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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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').
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, DAK1
::KanMX4) was crossed with
yJN008 (W303, MAT
,
DAK2
::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
gpd1
gpd2
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.
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RESULTS |
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
dak1
), 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).
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Table II
Dihydroxyacetone kinase activity in cell extract from different strains
For further details about the strains see information in Table I.
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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).

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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 dak1 dak2 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.
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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).

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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).
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Dihydroxyacetone Is Toxic to Yeast Cells--
The phenotypic
importance of the DAK genes was tested during a number of growth
conditions for the dak1
, dak2
, and
dak1
dak2
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
dak1
dak2
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.

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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.
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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
dak1
dak2
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
dak1
strain, whereas dak2
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).

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Fig. 4.
Growth on DHA as sole carbon and energy
source for the double deletion strain
dak1 dak2 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.
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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).

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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.
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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.

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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.
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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 dak1
dak2
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).
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).

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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.).
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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
dak1
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
glo1
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
glo1
deletion and the dak1
dak2
glo1
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 glo1
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.
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DISCUSSION |
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
dak1
dak2
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
(dak1
dak2
) 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.

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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).
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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.