 |
INTRODUCTION |
Glycerol production in the yeast Saccharomyces
cerevisiae is known to have fundamental physiological functions in
two distinct adaptive responses: osmoregulation and anaerobic redox
control (1). At high external osmolarity, yeast cells compensate for the loss of water by increased glycerol production (2). This adaptive
response is regulated, at least in part, by an osmosensing and
signaling system called the
HOG1 (high osmolarity
glycerol) MAPK pathway (3). Two putative osmosensors encoded by
SLN1 (1) and SHO1 (2), which communicate with a
MAPK module involving the MAPK Hog1p and its activator Pbs2p, control
the HOG pathway (3).
Cells that are transferred from aerobic to anaerobic conditions are
faced with the problem of catabolizing substrate molecules into a
balanced blend of oxidized and reduced end-products, to maintain
intracellular redox balance. Fermentation of glucose to ethanol is a
redox inert process in the sense that the NADH produced in glycolysis
is reoxidized by conversion of pyruvate to ethanol and CO2.
However, the assimilation of sugar to biomass generates excess NADH,
which is re-oxidized by diversion of part of the sugar substrate to the
more reduced end product, glycerol (4-6). Glycerol production is
essential for S. cerevisiae to dispose of surplus reducing
power under anaerobic conditions, because mutants with a blocked
glycerol production abruptly stop glucose catabolism when shifted to
anoxic conditions (7, 8).
Glycerol is produced by reduction of the glycolytic intermediate
dihydroxyacetone phosphate to glycerol 3-phosphate (G3P) followed by a
dephosphorylation of G3P to glycerol. The first step is catalyzed by
NAD-dependent glycerol 3-phosphate dehydrogenase (GPD),
which is encoded by the two isogenes GPD1 and GPD2.
GPD1 encodes the major isoform in aerobically growing cells. The
expression of this isogene, which is required for growth at high
osmolarity (9), is induced by hyperosmotic stress and is a target gene for the HOG pathway (10). The second isogene, GPD2, is
required for glycerol production in the absence of oxygen, and its
expression is stimulated under these conditions (8). Hence, the two
GPD genes have defined physiological roles in the adaptation
of S. cerevisiae to high osmolarity and anoxic conditions.
We previously reported the purification and characterization of two
isoforms of a glycerol 3-phosphatase (11). In this work we have studied the expression of the corresponding genes, GPP1
(RHR2) and GPP2 (HOR2) and the
phenotype of the deletion mutants. We report that the GPP
isogenes are differentially expressed under conditions of osmotic and
anaerobic stress and that the expression of GPP1 is under
strong influence of protein kinase A (PKA) activity. We also show that
the two isogenes have interchangeable functions under osmotic stress
but that deletion of GPP1 results in an anaerobic growth
defect. This study also reveals a role for glycerol metabolism in
oxidative stress protection, because the
gpp1
/gpp2
deletion mutant is hypersensitive
not only to osmotic stress and anoxic conditions but also to the
oxidant paraquat.
 |
EXPERIMENTAL PROCEDURES |
Strains, Media, and Yeast Genetic Methods--
S.
cerevisiae strains and genotypes are listed in Table
I. Cells were routinely grown at 30 °C
in either YPD medium supplemented with 120 µg/ml adenine or in a
synthetic yeast nitrogen base (YNB) medium (Difco) supplemented with
2% glucose and necessary amino acids and nucleotides, to a final
concentration of 120 µg/ml for each. Liquid cultures were grown in
250-ml E flasks on a rotary shaker at 200 rpm and were inoculated with
1% (v/v) from an overnight YNB culture. Media used for anaerobic
incubations were supplemented with ergosterol (10 mg/liter) and Tween
80 (420 mg/liter) (12, 13). Anaerobic growth in liquid medium was
performed either in 100-ml serum flasks (Bellco Glass Inc.) equipped
with rubber stoppers that were tightened with aluminum or screw cap
sealing. Prior to anaerobic incubation, culture media were flushed for 10 min with nitrogen gas (AGA plus, Sweden) containing less than 5 ppm
of oxygen. Alternatively, anaerobic growth was followed in 125-ml
sealed E flasks containing 25 ml of medium that was continually flushed
with nitrogen gas. Anaerobic incubation of diluted cell suspensions
spotted to agar plates were conducted in anaerobic jars made anoxic by
developing hydrogen gas in the presence of a palladium catalyst. Cell
density was estimated by measuring optical density at 610 nm in 1-cm
cuvettes (OD610), using a Novaspec II photometer (Amersham
Pharmacia Biotech, Sweden). Standard yeast genetic methods were used
throughout (14, 15).
Escherichia coli DH5
(16) was used for cloning and
amplification of DNA. Bacterial cultures were grown in 2 × LB
medium (17).
Cloning and Disruption of GPP1 and GPP2--
The GPP2
gene was isolated from the S. cerevisiae lambda clone 6592 (GenBankTM accession number U188139). A 2574-bp
BamHI/SpeI fragment encompassing the 753-bp ORF
plus 1118 bp upstream of the translational start was isolated and
integrated into the BamHI/XbaI site of the pRS316
vector. The GPP1 gene was isolated from a yeast genomic
library in the pCS19 vector (18, 19) by colony hybridization using
standard techniques and the GPP1-specific 5'-TGT GGT CAA AGG
CAT TGC GAT GG-3' oligonucleotide (20) as a probe. The 2815-bp XbaI/SalI fragment covering the 753-bp
GPP1 ORF and 1199 bp upstream of the translational start was
cloned into the pRS316 vector. For multicopy expression
NotI/SalI (GPP1) or
NotI/ClaI (GPP2) fragments were transferred from
the centromeric pRS316 to the 2µ vector pRS326.
Deletion of the GPP1 gene was accomplished by the long
flanking homology PCR-targeting technique (21, 22). In the first step,
a set of primers (5'-TGTGTGAGTTCCTCTTTTCTT-3' and
5'-TCAAAGGCATTGCGATGGTT-3') was used to amplify 263 bp of genomic DNA
from S. cerevisiae W303, upstream from the third codon in
the GPP1 ORF. A second set (5'-CGCTAAGGATGACTTGTTGA-3' and
5'-CTCTAACTTCTCGTCGTACT-3') was used to amplify a 358-bp fragment from
the ninth codon in the GPP1 ORF upstream the stop codon. The
5'-end of the primers adjacent to the insertion site carried 25 nucleotide extensions homologous to the 5' and 3' regions of the
hisGMX6 or kanMX4 disruption cassette of plasmid
pFA6a-hisGMX6 and pFA6-kanMX4 (21). In the second
PCR reaction, pFA6a-hisGMX6 and pFA6-kanMX4 were
used as templates and the 5' and 3' homologous regions of the first PCR
reaction were fused to the disruption cassette by serving as primers
together with the upstream forward and downstream reverse primers of
the flanking regions, thus producing the ORF targeting cassette. This
cassette was transformed into a haploid S. cerevisiae W303
strain, and independent transformants were selected for verification of
GPP1 replacement. Using a set of primers (forward:
5'-CAAGCAGGAAATCCGTATCA-3' and reverse 5'-TCATATGGAGCAATCCCACT-3') hybridizing upstream and downstream, respectively, of the disruption cassette chromosomal DNA was amplified. The length of the PCR products
was verified by agarose-gel electrophoresis. The GPP2 ORF
was disrupted in a similar way using a set of primers
(5'-CAAGTGAGGACTTTTCGGAT-3' and 5'-GTAGTCAATCCCATTCCGAA-3') to amplify
a 346-bp fragment upstream from the fourth codon in the ORF. The second
set (5'-GGACGATCTGTTGAAATGGT-3' and 5'-CCTGTCCACTTTCAAGTTGCT-3') was
used to amplify a 287-bp fragment from the seventh codon in the
GPP2 ORF downstream the stop codon. Correct integration of
the disruption modules into the GPP1 and GPP2
alleles was verified by PCR.
The gpp1
/gpp2
double mutant was constructed
by crossing of the single mutants. Tetrads gave rise to viable double
mutants on aerobic incubation in complete medium.
GPD1 and GPD2 were overexpressed from their own
promoters in the YEplac112 (23) multicopy plasmid, transformed into the gpd1
/gpd2
mutant.
Northern Blotting--
Total RNA was isolated by standard
procedures at indicated time points from cultures treated as described
in the figure legends. RNA samples were screened on ethidium bromide
agarose gels and quantified by spectrophotometry, using the Beckman
DU65 (Beckman Instruments) nucleic acid program. Samples containing 15 µg of total RNA were denatured and run on low formaldehyde (2.5%
v/v) agarose gels at 10 V/cm for 75 min, tested for quality at 254 nm
on thin-layer chromatography plates, and blotted to positively charged
nylon membranes (Roche) by capillary transfer, using 10× SSC as
transfer buffer. Blotted filters were cross-linked by 1-min exposure to
low wavelength UV and baked at 80 °C for 2 h. Prehybridization was performed for 3-4 h at 55 °C in 5× SSC, 10 mM
sodium phosphate (pH 6.5), 10 × Denhardt's solution, 2% SDS,
and 100 µg/ml herring sperm DNA.
Hybridization was performed at 55 °C for 18-20 h using the same
solution supplemented with 10% polyethylene glycol 4000 and having
labeled oligonucleotides added at 5 ng/ml. The filters were washed
twice in 1 × SSC/1% SDS, for 20 min at room temperature and once
for 15 min at the hybridization temperature. Membranes were stripped
for rehybridization by shaking in sterile water at 80 °C for 10 min.
To quantify transcript levels, signal intensity was quantified with a
Molecular Dynamics PhosphorImager and normalized to that for
ACT1 or IPP1 mRNA. For most experiments two
or more independent RNA blot analyses were performed for each growth
condition and transcript examined.
The oligonucleotides used were 5'-labeled with 25 mCi of
[
-32P]ATP (Amersham Pharmacia Biotech) and 5 units of polynucleotide kinase (Roche Molecular Biochemicals) per 50 ng
of probe, left at 37 °C for 30 min. Unincorporated ATP was displaced
using a Sephadex G-50 (Amersham Pharmacia Biotech) mini column.
Sequences of oligonucleotides used were: 5'-TGTGGTCAAAGGCATTGCGATGG-3'
to probe for GPP1 mRNA, and
5'-CTTGCTCATTGATCGGATATCCTAA-3' to assay for GPP2 mRNA.
Oligonucleotides used to probe for ACT1 and IPP1 were 5'-ATCGATTCTCAAAATGGCGTGAGG-3' and 5'-TGT CTG GTA GTG TAG GTC ATT
AGT-3', respectively. The specificities of the oligonucleotides used
for probing GPP1 and GPP2 mRNA were
controlled using the gpp1
and gpp2
strains.
Analysis of Glycerol and Glycerol 3-Phosphate--
Sampled cell
suspensions of 1 ml were analyzed for total (intra- plus extracellular)
glycerol by boiling for 10 min followed by centrifugation and enzymatic
determination of the glycerol content of the supernatant using a
commercial glycerol analysis kit (Roche Molecular Biochemicals). For
glycerol 3-phosphate analysis, cells from 10-ml samples were collected
by filtration (0.45 µm, Millipore). Filters were transferred to 0.5 M trichloroacetic acid containing 17 mM
EDTA and kept on ice for 15 min. The trichloroacetic acid was removed
by repeated extraction with 4 ml of water-saturated diethylether.
Residual diethylether was removed by percolation with water-saturated
nitrogen gas for 10 min (24). The extract was cleared by
centrifugation, and the glycerol 3-phosphate content was determined by
the enzymatic method described by Bergmeyer (25) using a Cobas FARA analyzer.
Preparation of Cell-free Extracts--
Cells were washed twice
in TRED buffer (10 mM triethanolamine, 1 mM
EDTA, pH 7.5) and resuspended in the same buffer plus 1 mM
dithiothreitol and a protease inhibitor mix (Complete, Roche) added in
amounts as recommended by the supplier. Extracts were prepared by
disruption of cells using acid-washed glass beads and subsequent
centrifugation (14,000 × g for 15 min). The obtained supernatants were desalted by gel filtration according to procedures described previously (26).
Protein Determination--
For enzyme assays protein
concentration was determined by the method of Bradford (Bio-Rad),
whereas metabolite levels were related to cell protein using the Lowry
method for protein determination. Bovine serum albumin was used as
standard for both methods.
Enzyme Assay--
Glycerol 3-phosphatase was assayed as
described previously (11). Briefly, cell-free extracts were incubated
in 20 mM Tricine-HCl (pH 6.5), 5 mM
MgCl2, and 10 mM DL-glycerol
3-phosphate in a total volume of 1.0 ml. After starting the reaction,
samples of 90 µl were withdrawn at different time points and the
reaction was stopped by adding 10 µl of 50% HClO4.
Inorganic phosphate was analyzed according to a previous study (27),
and the reaction rate was calculated from the slope of a linear plot of
released phosphate versus time. All glassware used was
immersed overnight in 1 M HCl and rinsed thoroughly in
distilled water, to eliminate phosphate contamination.
Western Blot Analysis--
Proteins were separated by SDS-PAGE
in 10% acrylamide gels at 125 V for 1.5 h using a Mini-PROTEAN II
electrophoresis system (Bio-Rad) and transferred to Hybond membranes
(Amersham Pharmacia Biotech, UK), according to the manufacturers'
protocols. The membranes were incubated for 1 h at room
temperature in 20 mM Tris, pH 7.6, 0.8% NaCl, 0.1% Tween
20 (buffer A) supplemented with 5% blocking reagent (Amersham
Pharmacia Biotech, UK) and then for 1 h with rabbit anti-Gpp
antibody diluted 1:5000 in buffer A. The Gpp antibodies were kindly
provided by Dr. Dunn-Coleman, Genencor International (Palo Alto, CA).
Membranes were washed for 15 min followed by 2 × 5 min in buffer
A. The membranes were probed for 1 h with a 1:15,000 dilution of
anti-rabbit peroxidase-linked antibodies (Amersham Pharmacia Biotech,
UK) in buffer A. Blots were washed as before, and antibody detection
was performed using the ECL labeling system (Amersham Pharmacia
Biotech, UK).
 |
RESULTS |
Loss of GPP Genes Results in Osmosensitivity and Poor Growth under
Anaerobic Conditions--
Because glycerol production is strictly
required by S. cerevisiae for osmoregulation and anoxic
redox adjustments (8), we examined the importance of the glycerol
3-phosphate genes GPP1 and GPP2 for growth at
high osmolarity and anaerobiosis. The single ggp1
and
ggp2
mutants showed no obvious growth defects on aerobic incubation in YNB medium, whereas the
gpp1
/gpp2
double mutant grew somewhat more
slowly under these conditions (Fig.
1A). The growth of the single
gpp1
and gpp2
mutants also appeared
indistinguishable from that of the wild type under NaCl stress, whereas
the gpp1
/gpp2
double mutant was strongly
inhibited by increased salinity (Fig. 1, A and
B), or osmotically comparable concentrations of sorbitol (data not shown).

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 1.
A, growth of the wild type (W303-1A)
strain, the gpp1 , gpp2 , and
gpp1 /gpp2 mutants in liquid YNB medium
( ) and YNB medium supplemented with 0.7 M NaCl ( ).
B, growth of the wild type strain, the gpp1 ,
gpp2 , and gpp1 /gpp2 mutants
on YPD medium containing 0, 0.3, 0.5, or 1.0 M NaCl. Cells
were grown overnight in liquid YNB medium, cell density was adjusted to
A610 = 1.0, and cells were spotted in serial
10-fold dilutions. Plates were scored after 3 days of incubation.
C, GPP1 and GPP2 mRNA levels in
wild type cells incubated in YNB media for 4 h with various
concentrations of NaCl. ACT1 mRNA served as internal
control. The graph shows quantified mRNA levels for GPP1
( ) and GPP2 ( ) relative to those of ACT1,
the highest mRNA level being set to 1.
|
|
Under anaerobic conditions (Fig. 2,
A and B) cells lacking GPP1 displayed
a much prolonged lag phase and a decreased exponential growth rate,
whereas gpp2
mutants grew like the wild type strain. Growth of the gpp1
/gpp2
mutant was severely
inhibited by transfer to anaerobiosis and only on prolonged incubation
growth occurred in liquid medium (Fig. 2A). The mechanism
underlying this slow adaptation to anaerobiosis is unexplored, although
an involvement of the recently described dihydroxy acetone
pathway (20) for glycerol production is possible.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 2.
A, growth of the wild type (W303-1A)
strain ( ), the gpp1 ( ), gpp2 ( ),
and gpp1 /gpp2 ( ) mutants in liquid YNB
medium under anaerobic conditions. B, growth of the wild
type strain, the gpp1 , gpp2 , and
gpp1 /gpp2 mutants when spotted in serial
10-fold dilutions onto YNB medium incubated in an anaerobic jar. Plates
were scored after 2 days of incubation. C, GPP1
mRNA levels in wild type cells incubated in YNB medium and then
shifted to anaerobic conditions. Cells were shifted to new conditions
at time point zero. ACT1 mRNA served as internal
control. The graph represent mRNA levels for GPP1
relative to those of ACT1, the highest relative mRNA
level being set to 1.
|
|
Glycerol Production Is Blocked in a gpp1/gpp2 Double Mutant but
Little Affected in gpp Single Mutants--
To clarify the role of the
GPP genes in glycerol biosynthesis, we analyzed glycerol
production of wild type and mutant cells following transfer to
increased osmolarity or anaerobiosis (Fig. 3). As expected from the growth
phenotype, the two single mutants showed stimulated glycerol production
much like the wild type, whereas the double mutant did not produce any
significant amounts of glycerol under any conditions. Unexpectedly, the
gpp1
mutant produced glycerol like the wild type under
anoxic conditions, despite the growth defect of this mutant under these
conditions (see above).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 3.
Kinetics of total glycerol production by wild
type (W303-1A), the gpp1 , gpp2 , and
gpp1 /gpp2 strains after shift
to YNB medium containing 0 M ( ), 0.5 M
( ), or 1.0 M ( ) NaCl, or YNB medium incubated under
anaerobic conditions ( ). Cells were grown to
A610 = 0.8-1.0, collected by centrifugation,
and suspended at half of the original cell density with fresh YNB
medium containing an appropriate concentration of NaCl.
|
|
Expression of Both GPP Genes Is Affected by Osmotic Shock Whereas
Expression of GPP1 Is Also Influenced by Anaerobic Conditions and PKA
Activity--
We then analyzed the extent to which the observed
glycerol response is associated with changes in GPP gene
expression. Northern analysis (Fig. 1C) confirmed previous
reports of osmotic induction of the GPP gene expression (11,
28, 29), demonstrating a weak response of the GPP1 gene,
whereas GPP2 expression was strongly induced in a salt
concentration-dependent fashion. After shift to anaerobic
conditions, the mRNA level of GPP1 showed a transient increase (Fig. 2C), whereas that of GPP2 remained
unaltered (not shown). The induction profiles of GPP1 or
GPP2 in mutants carrying only one of the isogenes were
similar to those observed for wild type cells (not shown), indicating
that loss of one isoform does not markedly affect expression of the other.
The PKA pathway plays a role in integrating growth control with
environmental stress (30, 31), and works antagonistically with the HOG
pathway on a variety of osmoresponsive genes (32). Although it was
previously reported that the osmotic induction of GPP2 is
PKA-independent (28, 29, 33), we noted that the basal GPP1
mRNA level is strongly dependent on PKA activity. This PKA effect
is in contrast to a previous report (28) but in agreement with other
findings (34). In a tpk1w1
tpk2
tpk3
strain having a low PKA activity
(35), GPP1 expression was severely decreased but still
responsive to osmotic stress (Fig. 4).
Attenuated PKA activity also did not affect the anaerobic response of
GPP1 gene expression (data not shown). Apparently, the PKA
pathway exerts an activating or derepressing effect on GPP1
expression as was previously noted for that of yeast ribosomal protein
genes (36) and the AP-1 factor-encoding GCN4 gene (37).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 4.
GPP1 mRNA levels in S. cerevisiae wild type strain SP1 (TPK1 TPK2
TPK3), S7-7A (TPK1 tpk2
tpk3 ), and S18-1DA
(tpk1w1 tpk2
tpk3 ) cells shifted to fresh YNB medium
containing 0 or 1 M NaCl. Experiments were initiated
with cells that were grown to A610 = 0.8-1.0
and diluted to half the original cell density with fresh YNB medium
containing an appropriate concentration of NaCl. Cells were sampled for
RNA extraction at the indicated times. IPP1 mRNA served
as internal control. The graphs show quantified GPP1
mRNA levels relative to those of IPP1 for the different
strains, and the highest level for each set of experiments was adjusted
to 1.0.
|
|
Determination of the specific enzyme activity of Gpp1p and Gpp2p
following transfer of cells to increased osmolarity or anaerobic conditions (Fig. 5), indicates that the
Gpp1p isozyme contributes between 70-90% of the glycerol
3-phosphatase activity. Because Gpp2p activity does not increase after
shift to anoxic conditions, this enzyme is responsible for only about
10% of the phosphatase activity in anaerobically grown cells.
Moreover, the absence of activity in the
gpp1
/gpp2
mutant confirms that the enzyme
assay specifically detects glycerol 3-phosphatase and that this
activity is responsible for the observed dephosphorylation of G3P.
Taken together, measurements of transcript levels and specific enzyme activity suggest that a major control on the cellular glycerol 3-phosphatase activity is exerted at the mRNA level.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 5.
Changes in glycerol 3-phosphatase-specific
activity of wild type (W303-1A), gpp1 ,
gpp2 , and
gpp1 /gpp2
strains incubated in YNB medium containing 0 M ( ),
0.5 M ( ), and 1.0 M ( ) NaCl, or in YNB
medium incubated under anaerobic conditions ( ). Experiments
were initiated with cells that were grown to
A610 = 0.8-1.0 and diluted to half the original
cell density with fresh YNB medium containing an appropriate
concentration of NaCl. Samples of 50 ml were taken at the indicated
time points and analyzed for glycerol 3-phosphatase specific
activity.
|
|
Overexpression of GPP1 or GPP2 Does Not Increase Glycerol
Production--
To examine the role of glycerol 3-phosphatase in
controlling the metabolic flux to glycerol, we introduced multicopy
plasmids carrying either GPP1 or GPP2 under the
control of their native promoters in wild type cells. The transformed
cells showed a 50- to 100-fold increased expression of either isoform
as demonstrated by enzyme activity measurements (Fig.
6A) or immunoblot analysis (Fig. 6C). Despite the more than 10-fold increase in
phosphatase activity, there was no significant change in the glycerol
production by cells cultured in basal medium (Fig. 6B).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 6.
A, glycerol 3-phosphatase specific
activity of the wild type (W303-1A) strain transformed with the
multicopy plasmid pRS326 (Wt) or the same plasmid containing
either the GPP1 or GPP2 gene controlled by its
own promoter. Enzyme activity was analyzed using exponentially growing
cultures (A610 = 0.8-1.0) grown aerobically in
YNB medium containing 0 M (black bars); 0.5 M (gray bars) or 1.0 M NaCl
(open bars), or YNB medium incubated under anaerobic
conditions (striped bars). B, effect on glycerol
production by overexpression of GPP1 and GPP2.
Samples from exponentially growing cells transformed and cultured
aerobically in YNB medium without NaCl as in A, were
analyzed for total glycerol. C, Western blot analysis of
GPP1 and GPP2 overexpression. Crude extract
prepared from equal aliquots of exponentially growing cells transformed
as in A and cultured aerobically in YNB medium containing 0 or 0.7 M NaCl, was analyzed using anti-GPP
antibodies.
|
|
Intracellular G3P Levels Are Strongly Affected by Phosphatase
Activity--
To discover whether loss of GPP genes leads
to intracellular accumulation of G3P, we determined the G3P pool in
wild type and mutant cells following exposure to environmental changes. This analysis pointed to a minor influence of GPP2 on the
G3P level under nonstressed conditions (Fig.
7A). During anaerobic growth
or under osmotic stress, the G3P level of the gpp2
mutant became 4- to 6-fold higher than in the wild type strain. Deletion of
GPP1 caused, on the other hand, an immediate 4-fold increase of the G3P level, and this level increased another 2-fold after shift
to anaerobic conditions. In the gpp1
/gpp2
mutant, the G3P pool was about 40-fold increased in aerobically grown
cells irrespective of salinity, whereas anaerobic incubation provoked a
progressive further accumulation to a level about 100-fold higher than
that of the wild type. These results show that the size of the G3P pool
is sensitively dependent on the glycerol 3-phosphatase activity.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 7.
A, glycerol 3-phosphate accumulation by
wild type (W303-1A), gpp1 , gpp2 ,
gpp1 /gpp2 , and
gpp1 /gpp2 /gut2 strains after
shift to YNB 0 M ( ); 0.5 M ( ) and 1.0 M ( ) NaCl; or in YNB medium incubated under anaerobic
conditions ( ). Cell cultures were grown in YNB medium to
A610 = 0.8-1.0, and experiments were started
with cells that were diluted to half the original cell density with
fresh YNB medium containing an appropriate concentration of NaCl.
Samples were taken at the indicated time points and analyzed for
intracellular glycerol 3-phosphate. B, anaerobic growth and
intracellular glycerol 3-phosphate concentration of wild type cells
( ) and the gpp1 /gpp2 mutant with ( )
and without ( ) 10 mM acetaldehyde.
|
|
We also noted that G3P accumulation in the double mutant was much
higher after transfer to anoxic conditions than after shift to 0.5 M NaCl, whereas both conditions gave rise to a similar increase of glycerol production in wild type cells (cf. Fig.
3). As a possible explanation for this effect we could rule out aerobic reoxidation of G3P via the GUT2-encoded mitochondrial G3P
dehydrogenase (38, 39), because the salt-induced accumulation of G3P in a gpp1
/gpp2
/gut2
triple
mutant was similar to that of a gpp1
/gpp2
double mutant (Fig. 7A). We next considered the possibility
that the anaerobically induced isoform of glycerol-3-phosphate
dehydrogenase, Gpd2p, allows for stronger G3P accumulation than the
osmostress-induced Gpd1p isozyme, by being less sensitive to product
(G3P) inhibition. To test this idea we overexpressed GPD1 or
GPD2 in gpd1
/gpd2
double mutants
and assayed the glycerol-3-phosphate dehydrogenase activity in extracts
in the presence of 5 and 10 mM G3P. These measurements
demonstrated, however, that both isoforms responded very similarly to
increased product concentrations (data not shown). We finally examined
whether anaerobic accumulation of NADH contributes to the massive
anoxic expansion of the G3P pool in
gpp1
/gpp2
mutants. To this end, the G3P
pool was analyzed in double mutants following anaerobic incubation in
the presence or absence of 10 mM acetaldehyde (Fig.
7B). S. cerevisiae efficiently reduces added acetaldehyde to ethanol (8), leading to regeneration of
NAD+ from NADH. After addition of acetaldehyde, the G3P
level decreased to about 50% and growth resumed. Following incubation,
G3P levels increased again and growth gradually declined. These effects
were clearly due to consumption of added acetaldehyde, because repeated addition released growth inhibition (data not shown). Hence, we conclude that an increased NADH/NAD+ ratio constitutes a
strong driving force for the anaerobic accumulation of G3P in
gpp1
/gpp2
mutants.
Interplay between Gpd and Gpp Isozymes for Tolerance to Osmotic and
Anaerobic Stress--
To examine a possible interplay between the Gpd
and Gpp isozymes in the glycerol production pathway, we constructed the
four possible gpd/gpp double mutants and analyzed
their growth behavior at high salinity and anaerobiosis (Fig.
8). Growth was limited at high salinity
only in cells lacking GPD1, whereas under anaerobic conditions growth limitation was seen only for cells lacking
GPD2. The anaerobic plate test used was not sufficiently
sensitive to detect growth changes due to the absence of
GPP1 (cf. Fig. 2). Nevertheless, the result
supports the notion that flux in the glycerol pathway is primarily
controlled at the level of glycerol 3-phosphate dehydrogenase.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 8.
Growth phenotype of double mutants deleted
for one of the GPD genes and one of the GPP
genes in the four possible pairwise combinations. Cells were
spotted in serial 10-fold dilutions onto YPD medium containing 0 M and 1.0 M NaCl, or on YPD medium incubated
under anaerobic conditions. The two bottom series show as controls the
gpd1 and gpd2 single mutants (see
text).
|
|
Diminished GPP1 Expression in Cells Cultured on a Nonfermentable
Carbon Source--
Cultured on the nonfermentable carbon source,
ethanol, S. cerevisiae increased GUT2 gene
expression (38, 39) leading to respiratory oxidation of produced G3P
via the G3P shuttle (40). We therefore examined the fate of the
GPP1 and GPP2 transcript levels in cells cultured
on ethanol as the sole carbon source. Although the GPP2
mRNA level appeared to be insensitive to the carbon source, the
GPP1 mRNA level dropped to about one-third of that
observed in cells cultured on glucose (Fig.
9A). Following exposure to
increased extracellular osmolarity, both GPP1 and GPP2 mRNA levels increased, demonstrating that the
osmotic response operates under these conditions. Overexpression of
GPP1 or GPP2 did not increase the osmotolerance
of ethanol-grown cells (Fig. 9B).

View larger version (48K):
[in this window]
[in a new window]
|
Fig. 9.
A, GPP1 and GPP2
mRNA levels in wild type (W303-1A) cells cultured to
A610 = 0.6 on glucose (YNB medium) or YP medium
containing (2% w/w) ethanol, supplemented with 0 M or 0.3 M NaCl. ACT1 mRNA served as internal
control. Transcript levels normalized to control mRNA are given as
relative values, the highest level for each transcript adjusted to 1.0. B, growth of the wild type strain (Wt) or the
same strain transformed with the multicopy plasmid pRS326 containing
either the GPP1 or GPP2 gene, or the
gut2 mutant when spotted in serial 10-fold dilutions onto
YP medium containing (2% w/w) ethanol or glucose and supplemented with
0 M or 0.6 M NaCl. Plates were scored after 3 days (glucose) or 5 days (ethanol) of incubation.
|
|
Loss of GPP Genes Results in Sensitivity to Oxidative
Stress--
Because glycerol production appears to be crucial for
redox regulation, and glycerol has been suggested as a radical
scavenger (41), we explored a possible role of the GPP genes
under conditions of oxidative stress. A plate test (Fig.
10A) revealed
hypersensitivity of the gpp1
/gpp2
mutant to
the superoxide anion-generating agent paraquat, whereas the single
mutants grew like wild type. Because a
gpd1
/gpd2
mutant also showed increased
sensitivity, glycerol metabolism per se seems required for
tolerance to paraquat. The difference in sensitivity between the two
double mutants might be due to the accumulation of G3P in the
gpp1
/gpp2
mutant, which may increase
vulnerability to oxidative conditions. However, this accumulation does
not generate increased sensitivity by affecting the electron flow to
the respiratory chain via Gut2p, because a
gpp1
/gpp2
gut2
triple mutant
is equally sensitive to paraquat as a
gpp1
/gpp2
mutant (data not shown).

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 10.
A, tolerance to oxidative stress
conditions of wild type (W303-1A) cells and the gpp1 ,
gpp2 , gpp1 /gpp2 ,
gpd1 /gpd2 mutants spotted in serial 10-fold
dilutions onto YPD medium containing 0.57 mg/ml paraquat. Plates were
scored after 5 days of incubation. B, GPP2
mRNA levels following exposure to paraquat in the W303-1A strain
and its isogenic msn2 /msn4 mutant and the
SP1 (TPK1 TPK2 TPK3) strain and its isogenic S7-7A
(TPK1 tpk2 tpk3 ) and S18-1DA
(tpk1w1 tpk2 tpk3 )
mutants. Experiments were initiated with cells that were grown to
A610 = 0.8-1.0 and diluted to half the original
cell density with fresh YNB medium containing paraquat to give a final
concentration of 0.16 mg/ml. IPP1 mRNA served as
internal control. The graphs show quantified GPP2 mRNA
levels relative to those of IPP1 for the different strains,
and the highest level for each set of experiments was adjusted to
1.0.
|
|
Consistent with the sensitivity of the
gpp1
/gpp2
double mutant to oxidative
stress, exposure to paraquat elicited increased expression of
GPP2 (Fig. 10B). Expression of GPP1
was not markedly affected (data not shown). The stress-response element
(STRE)-binding transcription factors Msn2p and Msn4p have been reported
to mediate, among others, induction of gene expression by oxidative
stress (42), and their nuclear localization is controlled by PKA (43). Although the promoter of GPP2 contains a single STRE element
(at position
272 nt), mutants lacking the genes MSN2 and
MSN4 or having attenuated PKA activity showed a similar
degree of induction, although with slightly delayed kinetics (Fig.
10B).
 |
DISCUSSION |
We here report cloning and characterization of GPP1 and
GPP2, two yeast isogenes for glycerol 3-phosphatase. Mutants
deleted for both genes fail to produce glycerol, exhibit strong
osmosensitivity, and arrest growth when shifted to anaerobic
conditions, demonstrating that glycerol 3-phosphatase is essential for
glycerol production and confirming the vital importance of the glycerol
pathway for tolerance to osmotic and anaerobic stress.
GPP1 and GPP2 Have Redundant Roles in Osmoadaptation Whereas GPP1
Is Specifically Required for Adaptation to Anaerobic
Conditions--
The observation that expression of both
GPP1 and GPP2 is stimulated by osmotic stress
suggested involvement of both genes in the osmoregulatory glycerol
response. This notion was verified by the finding that neither the
gpp1
nor the gpp2
single mutants exhibited
osmosensitivity (Fig. 1A). Thus, the strongly homologous GPP isogenes appear to be functionally exchangeable when
expressed at sufficiently high level. The osmostress-induced expression of number of genes is reported to depend on the HOG pathway
and/or the PKA pathway (28, 32, 44, 46, 47). Previous studies have
shown that the osmostress-induced expression of GPP1 and GPP2 is HOG pathway-dependent (11, 28, 29),
whereas the hyperosmotic activation of GPP2 transcription
was reported to be independent of PKA activity (33) and the STRE
binding, general stress-response factors Msn2p and Msn4p (48). The
present work shows that the osmotic response of GPP1
expression is also largely PKA-independent, whereas the basal
expression of this gene is strongly dependent on PKA activity. Hence,
PKA activity influences cellular glycerol 3-phosphatase activity by
affecting GPP1 expression but does not significantly
contribute to altering GPP gene expression in response to
changed medium osmolarity.
Although the GPP isogenes have redundant roles in
osmoregulation, the anaerobic growth defect of the gpp1
mutant (Fig. 2) shows that full adaptation to anoxic conditions is
dependent on the GPP1 gene. This is consistent with the
observations that the weakly expressed GPP2 gene does not
respond to anoxic conditions and is responsible for only a small
fraction of the total glycerol 3-phosphatase activity. Following a
shift to anaerobic conditions, the gpp1
mutant increases
its G3P level to about 17 mM (calculated from data of Fig.
7A), approximately a 15-fold increase as compared with wild
type levels. Considering that Gpp2p has a Km value
for G3P at 3.9 mM (11), the elevated G3P concentration will
substantially enhance the catalytic activity of the Gpp2p isoenzyme,
which might explain the surprising finding that the gpp1
mutant maintains wild type glycerol production (Fig. 3A), despite its low phosphatase content. The observed anaerobic growth defect might therefore be associated with the accumulation of G3P
rather than result from poor NADH reoxidation. In fact, the massive
NADH-driven anaerobic buildup of G3P in the
gpp1
/gpp2
mutant seems to be the prime
reason for the anoxic growth arrest of this mutant. This notion is
supported by the observation that acetaldehyde addition decreases the
G3P accumulation of the double mutant and alleviates its growth
inhibition (Fig. 7B), whereas addition of acetoin,
which, similar to acetaldehyde, prevents excessive NADH accumulation
(7), causes no reduction of the G3P pool, and does not relieve the
anaerobic growth arrest (data not shown). Hence, accumulation of G3P
rather than NADH seems responsible for the anaerobic growth inhibition
of the double mutant.
Glycerol 3-Phosphatase Is Not Rate-limiting in the Glycerol
Metabolism--
In wild type cells the G3P phosphatase did not appear
to limit the flux to glycerol under basal growth conditions, because overproduction of either of the GPP isozymes did not significantly enhance glycerol production. However, because several pathways compete
for G3P the cell has to coordinate the flux from G3P to glycerol with
that to glycerolipids (49, 50) and the G3P shuttle (Fig.
11). Although the flux to glycerolipids
is probably low (cf. ref. 51), the G3P shuttle efficiently
competes for G3P in cells grown on nonfermentable carbon sources. Cells
cultured on ethanol induce Gut2p activity due to derepression of
GUT2 (38, 39), and do not produce significant amounts of
glycerol (40). Thus, the derepressed cells direct the reducing
equivalents to the respiratory chain rather than having them sacrificed
on glycerol, which appears to be the rationale behind the diminished
GPP1 expression in cells cultured on ethanol. The cAMP-PKA
pathway may contribute to this control, because cells cultured on
ethanol have low cAMP levels (52), and diminished PKA activity
decreases GPP1 expression (Fig. 4). A requirement for low
GPP1 expression in cells growing on nonfermentable
substrates may explain why GPP1 is regulated oppositely to
most of the PKA target genes studied, which being involved in
stationary phase functions or stress responses, become transcriptionally activated by decreased PKA activity (30, 31). However, when exposed to hyperosmotic stress, cells cultured on ethanol
increase expression of both GPP1 and GPP2 and
this induction appears fully sufficient for an appropriate
osmoregulatory glycerol response, because overexpressing either the
GPP1 or GPP2 gene does not further enhance
osmotolerance. It thus appears that glycerol-producing wild type cells,
despite the complex metabolic requirements, maintain the phosphatase
activity at levels giving this reaction little rate control in the
glycerol pathway.
A Role for the Glycerol Metabolism in Oxidative Stress
Protection--
Wong and colleagues (53) observed increased
sensitivity to reactive oxygen species of a S. cerevisiae
mutant, having defective glycerol production (9). They also noted that
sensitivity to oxidative stress was reversed in mutants engineered
to produce mannitol, a polyol that is reported to protect
phosphoribulokinase against inactivation by hydroxyl radicals in
isolated thylakoids (54). Consistent with these findings we observed
that mutants with completely blocked glycerol production display
increased sensitivity to the superoxide-producing agent paraquat.
Furthermore, we noted that oxidative conditions generated by either
paraquat (Fig. 10B) or 0.2 mM
H2O2 (data not shown) stimulated
GPP2 gene expression. Although glycerol has been shown to
serve as an efficient hydroxyl radical scavenger in vitro
(41), other protective mechanisms are equally plausible. It was
recently pointed out that glycerol metabolism might serve to generate
NADPH (20, 55), an important electron donor for several defense systems
against oxidative stress (56). A transhydrogenase function converting
NADH to NADPH (cf. Fig. 11) was suggested to result from
recycling of glycerol to dihydroxyacetone phosphate via two recently
identified enzymes, a putative NADPH-dependent glycerol
dehydrogenase and a dihydroxyacetone kinase encoded by the isogenes
DAK1 and DAK2 (20). Using a two-dimensional PAGE
gel approach Godon et al. (55) examined changes in protein expression during the adaptive response of S. cerevisiae to
H2O2 stress and noted, among other changes, an
increased relative amount of isozymes for each step in the suggested
glycerol cycle. In S. cerevisiae the transcription factors
Yap1p (57, 58) and Skn7p (59, 60) are specifically involved in the
oxidative stress response. A recent two-dimensional PAGE analysis
detected, however, only DAK1 expression was Yap1p- and
Skn7p-dependent among the genes encoding
glycerol-metabolizing enzymes. Furthermore, we observed that
GPP2 induction in response oxidative stress was largely
independent of the transcriptional factors Msn2p and Msn4p and PKA
activity (Fig. 10B), suggesting control by as yet
unidentified factors.