From the Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284-7760
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
Growth of Saccharomyces cerevisiae
with a fatty acid as carbon source was shown previously to require
function of either glucose-6-phosphate dehydrogenase (ZWF1) or
cytosolic NADP+-specific isocitrate dehydrogenase (IDP2),
suggesting dependence of Reducing equivalents in the form of NADPH are essential for many
enzymatic steps involved in biosynthesis of cellular macromolecules. NADPH is also the essential cofactor for glutathione- and
thioredoxin-dependent enzymes that constitute major
cellular defenses against oxidative damage (reviewed in Refs. 1 and 2).
In eucaryotic cells, the majority of these NADPH-dependent
reactions occur in the cytosol. Availability of the reduced form of the
cofactor requires maintenance of high steady-state cytosolic redox
ratios for NADPH:NADP+ and values as high as ~70:1 have
been reported (3).
Enzymatic sources of NADPH are relatively few in number. In a recent
study (4), we utilized gene disruption and phenotype analyses to
examine the relative contributions by the oxidative branch of the
hexose monophosphate pathway and by differentially compartmentalized
isozymes of NADP+-specific isocitrate dehydrogenases in
Saccharomyces cerevisiae. Results indicate that yeast cells
are remarkably flexible in terms of enzymatic sources of NADPH
for growth, since mutants containing multiple gene disruptions grow
well under various cultivation conditions. In fact, the only dramatic
growth phenotypes observed were the inability of strains containing
co-disruptions of genes encoding glucose-6-phosphate
dehydrogenase (ZWF1) and cytosolic NADP+-specific isocitrate dehydrogenase (IDP2)
to grow with fatty acids as a carbon source and the inability of
homozygous In this report, we further examine the fatty acid Disruption of the yeast ZWF1 gene has been reported to
result in methionine (and cysteine) auxotrophy and an increased
sensitivity to oxidizing agents including hydrogen peroxide and diamide
(7, 8). The Met These previous studies of ZWF1 function were conducted using yeast
cells grown with glucose as a carbon source, a condition that represses
expression of IDP2 (12, 13). We report here that, under conditions when
IDP2 is normally expressed, disruption of IDP2 or of
ZWF1 produces similar phenotypes and growth defects are
compounded by co-disruption of both genes. Also, we provide evidence
that, in addition to a requirement for either ZWF1 or IDP2 to support
growth with fatty acids as a carbon source, the absence of both enzymes
results in rapid loss of viability. We suggest that the cause of
lethality is probably oxidative damage due to accumulation of hydrogen
peroxide, a major byproduct of peroxisomal Yeast Strains and Growth Conditions--
Yeast strains used in
this study were the parental haploid strain S173-6B (MAT
For tabulation of viable cell numbers, yeast strains were cultivated in
YP glycerol/lactate medium for 24 h then diluted into YP medium
with a fatty acid carbon source, with no carbon source, or with oleate
plus 200 µg/ml Geneticin. Aliquots of cultures were taken at 12-h
intervals and several dilutions plated onto YP glucose plates. Viable
cells were scored as colonies after 2-3 days incubation at 30 °C.
For measurements of intracellular peroxides as described by Lee and
Park (18), 10-ml cultures grown for 24 h with fatty acid carbon
sources were divided and incubated for 30 min with or without 10 µM 2',7'-dichlorofluorescein acetate (Molecular Probes,
Eugene, OR). The cells were pelleted, washed twice with water, then
lysed with glass beads in 300 µl of water. The lysates were cleared
by centrifugation and various dilutions used for fluorescence
measurements at peak excitation and emission wavelengths of 504 and 524 nm for dichlorofluorescein.
Sensitivity to exogenous hydrogen peroxide was tested by plating
strains on YP glucose or ethanol plates containing 0, 0.5, 1.0, 2.0, or
4.0 mM H2O2. Methionine
auxotrophies were analyzed on YNB glucose or ethanol/glycerol plates
containing all supplements needed for growth in the presence or absence
of 20 µg/ml methionine.
Protein Analyses--
Whole cell protein samples were obtained
by glass bead lysis of cells harvested from cultures in logarithmic
growth (A600 nm = 0.8-1.0). Assays for
NADP+-specific isocitrate dehydrogenase activity were
conducted as described previously (15). Protein concentrations were
determined using the Bradford assay (20) with bovine serum albumin as
the standard. Specific activities are expressed as µmoles of NADPH produced per minute per milligram protein. Immunoblot analysis was
conducted with an antiserum prepared against yeast IDP1 (12) which
cross-reacts with yeast IDP2. Immunoreactivity was detected by the
enhanced chemiluminescent method (ECL, Amersham Pharmacia Biotech) and autoradiography.
Saccharomyces Genome Data base designations for various
proteins include: ZWF1 (YNL241C), IDP1 (YDL066W), IDP2 (YLR174W), IDP3
(YNL009W), CTA1 (YDR256C), CTT1 (YGR088W), POX1 (YGL205W), SOD1
(YJR104C), and TSA1 (YML028W).
S. cerevisiae cells are able to utilize fatty acids as
carbon sources because the acetyl CoA produced by peroxisomal
-oxidation on a cytosolic source of NADPH.
In this study, we find that
IDP2
ZWF1 strains
containing disruptions in genes encoding both enzymes exhibit a rapid
loss of viability when transferred to medium containing oleate as the
carbon source. This loss of viability is not observed following
transfer of a
IDP3 strain lacking peroxisomal isocitrate
dehydrogenase to medium with docosahexaenoate, a nonpermissive carbon
source that requires function of IDP3 for
-oxidation. This suggests
that the fatty acid
phenotype of
IDP2
ZWF1 strains is not a simple defect in
utilization. Instead, we propose that the common function shared by
IDP2 and ZWF1 is maintenance of significant levels of NADPH for
enzymatic removal of the hydrogen peroxide generated in the first step
of peroxisomal
-oxidation in yeast and that inadequate levels of the
reduced form of the cofactor can produce lethality. This proposal is
supported by the finding that the sensitivity to exogenous hydrogen
peroxide previously reported for
ZWF1 mutant strains is
less pronounced when analyses are conducted with a nonfermentable carbon source, a condition associated with elevated expression of IDP2.
Under those conditions, similar slow growth phenotypes are observed for
ZWF1 and
IDP2 strains, and co-disruption
of both genes dramatically exacerbates the
H2O2s phenotype. Collectively,
these results suggest that IDP2, when expressed, and ZWF1 have critical
overlapping functions in provision of reducing equivalents for defense
against endogenous or exogenous sources of
H2O2.
INTRODUCTION
Top
Abstract
Introduction
References
IDP2
ZWF1 diploid strains to sporulate.
growth
phenotype demonstrated by
IDP2
ZWF1 yeast disruption
mutants. This phenotype is not observed for mutant strains containing a
single gene disruption of ZWF1 or of IDP2, and
the mutant strain containing both gene disruptions is capable of growth
on acetate as a carbon source (4). These observations suggest that ZWF1
and IDP1 have some overlapping function in support of
-oxidation of
fatty acids, a process that is strictly peroxisomal in S. cerevisiae (reviewed in Refs. 5 and 6).
phenotype has been interpreted as a
deficiency in NADPH required for a thioredoxin-dependent
reaction in methionine/cysteine biosynthesis (9). The
H2O2s phenotype may reflect
insufficient levels of NADPH to combat oxidative stress. Results of
several recent studies support the importance of the hexose
monophosphate pathway in this stress response; Juhnke et al.
(10) report that H2O2s is a common
phenotype for yeast mutants with defects in enzymes of this pathway.
Based on results of complementation studies, Slekar et al.
(9) suggest that the methionine auxotrophy displayed by yeast strains
lacking the cytosolic Cu/Zn superoxide dismutase (SOD1) is due to a
drain of NADPH from biosynthetic pools and that increased flux through
the hexose monophosphate pathway can alleviate this auxotrophy in
SOD1 disruption mutants. In addition, Godon et
al. (11) reported recently that overall changes in expression of
various cellular proteins in yeast cells following exposure to hydrogen
peroxide suggest a major redirection of carbon flux from glycolysis
into the hexose monophosphate pathway.
-oxidation, and that
significant levels of cytosolic NADPH are normally required for
efficient degradation of that metabolite in vivo.
EXPERIMENTAL PROCEDURES
leu2-3, 112 his3-1 ura3-52 trp1-289, Ref. 14) and mutant
strains containing coding region deletions and URA3
insertions in IDP1, IDP2, or ZWF1 loci
constructed as described previously (4, 13, 15). The
IDP2
ZWF1 haploid strain was obtained by mating and
sporulation, and the
IDP3 mutant was constructed by
deletion/insertion of the selectable kanMX4 gene (4, 16). Strains were cultivated in liquid medium or on 2% agar plates with
rich YP medium (1% yeast extract, 2% Bacto-peptone) or with minimal
YNB medium (0.17% yeast nitrogen base, 0.5% ammonium sulfate, pH 6.0)
with supplements of 20 µg/ml to satisfy auxotrophic requirements for
growth. Carbon sources were glucose, ethanol, glycerol, or lactate
added to 2%. Fatty acids (oleate or docosahexaenoate) used as carbon
sources were added to 0.1% with 0.2% Tween 40 (17).
RESULTS AND DISCUSSION
-oxidation serves as a substrate both for energy metabolism and for
net carbon biosynthesis via the glyoxylate pathway (reviewed in Ref.
21). Growth of yeast on oleate ((9) oleic acid (C18:1)) results in proliferation of peroxisomes and in dramatic elevation of expression of
peroxisomal enzymes, including the
-oxidation enzymes (reviewed in
Refs. 5 and 21). Significant increases in expression are also reported
for peroxisomal catalase (CTA1, Ref. 22), which removes the
H2O2 generated by acyl-CoA oxidase (POX1)
during the first step in each round of the pathway (Fig.
1), and of peroxisomal NADP+-specific isocitrate dehydrogenase (IDP3) (17, 23).
Van Roermund et al. (17) and Henke et al. (23)
have convincingly demonstrated an essential and specific role for the
latter enzyme in
-oxidation of fatty acids like petroselinate ((6)
petroselenic acid (C18:1)) and docosahexaenoate ((4, 7, 10, 13, 16, 19)
docosahexaenoic acid (C22:6)) that require NADPH for reduction of
even-numbered double bonds by 2,4-dienoyl CoA reductase (Fig. 1). In
contrast to these peroxisomal enzymes, cytosolic catalase (CTT1, Ref.
22) and cytosolic IDP2, which is repressed by growth on glucose, are
present at similar levels in cells grown with a variety of
nonfermentable carbon sources including ethanol (12) or oleate (Fig.
2). Levels of glucose-6-phosphate
dehydrogenase (ZWF1) activity are essentially constitutive with respect
to carbon source (4, 8) as are levels of mitochondrial IDP1 (Ref. 12
and Fig. 2).
View larger version (20K):
[in a new window]
Fig. 1.
Potential contributions of cytosolic NADPH to
peroxisomal -oxidation in yeast.
Following acyl-CoA synthesis, the first step in peroxisomal
-oxidation of a fatty acid in S. cerevisiae is conversion
to an enoyl CoA by acyl-CoA oxidase (POX1) with concomitant
production of hydrogen peroxide. Subsequent steps to produce acetyl CoA
and an acyl-CoA shortened by two carbons are not shown.
-Oxidation
of fatty acids with even-numbered double bonds (e.g.
docosahexaenoate) requires two ancillary enzymes, 2,4-dienoyl-CoA
reductase (1) and
3,
2-enoyl-CoA isomerase
(2).
-Oxidation of fatty acids with odd-numbered double
bonds (e.g. oleate) may proceed via two pathways (6); one
requires the same ancillary enzymes, but the second, as shown, requires
isomerase but not reductase function. The NADPH required by the
reductase is provided by peroxisomal NADP+-specific
isocitrate dehydrogenase (IDP3; Refs. 17 and 23). Removal of
the H2O2 produced during each round of
-oxidation is attributed to peroxisomal catalase (CTA1);
ancillary roles in this process are proposed for cytosolic catalase
(CTT1) and thiol-dependent peroxidases
(thioredoxin peroxidase, TPX, and glutathione peroxidase,
GPX). Other abbreviations are: TRR, thioredoxin
reductase; GLR, glutathione reductase; IDP2,
cytosolic NADP+-specific isocitrate dehydrogenase, and
ZWF1, glucose-6-phosphate dehydrogenase.
View larger version (32K):
[in a new window]
Fig. 2.
Expression of IDP2 and IDP1 with oleate as
the carbon source. Immunoblot analysis was conducted with an
antiserum that reacts with yeast IDP1 and IDP2 polypeptides (12) using
whole cell protein extracts (25 µg/ml) from the parental strain grown
on YP medium with glucose (lane 1) or from strains grown
with oleate as the carbon source: parental (lane 2),
IDP1 mutant (lane 3),
IDP2
mutant (lane 4), and
IDP3 mutant (lane
5). Below each lane is shown an average of values from two
independent experiments for NADP+-specific isocitrate
dehydrogenase specific activity. Under these conditions, the antiserum
does not react with the IDP3 polypeptide, presumably because the
isozyme contributes only ~15% of the total cellular activity in
oleate-grown cells (23).
We previously compared rates of growth with various fatty acids as
carbon sources for yeast strains containing disruptions of
IDP and/or ZWF1 loci (4). Other than replication
of the growth phenotypes (e.g. petroselenate)
described by others for strains with IDP3 gene disruptions
(17, 23), the only dramatic growth phenotype observed with various combinations of these gene disruptions was an inability of strains containing disruptions of both IDP2 and ZWF1 loci
to grow with any fatty acid (stearate, oleate, or petroselinate) as the
carbon source.
We further investigated this growth phenotype of
IDP2
ZWF1 strains by analyzing viable cell numbers as
described under "Experimental Procedures" following a shift of
cultures from medium with glycerol/lactate to medium with oleate as the
carbon source. As illustrated in Fig.
3A, logarithmic-phase doubling
times of ~8 h with oleate were measured for the parental strain,
whereas the mutant strain containing both gene disruptions exhibits a
rapid decline in viability when shifted to oleate as carbon source. The
decrease in viable cell numbers for the
IDP2
ZWF1
strain contrasts sharply with results obtained following a shift of the
parental culture to medium with no added carbon source (Fig.
3A); the parental culture ceases to grow after an initial
doubling but exhibits no loss of viability over this 60-h period.
Instead, the decrease in viability for the
IDP2
ZWF1
mutant strain is similar to but slower than the lethality produced by
shifting a parental culture to oleate medium containing Geneticin
(G418) at levels used for selective protocols. The calculated
t1/2 values for loss of viability are ~16 h for
the
IDP2
ZWF1 strain in medium with oleate and ~4 h
for the parental strain in medium with oleate plus Geneticin.
|
These results suggest that transfer of the IDP2
ZWF1
strain to medium with a fatty acid carbon source activates an
endogenous cellular mechanism that results in lethality. The time lag
(~12 h) before loss of viability is measurable (Fig. 3A),
relative to that observed with the parental strain with Geneticin, may be due induction of this mechanism as the cells adapt to oleate as a
carbon source. Thus, a potential cause of lethality in the
IDP2
ZWF1 strain is accumulation of toxic levels of
H2O2 during the process of
-oxidation (Fig.
1), which is induced by this shift. To examine this possibility,
similar shifts were conducted using a
IDP3 mutant strain.
Counts of viable cell numbers (Fig. 3B) give a doubling time
of ~7 h with oleate, a permissive carbon source for this strain,
since
-oxidation does not require a peroxisomal source of NADPH (17,
23). In contrast, when shifted to medium with docosahexaenoate, a
nonpermissive fatty acid carbon source, growth of the
IDP3 strain (Fig. 3B) resembles that of the
parental strain shifted to no carbon source (Fig. 3A). The
parental strain exhibits a doubling time of ~11 h with
docosahexaenoate (Fig. 3B). Significantly, there is little
loss of viability for the
IDP3 mutant strain in
docosahexaenoate medium over the time frame of this experiment. In this
mutant strain, as illustrated in Fig. 1, docosahexaenoate is expected
to be a substrate for acyl-CoA synthetase and oxidase but to undergo no
further metabolism in the absence of NADPH normally generated by IDP3.
Van Roermund et al. (17) have demonstrated an accumulation
of the enoyl CoA product of acyl oxidation in a IDP3
strain incubated with docosahexaenoate, suggesting that some
H2O2 is also produced. The absence of lethality
for the
IDP3 strain in medium with docosahexaenoate as
carbon source may thus be due to the presence of normal cellular
mechanisms (e.g. ZWF1 and IDP2) for removal of this toxic
intermediate and/or to reduced production of this metabolite by
acyl-CoA oxidase. To try to distinguish between these possibilities, we
examined growth responses of a strain containing disruptions of
IDP2, ZWF1, and IDP3 loci. As illustrated in Fig. 3B, a shift of the
IDP2
ZWF1
IDP3 strain to docosahexaenoate
medium produces a decrease of approximately 20% in viable cell numbers
after 12 h, but these numbers do not change significantly over the
time course of the experiment. In contrast, the
IDP2
ZWF1
IDP3 mutant strain exhibits a
significant loss of viability (t1/2
12 h)
following a shift to oleate medium. Our interpretation of these results is that this loss of viability is due to production of
H2O2 in multiple rounds of
-oxidation when
oleate is the substrate. The absence of this dramatic loss of viability
for the mutant strain when docosahexaenoate is the substrate suggests
that production of H2O2 may be limited, perhaps
due to product inhibition of oxidase activity.
To try to compare intracellular levels of H2O2,
we used 2',7'-dichlorofluorescein diacetate, a fluorescent probe
previously used to measure cellular oxidant production in mammalian
(24) and yeast cells (18, 25). This compound is believed to cross cell
membranes by passive diffusion, to be trapped after deacetylation by
intracellular esterases, and to undergo oxidation by reactive oxygen
species to form a fluorescent dichlorofluorescein derivative. Measurements were made with cultures grown with a fatty acid for 24 h, a time chosen to ensure adaptation to this carbon source but
prior to any significant decrease in cell number (Fig. 3). As shown in
Table I, two different series of
experiments produced different absolute but similar relative
fluorescence values. These values are consistent with significant
increases in intracellular oxidant levels in IDP2
ZWF1
and
IDP2
ZWF1
IDP3 strains cultivated with oleate and
a slight increase in these levels in the
IDP3 strain
cultivated with oleate. Fluorescence differences were not measurable
with other strains and growth conditions. Thus, increased fluorescence
appears to correlate with conditions that lead to loss of culture
viability.
|
Collectively, these results suggest that either ZWF1 or IDP2 is
essential to prevent the loss of viability that otherwise occurs during
-oxidation in yeast cells. We propose that the shared function is
maintenance of high cytosolic redox ratios to ensure efficient function
of NADPH-dependent enzymes involved in degradation of
H2O2 or in prevention of oxidative damage from that metabolite and that these thiol-dependent reactions
are an essential adjunct to catalase function (Fig. 1).
One correlate of this proposal is that IDP2, when expressed, may
alleviate some of the detrimental effects (e.g.
H2O2s) reported previously for
disruption of ZWF1 (8) and, conversely, that co-disruption
of IDP2 may exacerbate these effects. This was tested by
comparing growth of parental and mutant strains on plates with YP
medium containing as the carbon source either glucose, which represses
expression of IDP2, or ethanol, which is conducive to IDP2
expression (12). Effects on growth with both carbon sources of addition
of H2O2 was tested by titration on a series of
plates; results with concentrations (1 mM) producing definitive effects are illustrated in Fig.
4A. As expected, on plates
with glucose as the carbon source, the
H2O2s phenotypes are comparable for
ZWF1 and
IDP2
ZWF1 strains, whereas the
IDP2 strain exhibits resistance equivalent to that of
the parental strain. With ethanol as a carbon source, however, both the
ZWF1 strain and the
IDP2 strain exhibit
reduced growth (colony size) relative to the parental strain with
concentrations of H2O2 that clearly eliminate
growth of the
IDP2
ZWF1 strain. These results suggest a
"synthetic lethal" relationship between these cytosolic sources of
NADPH for protection from exogenous H2O2 when
both are expressed. Interestingly, despite this apparently essential
function, levels of ZWF1 are elevated at most 2-fold (11) and levels of
IDP2 are not significantly elevated (data not shown) in cells exposed
to H2O2 under conditions that result in over
10-fold increases in levels of other proteins with antioxidant functions including cytosolic catalase and thioredoxin reductase (11).
|
Since ZWF1 mutant strains are also reported to be
methionine auxotrophs on glucose medium, we planned similar experiments to examine potential compensation by IDP2 on nonfermentable carbon sources. However, as illustrated in Fig. 4B, while the
methionine auxotrophies of
ZWF1 and
IDP2
ZWF1 mutant strains are clearly apparent on
plates with glucose as the carbon source, no obvious growth phenotypes
are exhibited by any of the mutant strains on plates with
glycerol/ethanol as the carbon source. Thus, methionine biosynthesis on
nonfermentable carbon sources apparently does not require ZWF1 and/or
IDP2. Since a specific reaction in this biosynthetic pathway, that
catalyzed by NADPH/thioredoxin-dependent phosphoadenyl
sulfate reductase, is believed to be the site affected by disruption of
ZWF1 in glucose-grown cells (9), it would appear that there
is an alternative source(s) of NADPH for this reaction under other
growth conditions.
In this report, we provide evidence that the process of -oxidation
in peroxisomes can be lethal in the absence of major sources of
cytosolic NADPH. The most logical cause of lethality is damage resulting from accumulation of H2O2, a
stoichiometric product of each round of oxidation. Accumulation of
H2O2 is believed to be particularly hazardous
because of the increased potential for generation of hydroxyl radicals
(·OH) by the iron-mediated Fenton reaction (26). We propose that maintenance of a significant cytosolic pool of the reduced form of the
NADP(H) cofactor is an essential adjunct to catalase function during
this challenge and that the most likely fate of the reducing equivalents may be delivery to
thioredoxin/glutathione-dependent peroxidases via
respective reductases for enzymatic removal of H2O2 (Fig. 1). For S. cerevisiae, a
thioredoxin peroxidase (also called thiol-specific antioxidant protein)
has been described and kinetically characterized (27, 28). Based on the
Saccharomyces Genome Data base, there are several other open
reading frames potentially encoding thioredoxin and glutathione
peroxidases that to date have not been otherwise characterized. Because
of the current implication of a role for NADPH in protection of the
cell from adverse consequences of endogenous metabolism, further study of expression and compartmental localization of these
thiol-dependent enzymes is of significant interest.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Sondra Anderson for excellent technical support and Dr. Mark Panda for his assistance with fluorescence measurements.
![]() |
FOOTNOTES |
---|
* This work was supported by an award for pilot research from the Research Resources Program for Medical Schools of the Howard Hughes Medical Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 210-567-3782;
Fax: 210-567-6595; E-mail: henn{at}uthscsa.edu.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|