the indicated DHA concentrations in a total volume of 50 µl for 0, 10, 20, 40, and 60 s. Uptake was found to be linear with time during the period measured (the mean correlation coefficient of the linear regression for all uptake experiments was calculated to be 0.966 ± 0.092 (n = 27)). The rate of DHA uptake was taken as the slope of the linear curve fitted through data at the five different time points.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The DHA Kinase Activity Is Linked to the YML070W/DAK1 Gene-- In order to experimentally link the putative DAK-encoding genes, YML070W (DAK1) and YFL053W (DAK2), to the salt stress-induced DAK enzymatic activity in S. cerevisiae (8), deletion mutants were constructed, either in single or in combination (Table I). Gene deletions were produced in diploids that were subsequently sporulated to obtain the corresponding haploid with the gene(s) deleted. It was clear that none of the gene deletions, either in single or double knock-out strains, resulted in lethality; nor could we in the mutants observe any sporulation defect (data not shown). In addition, DAK1 and DAK2 overexpression strains were constructed, carrying a multicopy plasmid with the YML070W/DAK1 or the YFL053W/DAK2 gene expressed behind the strong and constitutive TPI1 promoter (Table I).

The wild type strain YJN009-1A (a W303-1A isogenic derivative) displayed an easily detectable DHA kinase activity of 4.3 units/mg protein under basal growth conditions (Table II). This is an activity value in line with earlier reported data on another wild type laboratory strain, SKQ2n, which during nonsaline growth exhibits a DAK activity of 7.6 units/mg protein (8). The mutant where the YML070W/DAK1 gene was deleted (designated dak1Delta ), exhibited no detectable DAK activity in either control or salt stress medium even after the addition of increased amount of cell extract to the assay (we estimated that the DAK activity of this mutant strain was at least 1000-fold lower than in the wild type). Thus, the YFL053W/DAK2-encoded product does not contribute significantly to the overall DAK activity, at least under these growth conditions. This was further supported by the fact that deletion of the YFL053W/DAK2 resulted in no detectable difference in the recorded DAK activity compared with the wild type (data not shown).

                              
View this table:
[in this window]
[in a new window]
 
Table II
Dihydroxyacetone kinase activity in cell extract from different strains
For further details about the strains see information in Table I.

Overexpression of the YML070W/DAK1 gene product in a DAK double deletion strain resulted in a roughly 250-fold enhanced specific DAK activity compared with the wild type to 1040 units/mg protein during growth in basal medium (Table II). On the contrary, the overexpression construct carrying the YFL053W/DAK2 gene did not, during growth on glucose, display any detectable DAK activity. We conclude that the open reading frame YML070W/DAK1 encodes a dihydroxyacetone kinase in S. cerevisiae.

Kinetic Properties of Dak1p-- The kinetic properties of Dak1p were initially tested on crude extracts obtained from the wild type yJN009-1A grown in 0.7 M NaCl medium. Proper kinetic measurements were technically problematic at low substrate concentrations, because the obtained measurements were generally low and close to the background. However, it was estimated that the Dak1p Km for DHA was roughly in the range 10-20 µM. To obtain more reliable experimental data, extracts obtained from the strain overproducing the Dak1 protein were used for subsequent analysis of kinetic properties. As can be seen in Fig. 1A, the Km for DHA (at a fixed concentration of ATP of 10 mM) was determined to 22 µM. This value of Km for DHA was thus in agreement with the Km estimate using crude extracts obtained from the wild type strain. The Km value determined for ATP (at a fixed concentration of DHA of 4 mM) was 0.49 mM (Fig. 1B).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1.   Kinetic properties of the Dak1 protein. DAK activity was measured by a coupled reaction to glycerol-3-phosphate dehydrogenase (GPD). Values represent means of measurements on three independent transformants of dak1Delta dak2Delta with the pYX212-DAK1 overexpression plasmid (strain pTPI1-DAK1), and error bars indicate S.D. (all data points have error bars). Data are represented as Hanes plots. A, the Km and Vmax for DHA was determined by measurements in the range of 40-800 µM DHA at a constant concentration of ATP of 10 mM. B, the Km and Vmax for ATP were determined by measurements in the range 0.1-5 mM ATP at a constant concentration of DHA of 4 mM.

Native DAK Appears to Be a Dimer-- The DAK activity from Schizosaccharomyces pombe IFO0354 has been shown to be resolved in two different peaks of different molecular weight after gel filtration (26). The extract from the strain overexpressing Dak1p was subjected to gel filtration, and contrary to S. pombe the DAK activity in this species was eluted as one single peak (Fig. 2A). The molecular mass of this native form of DAK was estimated to be 115 kDa (Fig. 2B), which fits well with the assumption that the native conformation of DAK in S. cerevisiae could be as a homodimer, since the theoretical value for the Dak1 protein is 62 kDa and its mass has earlier been experimentally determined to be 59.5 kDa (8). However, we cannot at present exclude extensive modifications or heterodimer formation from our data. In support of the homodimer conformation of DAK in S. cerevisiae are the earlier reports on highly purified DAK from C. freundi and H. polymorpha, which migrated with an apparent mass of roughly double the respective DAK protein and was clearly shown to be composed of only DAK monomers (27, 28).


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2.   Gel filtration on a Superdex column of cell extract from a strain overexpressing DAK1. A, the relative DAK activity (circles) is revealed in one single peak. A line without symbols indicates absorbance at 280 nm (A280), which is continuously recorded and reflects the total amount of protein. B, the estimate for the native molecular mass of Dak1p is roughly 115 kDa. Calibration proteins (see "Experimental Procedures") are indicated with filled symbols and plotted versus the partition coefficient (Kav).

Dihydroxyacetone Is Toxic to Yeast Cells-- The phenotypic importance of the DAK genes was tested during a number of growth conditions for the dak1Delta , dak2Delta , and dak1Delta dak2Delta deletion strains. No growth defect on glucose, galactose, and ethanol media (either carbon source administered at a concentration of 2% (w/v)) was recorded between the wild type and the mutants (data not shown). Growth on DHA as the sole carbon and energy source was initially also tested at 2% (w/v) DHA (~0.2 M DHA); however, even after 7 days of incubation, no growth could be detected by optical recording. To test for viability changes during this DHA treatment, the 7-day cultures were diluted and spotted on rich medium plates (YPD). It was found that DHA as the sole carbon source not only precluded growth but killed the cells (Fig. 3A); the surviving fraction after 7 days of incubation is less than 0.01% (initial cell density of 5 × 105 cells/ml), whereas incubation with glycerol or without the addition of a carbon source showed no diminished viability. The kinetics of DHA-dependent killing of the cultures was subsequently tested. Cellular toxicity from 0.2 M DHA exposure was clearly apparent; the wild type rapidly declined in viable cell numbers after about 90 h of exposure (Fig. 3B). It was also clear that the cellular DHA sensitivity was linked to the DAK genes, since the double mutant dak1Delta dak2Delta was much more sensitive than the wild type, whereas the strain overexpressing DAK1 displayed drastically enhanced resistance. However, in all cases, the apparent slope of the decline was roughly similar, indicating a two-step process in the killing: (i) an initial phase where death is initiated, which is strictly dependent on the presence of DAK, and (ii) the subsequent phase of cell decline, which is independent of DAK.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3.   DHA is toxic to yeast. A, wild type cultures at a cell density of 5 × 105 cells/ml were incubated for 7 days in YNB medium with 0.2 M DHA or 0.2 M glycerol or without the addition of a carbon source. After incubation, cultures were diluted and plated on YPD agar plates (as 10-µl drops). Plates were incubated at 30 °C, and growth was recorded after 2 days of incubation. B, cellular toxicity to DHA is related to the presence of DAK1. Exponentially growing cells in basal growth medium (YNB; 0 M NaCl) of wild type, DAK double deletion strain (yJN009-1D) and the strain overexpressing DAK1 (yMM006) were transferred to the same medium containing 0.2 M DHA and incubated at 30 °C. At the indicated time points, aliquots were plated onto YPD plates. For all cultures, the colony forming unit value at T = 0 is set to 100%. A typical result is shown.

Yeasts Grow Well on DHA If DAK1 or DAK2 Is Overexpressed-- The strain overexpressing DAK1 displayed a small increase in cell density during the DHA treatment (Fig. 3B), probably indicating a low level of proliferation. Since DHA is toxic at 0.2 M, the concentration of DHA was decreased to 50 mM, and growth was recorded (Fig. 4). In this analysis, also the strain that carries the overexpression construct for YFL053W/DAK2 was included. Strains overexpressing either DAK1 or DAK2 grew well on 50 mM DHA as the sole energy and carbon source with a generation time of about 5 h. The wild type slightly increased cell density under these low DHA conditions, whereas the dak1Delta dak2Delta mutant did not exhibit any growth at all. The slow growth of the wild type appeared dependent on the DAK1 gene, since no growth was recorded in the dak1Delta strain, whereas dak2Delta displayed identical growth to the wild type (data not shown). The DAK activity was only slightly enhanced in the wild type during DHA growth, from 4.3 to 7.5 units/mg protein (Table II). In the double deletion strain, no DAK activity could be recorded even after prolonged incubation in DHA, indicating that no additional DAK genes besides these two sequence homologues appear to be present in the yeast genome. The DHA growth of the DAK2-overexpressing strain was something of a surprise, since no DAK activity was earlier recorded during glucose growth in this strain. Subsequently, the DAK activity was measured in DHA-grown cells, and under these DHA-dependent growth conditions the strain overexpressing DAK2 exhibited even higher specific activity compared with the DAK1-overproducing strain, 140 units/mg protein versus 117 units/mg protein (Table II). Kinetic analysis of the DAK activity in the strain where DAK2 was overexpressed during DHA growth revealed that also the Dak2 protein displayed a high affinity for DHA and ATP; Km(DHA) = 5 µM and Km(ATP) = 0.1 mM (data not shown). Thus, both DAK enzymes exhibited Km values for ATP that are well below the reported intracellular ATP concentration attained under a wide array of growth conditions; the ATP concentration covers a range of roughly 1-5 mM (29).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4.   Growth on DHA as sole carbon and energy source for the double deletion strain dak1Delta dak2Delta as well as the same strain overexpressing either DAK1, pTPI1-DAK1, or DAK2, pTPI1-DAK2. Overnight cultures from glucose minimal medium were harvested, washed, and inoculated to basal growth medium, YNB 0 M NaCl, containing 50 mM DHA as the sole carbon and energy source, and growth was recorded. A typical result is shown.

Compared with glucose-grown cells, the level of overexpression of DAK1 during DHA growth was strongly reduced (1040 units/mg during glucose growth, which decreased to only 117 units/mg when DHA was the sole carbon and energy source). Neither the mechanistic reason for the carbon source variability of the DAK1-encoded activity nor the fact that measurable activity of the Dak2 protein was fully dependent on utilization of DHA is presently understood (recall that the expression of both genes is governed by the TPI1 promoter). However, the data from DHA grown cells strongly suggests that not only YML070W/DAK1 but also the YFL053W/DAK2 gene encodes a DAK in S. cerevisiae. In addition, it can be concluded that high level expression of DAK is essential and apparently the only prerequisite to circumvent the toxic effect from DHA and will provide the cells with a high capacity for growth on DHA as the sole energy and carbon source.

Glucose Suppression of DHA Toxicity-- The toxicity from high concentrations of DHA could be partially suppressed by the addition of another carbon source to the media. In particular, glucose efficiently restored good growth even in the presence of 0.2 M DHA (Fig. 5A), whereas the less favorable carbon source galactose did so to a lesser extent (Fig. 5B). Growth on the respiratory carbon source ethanol only marginally improved tolerance to DHA (data not shown).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 5.   The toxicity of DHA is not dependent on the presence of oxygen. Cultures of the wild type were incubated with (squares) or without (circles) 200 mM DHA under aerobic (open symbols) or anaerobic (filled symbol) conditions, the latter by flushing cultures with nitrogen (23). A, glucose as carbon and energy source. B, galactose as carbon and energy source. A typical result is shown.

The differential suppression of DHA toxicity by various carbon sources could be explained by different uptake capacities for externally added DHA in relation to the main carbon source utilized. However, kinetic analysis revealed no carbon source-dependent differences in DHA uptake capacity in the range 0.18-200 mM DHA (Fig. 6). Furthermore, the uptake of external DHA appears to pass through the membrane by simple diffusion and not to be protein-mediated, since we found no indications in this concentration range for saturation kinetics. Thus, the DHA uptake capacity cannot explain the differential carbon source suppression.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 6.   Uptake kinetics for DHA. Midlog cultures, grown on either glucose (open circles), galactose (closed circles), or ethanol (open squares) as carbon and energy source, were tested for rate of uptake (µmol/s × dry weight cells) of DHA in the range 0.1-200 mM. Measurements at 100 and 200 mM DHA were performed in triplicate independent samples, and error bars indicate S.D.

The marginal suppression of the DHA toxicity from addition of galactose or ethanol might be explained by an involvement in the toxicity mechanism from respiration, since both of these carbon sources will foster higher respiratory activity (30). This hypothesis was tested by comparing the DHA response of glucose or galactose grown cells under anaerobic conditions. We found that DHA hampered proliferation also under anaerobic conditions independent of carbon source (Fig. 5). In fact, under these anaerobic conditions, no growth in the presence of DHA could be recorded in galactose medium (Fig. 5B). In addition, the killing of cells from treatment with only 0.2 M DHA (no addition of other carbon sources) was not excluded by anaerobic incubation; cells died off as rapidly when oxygen was not present (data not shown). Apparently, neither oxygen nor respiration per se appears involved in the mechanisms behind DHA toxicity.

Phenotypic Growth Effects Relating to DAK during Saline Growth-- The DAK1 gene was initially identified in S. cerevisiae as induced during saline growth (8). To evaluate the importance of this observed change in DAK expression during salt stress, the dak1Delta dak2Delta double deletion strain was tested for growth defects in salt-containing media. We could not, however, score any phenotypic growth changes for the mutant compared with the wild type, either in the length of the adaptation phase or on growth rate, in the range 0-2.2 M NaCl (glucose-containing media; data not shown). In addition, the amount of produced and accumulated glycerol during saline growth in the double deletion strain was also the same as for the wild type (Table III). Since the effect from DHA toxicity was strongly dependent on the carbon source (see above), we also tested saline growth with galactose as carbon source. Even with this slightly poorer carbon source, there was no difference in growth properties between the wild type and the double deletion strain in media containing 1.4 M salt (data not shown).

                              
View this table:
[in this window]
[in a new window]
 
Table III
Glycerol production and accumulation during growth in medium containing 0.7 M NaCl

However, when an extra load of 0.2 M DHA was added to the salt medium, a clear effect from the deletion of DAK was observed. To examine this combined effect in more physiological detail, we screened the strains with different DAK expression for their DHA dependence, with or without 1 M NaCl and also for the effect from the different carbon sources glucose or galactose (Fig. 7). First, it is clearly apparent that glucose suppresses the DHA effect much more strongly than galactose (as indicated above), and only minor defects in growth were observed even at 0.2 M DHA in glucose media. However, in galactose media, even minor increases in DHA concentrations caused hampered growth behavior. Most importantly, in the galactose case, the effect was evidently dependent on the expression of DAK even without salt addition; at 0.2 M DHA, there was a significant defect for the wild type and in the double deletion (generation times in the range of 17-22 h) compared with any of the strains overexpressing DAK (generation times in the range 6-8 h).


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 7.   Salt addition aggravates the growth defect imposed by DHA addition, and this phenomenon is dependent on the presence of the DAK1 or DAK2 genes. Cultures were grown in glucose or galactose containing saline (1 M NaCl; B and D) or in control (0 M NaCl; A and C) YNB medium with the indicated concentrations of DHA in the range of 0-200 mM. Values indicate averages of duplicated experiments. The mean coefficient of variation for all samples was 2.5 ± 4.5% (S.D.).

Second, from the data in Fig. 7, it is clear that salt addition will strongly enhance the toxicity effect from DHA, independent of the carbon source utilized. In saline media, it is apparent that deleting the DAK genes resulted in hampered growth, whereas overexpression of either one of the DAK genes strongly improved salt growth in the presence of DHA. However, in all cases, overexpression of DAK1 was more beneficial than overexpression of DAK2. The differential behavior of the strains overexpressing DAK1 or DAK2 was most easily observed in saline galactose medium at 0.2 M DHA, where DAK1 overexpression resulted in only a slightly longer generation time compared with the situation without DHA, whereas the DAK2-overexpressing strain did not start to grow during the course of the experiment (96 h). No effect was found in glucose-containing media from deletion of DAK2 in the dak1Delta background. Since overexpression of DAK2 suppresses the toxic effects from salt and DHA, this indicated that this gene might not be sufficiently expressed under glucose growth. In summary, the DHA toxicity is strongly amplified during saline growth where the DAK1 gene product appears to play a more significant cellular role in the cellular detoxification mechanism(s).

DHA Toxicity: A Link to Methylglyoxal and Formaldehyde?-- It is unclear how DHA exerts its toxicity; it could either be directly involved in the primary toxic effect or alternatively DHA is converted to some toxic compound. A tempting candidate for DHA conversion is methylglyoxal that also is a three-carbon compound and which is known to be toxic at low (millimolar) concentrations. DHA is also reported to nonenzymatically convert to methylglyoxal (31). In addition, glyoxylase I, encoded by the GLO1 gene, which is involved in the detoxification of this metabolite, was recently shown to be induced by salt, and an increased production of methylglyoxal under these conditions was also demonstrated (32). We therefore tested whether abolishment of the GLO1 gene-dependent activity would make cells sensitive to DHA, thus providing evidence that DHA might be metabolized via this pathway. However, the glo1Delta strain exhibited similar sensitivity to 0.2 M DHA as the wild type, as did the strain overexpressing GLO1 (data not shown). In addition, the glo1Delta deletion and the dak1Delta dak2Delta glo1Delta deletion mutant grew well on salt; in fact, these strains and the wild type are indistinguishable in 1.4 M NaCl minimal glucose medium (data not shown). We also tested the opposite situation, if the DAK constructs were more or less sensitive to methylglyoxal. Neither the DAK1 overexpression strain nor the DAK double deletion strain differed in sensitivity to methylglyoxal compared with the wild type at 2 or 4 mM, whereas the glo1Delta made the cells highly sensitive (data not shown).

An alternative route for DHA toxicity is conversion to formaldehyde by some of the yeast transketolases Tkl1p or Tkl2p (33). The toxic effect from formaldehyde is well documented, and even very low amounts of this reactive compound have a devastating impact on yeast growth and survival. We tested the formaldehyde effect for different combinations of DAK1 and DAK2 alterations (deletions and overexpression) and found that in the range 0.1-2 mM formaldehyde, there was no significant effect on growth in normal glucose-containing medium (data not shown). Neither could we see any effect from deleting any of the genes TKL1, TKL2, TAL1, or YGL043C (a sequence homologue of TAL1), all potential candidates for this enzymatic step in S. cerevisiae. The TAL deletions also provided evidence that DHA is not toxic due to interference with this reaction in the pentose phosphate pathway, which transfers a DHA moiety.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

There Are Two Dihydroxyacetone Kinases in S. cerevisiae-- The genome of the yeast S. cerevisiae contains two putative DAK genes that encode proteins with 46% amino acid identity, YML070W/DAK1 and YFL053W/DAK2 (8). We show in this study that both Yml070w/Dak1p and Yfl053w/Dak2p are indeed dihydroxyacetone kinases. This was clearly substantiated for the DAK1-encoded protein, since a gene deletion abolished measurable DAK activity, whereas DAK1 overexpression led to radically higher levels of DAK activity during growth in glucose medium. In addition, the kinetic properties of the Dak1 protein in S. cerevisiae, with a here reported Km for DHA of 22 µM and for ATP of 0.49 mM, are similar to most of the earlier characterized DAKs (e.g. the methylotrophic yeast Hansenula polymorpha/Pichia angusta and the fission yeast S. pombe exhibit DAKs with Km(DHA) in the range of 11-52 µM and Km(ATP) in the range of 0.3-0.55 mM) (26, 28, 34). These values for the yeast enzymes are also in good agreement with Km(DHA) of DAKs from diverse bacterial species (27). The exception in the collection of DAKs is one of the two forms of DAK characterized in strain IFO0354 of S. pombe, which displays an unusually high Km for DHA of 3 mM (26). However, this enzyme exhibits similar affinities for DHA, glyceraldehyde, and glycerol, thus classifying this enzyme as a more general triose kinase (EC 2.7.1.28) and not a specific DHA kinase (EC 2.7.1.29). Overexpression of DAK1 enabled dak1Delta dak2Delta cells to grow well on DHA as a sole source of carbon and energy, further substantiating its role in DHA metabolism. This was also the case for the corresponding strain overexpressing the YFL053W/DAK2 gene. Under DHA utilization we could easily measure DAK activity in this DAK2-overproducing strain, and Dak2p revealed affinities for DHA and ATP in the same range as Dak1p. The mechanistic explanation for this differentially measured DAK activity in the DAK2 overexpression strain, in relation to the carbon source, is not known and is currently being investigated in our laboratory. However, the recorded DAK activity during DHA growth clearly proves the classification of this Dak2p enzyme as a DAK.

DHA Is Toxic-- It is clear from our study that DHA is toxic to yeast cells. Contrary to the intracellular metabolites methylglyoxal and formaldehyde, few indications are reported in the literature that DHA would be harmful. A decreased cell yield was reported for Zymomonas mobilis with increased concentrations of externally added DHA, although no cell death was reported and no mechanistic explanation was provided (35). There are several reports where methylglyoxal is shown to modify arginine and lysine residues of proteins by the nonenzymatic Maillard reaction both in vivo and in vitro (31, 36, 37). There is to our knowledge only one report where DHA has also been shown to react in a similar way with proteins; DHA was reported to react with bovine serum albumin in vitro to produce adducts that could be detected with anti-5-methylimidazolone antibodies (also used to detect the products from methylglyoxal treatment) (36). However, a complication in this analysis is that there are indications that DHA can be converted to methylglyoxal nonenzymatically (31).

The putative nonenzymatic direct conversion of DHA to this known toxic compound was also why we investigated the possible relation between DAK and methylglyoxal. We could not, however, find any link between the two. Maybe most importantly, a glo1 deletion strain, which is severely sensitive to low levels of methylglyoxal, displayed invariant DHA sensitivity compared with the wild type. An alternative possibility for methylglyoxal production would be that the DHA externally added is converted by DAK to DHAP, and an increased pool of the this triosephosphate should thus lead to enhanced methylglyoxal levels. In that case, a DAK double deletion (dak1Delta dak2Delta ) would improve resistance to DHA, which was contrary to the finding in our analysis (Fig. 3). Furthermore, overexpression of DAK1 strongly improved tolerance, indicating that keeping the DHA pool low in the cell is the key prerequisite for survival. Thus, we find it unlikely that DHA exerts its toxic effect via nonenzymatic or enzymatic conversion to methylglyoxal. We also investigated the link to another toxic compound, formaldehyde, which is know to be a weak mutagen and recombinogen in yeast. The main enzymatic system for formaldehyde detoxification is glutathione-dependent formaldehyde dehydrogenase encoded by the SFA1 gene (38). The link to DHA would in this case be enzymatic conversion to formaldehyde via transketolase Tkl1p or Tkl2p (33). However, the addition of formaldehyde in the range 0.1-2 mM hampered growth to a similar extent in strains lacking or overexpressing DAK. Furthermore, deletions of TKL1 or TKL2 did not improve tolerance to DHA.

In the pentose phosphate pathway, transaldolase transfers a three-carbon moiety of dihydroxyacetone. It is therefore conceivable that the addition of DHA could interfere with this reaction and that this could in turn cause the formation of undesired products or the reduced levels of necessary compounds. However, neither mutants of transaldolase or transketolase nor a deletion mutant in glucose-6-phosphate dehydrogensae (ZWF1; the first step of the oxidative branch of the pentose phosphate pathway) were altered in their sensitivity to DHA, thus making this hypothesis unlikely. We thus conclude that at present we cannot find any indications of the direct mechanisms or targets on cell toxicity via DHA.

Different Cellular Roles of DAKs-- There are currently 21 proteins, from all kingdoms except Archaea, which exhibit a BLAST score of greater than 200 with homology over at least 90% of the full length of the protein when compared with yeast Dak1p. The sequence relation of 17 of these are depicted in the nonrooted phylogenetic tree in Fig. 8 (only two of the four sequences from S. pombe and Arabidopsis thaliana were included). It is clear from this tree display that most of these DAK sequences are about 30-35% sequence-identical to the S. cerevisiae enzymes (e.g. 31% in the case S. cerevisiae Dak2p and the enzyme from the bacteria Citrobacter freundii). The exceptions are Dak2p in S. cerevisiae and the Dak2p from S. pombe, which have a higher degree of sequence identity (73%), and Dak1p and the recently identified corresponding enzyme from Zygosaccharomyces rouxii (39), which are 70% identical.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 8.   Phylogenetic tree of 17 of the 21 proteins that show best homology to Dak1p of S. cerevisiae (BLAST score above 200). The position of DAK1 and DAK2 from S. cerevisiae is displayed in boldface type. The tree is constructed by the use of multiple aligned sequences utilizing the ClustalW program (available on the World Wide Web at www.ebi.ac.uk).

An interesting observation about DAK is the multitude of cellular roles it has been ascribed in different cell systems. (i) The fission yeast S. pombe clearly depends on DAK for the catabolism of glycerol, the first step in the pathway being a NAD+-specific glycerol dehydrogenase and the second step being DAK (22), and this route for glycerol assimilation is also utilized in the bacteria C. freundii (27). (ii) DAKs have been characterized in methylotrophic yeast species, like H. polymorpha/P. angusta (28) and Pichia pastoris (40). These species are characterized by a highly efficient metabolism for the utilization of the one-carbon compound methanol, which is not utilized by most yeast species, and certainly not by S. cerevisiae. In methylotrophic yeast, the DAK enzyme has been shown to be essential during growth on methanol as the sole carbon and energy source, and in the process of assimilation of methanol, dihydroxyacetone is formed from formaldehyde and xylulose-5-phosphate by a specific dihydroxyacetone synthase and then further metabolized via DAK (33). (iii) In Escherichia coli, the DAK protein consists of three soluble protein subunits (41). Two of these are sequence-similar to the N-terminal and C-terminal part of the regular ATP-dependent DAKs from various organisms, whereas the third subunit has sequence resemblance to enzymes involved in the phosphotransferase system in bacteria. The phosphotransferase system mediates phosphorylation of carbohydrates at the expense of phosphoenolpyruvate via a phosphorelay cascade involving several proteins. It is reported that in E. coli, the DAK enzyme utilizes a phosphoprotein instead of ATP as phosphoryl donor. The cellular role of this phosphoenolpyruvate-linked DHA phosphorylation in E. coli via DAK is currently not clear but the system is hypothesized to be suitable as a sensor for intracellular metabolites (41).

We here report on yet another cellular role for DAK, in detoxification of DHA in S. cerevisiae. The DHA sensitivity was related to the level of expression of DAK. In addition, the low Km value for DHA for both Dak1p and Dak2p further strengthens the role of DAK in assuring a low internal pool of DHA. Thus, it can be concluded that the DAK enzyme has, during the course of evolution, been selected and adjusted to serve various physiological needs in diverse organisms. At this stage, it is not clearly apparent that these different specific roles are reflected in the sequence comparisons (Fig. 8). Part of the explanation for that could of course be that in some, or many, of these organisms DAK plays more than one cellular role. It should also be noted that DAK proteins have been identified in representatives of higher eucaryotes (Fig. 7), both in plants (A. thaliana and Lycopersicon esculentum, tomato) and in the nematode Caenorhabditis elegans and in mammals. The cellular role of DAK in these higher eukaryotes has not, to our knowledge, been analyzed. It will be of interest to investigate whether DHA is toxic also in multicellular species.

The Cellular Role of DAK and DHA in Stress Metabolism-- The increased expression of DAK1 in S. cerevisiae under saline conditions would indicate a stress-imposed metabolic production of DHA, which pool has to be strictly regulated by DAK. In addition, since DAK1 is up-regulated during cellular adaptation to a wide range of environmental conditions (6, 7), it appears as if increased DHA production could be a problem under many stresses. The most probable route for DHA formation during adaptation to and growth in saline media would be from conversion of the intracellularly accumulated glycerol. This compatible solute is produced in high amounts in response to osmotic stress and is accumulated at high stress magnitudes to even molar concentrations (14). A putative NADPH-dependent glycerol dehydrogenase has been identified that also was found to be induced under these stress conditions (8). However, externally added glycerol is not toxic to cells (Fig. 3), and glycerol is not accumulated to any significant levels under any of the other nonosmotic stresses like heat or oxidative stress. Some other metabolic paths could have a role in DHA production: (i) dephosphorylation of DHAP; (ii) formaldehyde conversion via transketolase Tkl1p or Tkl2p (33); (iii) conversion from some unknown metabolite (e.g. a stereospecific (2R,3R)-2,3-butanediol dehydrogenase was reported to, in a NADH-dependent manner, react with DHA with about 10% of the activity reported for its natural substrate) (42); and (iv) transketolase- and transaldolase-mediated reactions in the pentose phosphate pathway. However, we could not find indications of altered sensitivity to DHA in any of the tested deletion mutants for some of the relevant enzymes. Further analysis of DHA sensitivity will have to be performed in mutants with combinations of gene deletions as well as analysis of metabolic flux changes during stress conditions in order to clarify the DHA aspect of stress-imposed metabolic changes in the wide array of environments.

We found a clear stress-related phenotype for the DAK double deletion when DHA was externally added. However, without DHA addition, we could not score a growth phenotype, neither at very high salt or sorbitol concentrations (osmotic stress) nor under a number of other conditions of stress like high temperature or oxidative stress (data not shown). This is surprising but probably reflects the existence of alternative detoxification mechanisms of DHA. Any of the above described putative systems for DHA production could equally well be involved in the reversible reaction, DHA detoxification. In identifying such an alternative detoxification system, one should utilize the knowledge generated in this study about the suppressive effects on DHA toxicity from glucose but not galactose growth as well as the lack of correlation to respiration. Clearly, there is a link between DHA toxicity and general catabolism; however, we cannot at present distinguish between carbon source-dependent altered production or breakdown of DHA.

A number of alternative hypotheses besides DHA detoxification have been presented for the cellular role of DAK in S. cerevisiae. These include regulating the glycerol pool and acting as one link in a multienzyme transhydrogenase system (8) or being an important measure for balancing the ATP imbalance during sudden shifts in growth potential (15). In this study, we have found no experimental evidence for any of these suggestions; on the contrary, the glycerol pool in the DAK deletion mutant under saline growth appears identical to the wild type. Even if a role in detoxification of DHA is strongly supported by our data, we cannot at present totally exclude the possibility that the other DAK-dependent mechanisms proposed can be valid, at least under certain growth conditions.

    ACKNOWLEDGEMENT

The GLO1 plasmids were kindly provided by Dr. Yoshiharu Inoue.

    FOOTNOTES

* This work was supported by the Swedish Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. E-mail: anders. blomberg{at}gmm.gu.se.

Published, JBC Papers in Press, October 24, 2002, DOI 10.1074/jbc.M203030200

    ABBREVIATIONS

The abbreviations used are: DAK, dihydroxyacetone kinase; ORF, open reading frame; MES, 4-morpholineethanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Pearce, A. K., and Humphrey, T. C. (2001) Trends Cell Biol. 11, 426-433[CrossRef][Medline] [Order article via Infotrieve]
2. Grant, C. M. (2001) Mol. Microbiol. 39, 533-541[CrossRef][Medline] [Order article via Infotrieve]
3. Wolfger, H., Mamnun, Y. M., Mamnun, Y. M., and Kuchler, K. (2001) Res. Microbiol. 152, 375-389[CrossRef][Medline] [Order article via Infotrieve]
4. Piper, P., Calderon, C. O., Hatzixanthis, K., and Mollapour, M. (2001) Microbiology 147, 2635-2642[Free Full Text]
5. Ansell, R., Granath, K., Hohmann, S., Thevelein, J., and Adler, L. (1997) EMBO J. 16, 2179-2187[Abstract/Free Full Text]
6. Gasch, A. P., Spellman, P. T., Kao, C. M., Carmel-Harel, O., Eisen, M. B., Storz, G., Botstein, D., and Brown, P. O. (2000) Mol. Biol. Cell 11, 4241-4257[Abstract/Free Full Text]
7. Causton, H. C., Ren, B., Koh, S. S., Harbison, C. T., Kanin, E., Jennings, E. G., Lee, T. I., True, H. L., Lander, E. S., and Young, R. A. (2001) Mol. Biol. Cell 12, 323-337[Abstract/Free Full Text]
8. Norbeck, J., and Blomberg, A. (1997) J. Biol. Chem. 272, 5544-5554[Abstract/Free Full Text]
9. Boy-Marcotte, E., Lagniel, G., Perrot, M., Bussereau, F., Boudsocq, A., Jacquet, M., and Labarre, J. (1999) Mol. Microbiol. 33, 274-283[CrossRef][Medline] [Order article via Infotrieve]
10. Rep, M., Krantz, M., Thevelein, J. M., and Hohmann, S. (2000) J. Biol. Chem. 275, 8290-8300[Abstract/Free Full Text]
11. Godon, C., Lagniel, G., Lee, J., Buhler, J.-M., Kieffer, S., Perrot, M., Boucherie, H., Toledano, M. B., and Labarre, J. (1998) J. Biol. Chem. 273, 22480-22489[Abstract/Free Full Text]
12. Vido, K., Spector, D., Lagniel, G., Lopez, S., Toledano, M. B., and Labarre, J. (2001) J. Biol. Chem. 276, 8469-8474[Abstract/Free Full Text]
13. Jelinsky, S. A., and Samson, L. D. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1486-1491[Abstract/Free Full Text]
14. Blomberg, A., and Adler, L. (1992) Adv. Microbial Phys. 33, 145-212[Medline] [Order article via Infotrieve]
15. Blomberg, A. (2000) FEMS Microbiol. Lett. 182, 1-8[CrossRef][Medline] [Order article via Infotrieve]
16. Thomas, B. J., and Rothstein, R. (1989) Cell 56, 619-630[Medline] [Order article via Infotrieve]
17. Wach, A. (1996) Yeast 12, 259-265[CrossRef][Medline] [Order article via Infotrieve]
18. Wach, A., Brachat, A., Alberti-Segui, C., Rebischung, C., and Philippsen, P. (1997) Yeast 13, 1065-1075[CrossRef][Medline] [Order article via Infotrieve]
19. Inoue, Y., and Kimura, A. (1996) J. Biol. Chem. 271, 25958-25965[Abstract/Free Full Text]
20. Brachmann, C. B., Davies, A., Cost, G. J., Caputo, E., Li, J., Hieter, P., and Boeke, J. D. (1998) Yeast 14, 115-132[CrossRef][Medline] [Order article via Infotrieve]
21. Winzeler, E. A., Shoemaker, D. D., Astromoff, A., Liang, H., Anderson, K., Andre, B., Bangham, R., Benito, R., Boeke, J. D., Bussey, H., Chu, A. M., Connelly, C., Davis, K., Dietrich, F., Dow, S. W., El, Bakkoury, M., Foury, F., Friend, S. H., Gentalen, E., Giaever, G., Hegemann, J. H., Jones, T., Laub, M., Liao, H., Davis, R. W., et al.. (1999) Science 285, 901-906[Abstract/Free Full Text]
22. Gancedo, C., Llobell, A., Ribas, J. C., and Luchi, F. (1986) Eur. J. Biochem. 159, 171-174[Abstract]
23. Valadi, H., Månsson, Å., Adler, L., Blomberg, A., and Gustafsson, L. (2001) J. Microbiol. Methods 47, 51-57[CrossRef][Medline] [Order article via Infotrieve]
24. Warringer, J., and Blomberg, A. (2003) Yeast 20, 53-67[CrossRef][Medline] [Order article via Infotrieve]
25. Tamas, M. J., Luyten, K., Sutherland, F. C., Hernandez, A., Albertyn, J., Valadi, H., Li, H., Prior, B. A., Kilian, S. G., Ramos, J., Gustafsson, L., Thevelein, J. M., and Hohmann, S. (1999) Mol. Microbiol. 31, 1087-1104[CrossRef][Medline] [Order article via Infotrieve]
26. Yoshihara, K., Shimmada, Y., Karita, S., Kimura, T., Sakka, K., and Ohmiya, K. (1996) Appl. Environ. Microbiol. 62, 4663-4665[Abstract]
27. Daniel, R. K. S., and Gottschalk, G. (1995) J. Bacteriol. 177, 4392-4401[Abstract]
28. Kato, N., Yoshikawa, H., Tanaka, K., Shimao, M., and Sakazawa, C. (1988) Arch. Microbiol. 150, 155-159
29. Larsson, C., Nilsson, A., Blomberg, A., and Gustafsson, L. (1997) J. Bacteriol. 179, 7243-7250[Abstract]
30. Fraenkel, D. G. (1982) in The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression (Strathern, J. N. , Jones, E. W. , and Broach, J. R., eds) , pp. 1-37, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
31. Inoue, Y., and Kimura, A. (1995) Adv. Microb. Physiol. 37, 177-227[Medline] [Order article via Infotrieve]
32. Inoue, Y., Tsujimoto, Y., and Kimura, A. (1998) J. Biol. Chem. 273, 2977-2983[Abstract/Free Full Text]
33. Sakai, Y., Nakagawa, T., Shimase, M., and Kato, N. (1998) J. Bacteriol. 180, 5885-5890[Abstract/Free Full Text]
34. Marshall, J. H., May, J. W., Sloan, K., and Vasiliadis, G. E. (1986) J. Gen. Microbiol. 132, 2611-2614
35. Viikari, L., and Korhola, M. (1986) Appl. Microbiol. Biotechol. 24, 471-476
36. Uchida, K., Khor, O. T., Oya, T., Osawa, T., Yasuda, Y., and Miyata, T. (1997) FEBS Lett. 410, 313-318[CrossRef][Medline] [Order article via Infotrieve]
37. Ahmed, M. U., Brinkmann Frye, E., Degenhardt, T. P., Thorpe, S. R., and Baynes, J. W. (1997) Biochem. J. 324, 565-570[Medline] [Order article via Infotrieve]
38. Fernandez, M. R., Biosca, J. A., Norin, A., Jornvall, H., and Pares, X. (1995) FEBS Lett. 370, 23-26[CrossRef][Medline] [Order article via Infotrieve]
39. Wang, Z.-X., Kayingo, G., Blomberg, A., and Prior, B. A. (2002) Yeast 19, 1447-1458[CrossRef][Medline] [Order article via Infotrieve]
40. Luers, G. H., Advani, R., Wenzel, T., and Subramani, S. (1998) Yeast 14, 759-771[CrossRef][Medline] [Order article via Infotrieve]
41. Gutknecht, R., Beutler, R., Garcia-Alles, L. F., Baumann, U., and Erni, B. (2001) EMBO J. 20, 2480-2486[Abstract/Free Full Text]
42. González, E., Fernández, M. R., Larroy, C., Solà, L., Pericàs, M. A., Parés, X., and Biosca, J. A. (2000) J. Biol. Chem. 275, 35876-35885[Abstract/Free Full Text]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.