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Journal of Bacteriology, August 1999, p. 4719-4723, Vol. 181, No. 15
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Glucose Metabolism in gcr Mutants of
Saccharomyces cerevisiae
H.
Uemura1 and
D. G.
Fraenkel2,*
Section of Gene Engineering, Department of
Molecular Biology, National Institute of Bioscience and Human
Technology, Tsukuba-shi, 305 Japan,1 and
Department of Microbiology and Molecular Genetics, Harvard
Medical School, Boston, Massachusetts 021152
Received 22 February 1999/Accepted 18 May 1999
 |
ABSTRACT |
A gcr2 null mutant of Saccharomyces
cerevisiae grows well on glucose in spite of its lower level of
glycolytic enzymes between triose phosphates and pyruvate. A
quantitative analysis shows that these levels are adequate to the flux
but glycerate phosphates are elevated.
| |
TEXT |
Gcr1p and Gcr2p are elements
affecting transcription of glycolytic genes in Saccharomyces
cerevisiae (20, 26). Gcr1p is a DNA-binding protein
interacting with a consensus sequence in the promoter, Gcr2p interacts
with Gcr1p; both factors are needed for normal transcriptional
activation. Null and point mutants have decreased levels of most of the
glycolytic enzymes. The size of the effect depends on the enzyme,
growth medium, and strain background, with levels of phosphoglycerate
mutase and enolase being 5 to 10% of normal in gcr1 mutants
grown in the presence of glucose and less otherwise; like strains
completely blocked in single glycolytic reactions, gcr1
mutants do not grow on glucose (7). However, gcr2
mutants, which show a pattern of enzyme decreases qualitatively similar
to those of gcr1 (see also below) grow well on glucose
(25). The relative normality does not appear to reflect a
bypassing of the most-affected steps, for the gcr2 mutation
did not restore growth on glucose to a gpm1
(phosphoglycerate mutase) mutant (data not shown). Here we use a
resting-cell method (3) to assess glucose flux
(vglucose), enzyme levels, and metabolite levels
in gcr2 as well as gcr1 and gpm1 null mutants.
Strains.
DFY724 is a wild-type strain (MAT
leu2-3,112 ura3-52 his6 [strain 2845 in reference
27]); its isogenic derivatives are DFY725
(
gcr2-3::URA3 [strain YHU 3002-8C
in reference 27]), DFY726
(gcr1::URA3 [strain C179-15C in
reference 27]), and DFY727
(gpm1::LEU2). (DFY727 construction
was done by cloning GPM1 as a 2.3-kbp
SphI-AatI fragment from pPGM1
[29] [the open reading frame is bp 754 to 1497] into
pT7/T3alpha-18 [from Bethesda Research Laboratory], replacement of
its BglII-SalI fragment [bp 200 to 1647] by
LEU2 from YEp13 as a BglII-XhoI
fragment, and transplacement into DFY724 after digestion with
SphI and SacI.) Strain DFY730 carries a plasmid
with pGAL-GPM1 integrated in strain HD162-5C (MATa
gpm1::URA3 trp1-289 ura3-52 leu2-3,112
his3-1 MAL2-8c SUC2 GAL) (from J. Heinisch
[15], as was an isogenic GPM1 comparison
strain HD162-5A, here called DFY728). (For the pGAL-GPM1 plasmid, pL834-3, the
GAL1-GAL10 promoter [an EcoRI-XhoI fragment from pYEUra3 {Clontech}] was inserted in the multiple cloning site [EcoRI-SalI] of the URA3 plasmid
YIp352, and then GPM1, as an EarI-AatI
fragment from pGPM [EarI is 104 bp upstream of
the starting codon], was inserted in the SmaI site
downstream of the GAL1 promoter, giving a Gal-driven
transcript with a 5' noncoding region of 191 bp; integration was by
transformation after digestion with AatI in the
URA3 gene. As desired, strain DFY730 grew on galactose but
not glucose, while strain HD162-5C grew on neither.)
Resting-cell incubations.
As slightly modified from reference
3, washed cells were suspended at an
A580 of 100 in buffer B61 containing
cycloheximide (10 µg/ml) and antimycin A (2 µg/ml). After 30 min of
preincubation at 30°C with shaking, glucose was added to 1% and the
cultures were periodically sampled. For glucose use and ethanol and
glycerol formation, supernatants were used; for enzyme assay, pellets
from, e.g., 0.5-ml portions were frozen until use, while for
intracellular metabolites at the times indicated in Table 1 1-ml
portions were added to tubes containing 0.1 ml of 11.7 M perchloric
acid and 20 mM Na2EDTA, the contents of the tubes were
vigorously mixed for 30 s, and after at least 30 min at 
2°C
and additional mixing, the contents were neutralized with 0.282 ml of 3 M KHCO3; the supernatants were kept at 80°C until use.
In about half of the experiments all three types of data were obtained.
Metabolite assays.
Glucose, ethanol, glycerol,
glucose-6-phosphate, fructose-1,6-bisphosphate, and
glycerate-3-phosphate assays employed a Roche Cobas Bio Autoanalyzer
(as in reference 8) at 340 nm (for ethanol, 365 nm)
and standard endpoint techniques (23); prepared reagents were used for ethanol (Roche OnLine) and glycerol (Boehringer; samples
were pretreated 5 min at 85°C to remove ATPase activity.) For
improved sensitivity, glycerate-2-phosphate and phosphoenolpyruvate were assayed as ATP by a method modified from reference
19; the samples were pretreated with activated
charcoal (Sigma catalog no. C 4386), and the charcoal was removed with
spin filters (0.4-ml size; 0.22 µl porosity; Millipore MC).
Enzyme assays.
The cell pellets were suspended in a solution
containing 0.5 ml of 50 mM potassium phosphate (pH 7.4), 2 mM
Na2EDTA, 2 mM dithiothreitol, and 1 mM phenylmethylsulfonyl
fluoride; treated 50 s with a Mini Beadbeater (Biospec Products);
and centrifuged for 10 min. Three-microliter portions (undiluted and
1/10 and 1/100 dilutions) were assayed with an Autoanalyzer (as
described above) at 30°C, with 0.35-ml incubation volumes. The pH 7.1 buffer (see reference 30) (25 mM Tris, 20 mM KCl, 10 mM MgSO4) also contained 0.2 mM NADH (or, for
glucose-6-phosphate dehydrogenase or phosphoglucose isomerase, 0.2 mM
NADP), 1 mM substrate(s) unless specified otherwise, and auxiliary
enzymes from Boehringer (amounts [in micrograms per milliliter of
assay] are given in brackets) as follows: for glucose-6-phosphate
dehydrogenase, glucose-6-phosphate; for phosphoglucose isomerase,
fructose-6-phosphate and glucose-6-phosphate dehydrogenase
[2]; for triose-phosphate isomerase (0.25 mM), glyceraldehyde-3-phosphate and glycerol-3-phosphate dehydrogenase [1]; for glyceraldehyde-3-phosphate dehydrogenase, 0.5 mM glycerate-3-phosphate, ATP, and phosphoglycerate kinase
[2]; for phosphoglycerate kinase, 0.5 mM
glycerate-3-phosphate, ATP, and glyceraldehyde-3-phosphate dehydrogenase [20]; for phosphoglycerate mutase,
glycerate-2-phosphate, 0.2 mM glycerate-2,3-bisphosphate, ATP,
phosphoglycerate kinase [2], and
glyceraldehyde-3-phosphate dehydrogenase [20]; for enolase, glycerate-2-phosphate, ADP, pyruvate kinase
[6], and lactate dehydrogenase [2];
and for pyruvate kinase, phosphoenolpyruvate, ADP, and lactic
dehydrogenase [10].
Growth.
Enriched medium R61 (9) was supplemented
with uracil, 50 µg/ml, and 2% glucose or galactose with harvest at
an A580 of ca. 1 or on 0.2% lactic acid with
harvest at an A580 of ca. 10; as specified, in
some of the latter experiments, the medium was supplemented with 1%
glucose for a limited period before harvest. For gcr1 and
gpm1 strains the medium also contained 0.01% glycerol.
For growth, the basic parameters are growth rate and rate of substrate
utilization. For the parental wild-type strain DFY724 and
gcr2 null mutant DFY725 in enriched medium with glucose at 30°C, the growth rates were 0.53 and 0.31 h
1,
respectively, and the corresponding rates of glucose utilization (vglucose values) were 0.49 and 0.28 µmol/(min × mg of protein). Thus, the mutant does grow more
slowly than the parent but with similar yield. The data in Table
1 were obtained with
nongrowing cells. (The protocol [from reference 3]
uses cells grown as desired [not necessarily with glucose], suspended
in a buffer with cycloheximide and antimycin to prevent new protein
synthesis and respiration at a high-cell density so metabolites can be
obtained by direct acidification without a concentration step.) Under
such conditions the rate of glucose use is one-half or more of that in
growth. (By contrast with reference 3, however, in
the present experiments metabolite levels did not attain a plateau
before glucose exhaustion.) The main data sets, 1 and 2, are for
wild-type and gcr2 mutant strains grown on glucose. Both
strains used glucose at a similar rate and with an ethanolic
fermentation; slightly more glycerol was made in the mutant than in the
wild type. Enzyme assay values show that the later reactions of
glycolysis in the mutant have levels one-fourth to one-half those of
the wild type; with improved assays these values are somewhat higher
than those previously reported (25). Since in Table 1 enzyme
activities (Vs) are expressed in the forward glycolytic
direction and in the same units as vglucose, it
can be seen that even the most-affected steps in the mutant have
capacities (values of ca. 2.4) in excess of the rate of ethanol
formation (ca. 0.4), the latter being a measure of the net in vivo rate
of those two reactions.
The last five lines of Table
1 show intracellular metabolite
concentrations, both before and 10 min after glucose addition.
For the
hexose phosphates the patterns were similar in the wild
type and the
gcr2 mutant: marginal levels during starvation and
high
levels afterwards. For later metabolites of glycolysis, the
pattern was
more complicated, with significant amounts in the
starved cells (as
known, and perhaps related to insufficient fructose-1,6-bisphosphate
to
activate pyruvate kinase [
2,
21]). During glucose
metabolism,
in the wild-type strain those levels were unchanged, while
by
contrast in the
gcr2 mutant, as might be expected in view
of its
reduced enzyme levels, they were considerably
elevated.
The other data sets are less complete. With lactic acid instead of
glucose as the carbon source, in the wild-type strain (data
set 3) the
levels of lower enzymes of glycolysis were approximately
one-third of
those during growth on glucose and, perhaps coincidentally,
so was the
glucose flux under the resting-cell conditions; metabolite
levels were
low. Forty-five minutes of exposure to glucose prior
to harvest (data
set 3A) did not clearly increase levels of the
assayed enzymes, but
flux and metabolites significantly increased
in the test situation. For
both
gcr2 (data set 4) and
gcr1 (data
set 5)
strains, enzyme levels from gluconeogenic growth were in
the 2 to 10%
range, glucose use was marginal, levels of hexose
phosphates were low,
and levels of glycerate-3-phosphate were
high; several hours of prior
exposure to glucose (data sets 4A
and 5A) increased enzyme levels and
glucose flux, more for
gcr2 than for
gcr1, with
glycerate-3-phosphate levels remaining
high.
The most notable perturbations in
gcr2 and
gcr1
strains were of glycerate-3-phosphate, implying significant in vivo
impediment
at phosphoglycerate mutase; for comparison purposes, data
set
6 is for a
gpm1 structural gene mutant, which with
normal levels
of other enzymes and completely unable to grow on
glucose, gave
a
vglucose of 0, formed no
ethanol, but likewise accumulated high
levels of glycerate-3-phosphate
(see also reference
6). And
(not shown) a 3-h prior
exposure to glucose did not confer on
this mutant the ability to use
glucose in the resting-cell situation
either. Data set 8 is for a
strain with
GPM1 expression dependent
on galactose (data set
7 is for the isogenic wild type). Unfortunately,
the phosphoglycerate
mutase level was low even in cells grown
on galactose, so apart from
showing that the need for phosphoglycerate
mutase may be less on
galactose than glucose, the strain was not
useful as a means of
providing a range of levels of a single enzyme.
Nontheless, as
expected, the low enzyme level was associated with
low but not zero
vglucose and a high level of
glycerate-3-phosphate.
There was also a large relative increase in the
amount of glycerol
made (as also seen for the marginal glucose use by
gcr mutants
[data sets 4A and 5A]; glycerol formation is
an alternative route
for NADH reoxidation when acetaldehyde is
unavailable [
11]).
It is of interest that this strain
with nominally only a low phosphoglycerate
mutase level (the enolase
level was normal) contained unusually
high levels of
glycerate-2-phosphate; perhaps this finding is
related to the
inhibition of enolase by high-level glycerate-3-phosphate
(
31).
Qualitatively, the results show (i) how rapid glucose metabolism in a
wild-type strain goes with high levels of glycolytic
enzymes; (ii) the
compatibility of substantial decreases in certain
enzymes with normal
vglucose (e.g.,
gcr2 as grown on
glucose),
as also known in specific cases: 2% level of
phosphoglycerate
kinase and normal glucose flux (
24), and
0.7% level of phosphoglycerate
mutase activity allowing half the
normal growth rate on glucose
(
30); and (iii) the apparent
absence of a major bypass of the
lower reactions of glycolysis. It is
pertinent that two other
putative phosphoglycerate mutase genes as
tested have marginal
or no function (
15). Lower glycolytic
enzyme levels are associated,
however, with accumulations of their
substrates, and one speculation
is that although such accumulation is
not always clearly detrimental
(e.g., normal flux with high
glycerate-3-phosphate in the
gcr2 mutant; see also reference
10), it may be that glycolytic enzyme
activities are
normally in such apparent excess in part to avoid
effects like the
inhibition of enolase by glycerate-3-phosphate
cited
above.
It is tempting to ask whether the metabolite concentrations also make
quantitative sense in terms of the measured amounts
and known
parameters of the enzymes and the observed fluxes. There
is one case in
yeast for a reversible reaction where the fit was
adequate
(phosphoglucose isomerase [
32]). As shown in Table
2 calculations with the present data show
a poor fit of measured
values versus predicted values, although, as
also shown, relatively
small changes in certain individual entries
would remove the differences.
Unfortunately, aside from the
cancellation of errors, each entry
in such calculations has
limitations, including (i) the large
standard deviations of items in
Table
1; (ii) the fact that enzyme
parameters are not all well known,
or have a wide range of reported
values, or need correction for pH or
Mg
2+ (
17); (iii) the presence of isoenzymes for
several reactions
and limited knowledge of their individual parameters;
(iv) for
phosphoglycerate mutase, the absence of data on
glycerate-2,3-bisphosphate
concentration; (v) the use of normalized
values for protein content
and internal cell volume; (vi) the fact of
certain glycolytic
enzymes having a nominal active-site concentration
within a 0.1
factor of substrate concentration, requiring additional
correction
(
4,
28); and (vii) for reversible reactions the
limitation
of calculating net flux as a difference between two much
larger
numbers (Table
2). We also remark that in the present work
emphasis
was on
gcr mutant strains with several enzymes
affected; although
strain DF730 (Table
1, data set 8) was not useful
for the purpose,
varying a single enzyme in one strain would
nonetheless be preferred.
| |
ACKNOWLEDGMENTS |
We thank M. Koshio for strain construction, and D. Clifton, F. Rosen, J. Heinisch, W. de Koning and K. van Dam, and D. Fell and
S. Thomas.
This work was supported in part by the William F. Milton Fund (D.G.F.)
and the Science and Technology Agency of Japan (H.U.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Microbiology and
Molecular Genetics, Bldg. D1-121, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 432-1912. Fax: (617) 738-7664. E-mail: fraenkel{at}hms.harvard.edu.
| |
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Journal of Bacteriology, August 1999, p. 4719-4723, Vol. 181, No. 15
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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