 |
INTRODUCTION |
The fission yeast Schizosaccharomyces pombe efficiently
degrades L-malate to CO2 under aerobic
conditions and to ethanol and CO2 under anaerobic
conditions (1). Cells of S. pombe are not able to utilize
malate as the sole energy source or incorporate the malate into biomass
(2) and therefore require glucose or other carbon sources for the
energy-dependent transport and efficient degradation of
malic acid (3). Three enzymes are involved in malate degradation in
S. pombe, namely the malate transporter, malic enzyme, and
malate dehydrogenase (4). The transporter, encoded by the
mae1 gene (5), uses an H+-symport system for the
active transport of L-malate, and the NAD-dependent malic enzyme (EC 1.1.1.38) catalyzes the
oxidative decarboxylation of L-malate to pyruvate and
CO2. The mitochondrial malate dehydrogenase oxidizes
L-malate to oxaloacetate in the tricarboxylic acid cycle
and is responsible for 10% of the degradation of malate under aerobic conditions.
Molecular analysis of the S. pombe malic mae2
gene showed a high degree of homology with malic enzymes from various
organisms (6). Eight highly conserved regions were identified in malic enzymes, including the binding sites for L-malate and the
dinucleotide co-factors NAD(P)+ (7, 8). Although the
secondary structure of malic enzymes is highly conserved, the coenzyme
specificity (NAD+ or NADP+) and cellular
localization (cytosolic or mitochondrial) are strongly linked to their
regulation and metabolic function (9). Cytosolic NADP-dependent malic enzymes play an important role in
lipid metabolism in higher eukaryotes (10), whereas
NAD-dependent malic enzymes provide mitochondrial NADH
for electron transport or cytosolic NADH for reductive power for
other metabolic pathways (9).
The degradation of malate in yeasts such as Candida utilis
and Hansenula anomala that can utilize intermediates of the
tricarboxylic acid cycle as the only source of carbon was reported to
be subject to glucose repression and induction by L-malate
(11). However, no substrate induction or glucose repression was
observed in species such as S. pombe, Saccharomyces
cerevisiae, and Zygosaccharomyces bailii that cannot
utilize malate as the only source of carbon. A mitochondrial
NADP-dependent malic enzyme was recently cloned from
S. cerevisiae (12), and a role in the provision of
intramitochondrial NADPH or pyruvate under anaerobic conditions was
ascribed to the enzyme. DNA sequence analysis of the S. pombe malic enzyme gene (6) did not indicate the presence of a
mitochondrial targeting signal, suggesting that it functions in the
cytosol where it catalyzes the decarboxylation of malate to pyruvate.
This could serve to regulate the levels of intracellular malate for its
function in shuttle systems with oxaloacetate or aspartate or to
provide NADH or pyruvate for other metabolic pathways.
Because of the inability of S. pombe cells to utilize
L-malate as the only source of carbon and energy, the
effective transport and intracellular degradation of
L-malate by the yeast is intriguing. Transcription of the
malic enzyme gene was therefore studied to elucidate the
transcriptional regulation and physiological importance of malate
degradation in S. pombe. Our results showed increased levels
of mae2 transcription when cells were grown in high
concentrations of glucose (8%) or under anaerobic conditions. A
detailed analysis of the mae2 promoter enabled us to
identify three novel negative cis-acting elements, URS1,
URS2, and URS3, as well as two positive cis-acting elements
that have sequence similarity to the binding sites of
cAMP-dependent transcription factors.
 |
EXPERIMENTAL PROCEDURES |
Strains, Plasmids, and Media--
All strains and plasmids used
in this study are listed in Table I.
Yeast strains were cultured in SC
leu medium (0.17% yeast
nitrogen base without amino acids and ammonium sulfate (Difco
Laboratories, Detroit, MI), 0.5%
(NH4)2SO4, amino acid supplements,
and 2% glucose, unless stated otherwise). Media were supplemented with
0.2% L-malic acid (Sigma) when required. Escherichia
coli cells were grown in Luria broth with 200 mg/liter ampicillin
for plasmid selection.
View this table:
[in this window]
[in a new window]
|
Table I
Strains and plasmids used in this study
pMV refers to the native mae2 promoter and gene, and pMZ
refers to fusions of the mae2 promoter and lacZ
gene.
|
|
DNA Isolation, Transformation, and Analysis--
Standard
procedures were used for plasmid isolation and transformation of
competent cells of E. coli (13), S. pombe
leu
(14), or S. cerevisiae (15). Standard
procedures were used for DNA sequencing (13), and computerized analysis
(16) was done to identify putative regulatory elements. For primer
extention analysis, primers MV2 and MV3 (Fig. 1 and Table
II) were annealed to 50 µg of total RNA
and treated with Superscript Moloney murine leukemia virus reverse
transcriptase in the presence of [
-32P]dATP (17). The
products were loaded on a 6% acrylamide urea gel with DNA sequence
ladders obtained with the respective primers.
View this table:
[in this window]
[in a new window]
|
Table II
Primers used for mutation of putative cis-acting sites in the mae2
promoter
The respective annealing positions of the primers are given in Fig. 1.
|
|
Malic Enzyme Activity Assays--
Wild type and transformed
strains of S. pombe and S. cerevisiae were
cultured overnight in 10 ml of SC
leu medium with or
without 0.2% L-malate. Crude cell extracts and enzyme
assays were done as described by Osothsilp and Subden (18). Enzyme
activities are given as µmol of NADH produced/min/mg of protein as
determined by Bradford assays (Bio-Rad).
RNA Isolation and Northern Analysis--
Total RNA was isolated
from wild type S. pombe cells grown overnight in
SC
leu medium with 1% glucose to
A600 of 0.8. Cells were harvested and resuspended in fresh medium with 2% glucose or 2% glycerol, ethanol, with one set of glucose cultures sealed with mineral oil and incubated stationary so as to mimic anaerobic conditions. Incubation was continued, and total RNA was isolated (19) after 30, 60, and 90 min,
respectively. Samples of 15 µg of total RNA were subjected to
electrophoresis on a 0.8% formaldehyde agarose gel (13) and transferred to a Nylon membrane (MSI, Westboro, MA). The
510-bp1
BglII-EcoRV fragment of the mae2 gene
and 1.2-kilobase ClaI fragment of the S. cerevisiae
ACT1 gene served as probes.
Cloning of Unidirectional Deletion Fragments and lacZ
Fusions--
Exonuclease III deletion fragments of the mae2
promoter (6), plasmids pMV1 to pMV18 (Table I), were selected to
construct mae2 promoter-lacZ fusions. These
plasmids were digested with PstI and ApaI (Fig.
2) to replace the mae2 open reading frame (ORF) and
3'-flanking region with a 3.7-kilobase PstI-ApaI
fragment containing the lacZ ORF from YEp356, resulting in
mae2-promoter lacZ fusions (pMZ1 to pMZ18).
-Galactosidase Assays--
Yeast cells transformed with the
respective pMZ plasmids were inoculated into 10 ml of
SC
leu with 0.2% glucose, 2% glucose, 8% glucose, or
2% raffinose as the carbon source. L-Malate was added at
0.2% when required, and anaerobic conditions were obtained with
mineral oil and and incubation without shaking. Cultures were grown to
A600 of 0.8 to 0.9, and
-galactosidase assays
were performed with permeabilized cells (13). Assays were done in
duplicate with samples representing 8 µl/ml of original cell culture
on at least three transformants from different transformation sets.
Site-directed Deletions and Mutations--
Various deletions
within the 480 bp upstream of the mae2 ORF were constructed
with restriction enzyme digestions of pMV46 as indicated in Table I.
Restriction enzyme overhangs were filled with dNTPs and Klenow enzyme
prior to religation, and standard procedures were used for subsequent
cloning (13). Nine putative cis-acting elements identified
in the mae2 promoter region (Fig. 1) were mutated using
polymerase chain reaction primers to alter the native sequence to a
BstEII restriction site (GGTCACC). For example, mutation of
the GTTGATTGG sequence at nt
298 was introduced into plasmid pMV53
(Fig. 2) with two sets of primers, primers MV24 and MV26 (yielding
fragment A) and primers MV25 and MV27 (fragment B). Both fragments were
digested with BstEII and ligated, and the product was
amplified with primers MV24 and MV27. The product was digested with
SphI and SalI and cloned into the native SphI, SalI sites of pMV46.
In pMV54, the TCATTCATTT sequence at nt
244 was mutated in a similar
way with primers MV24 and MV29, and MV28 and MV27 (Fig. 1), and mutation of AAAATTGCGAG at nt
202 in pMV55 was obtained with primers MV24 and MV30. Plasmid pMV61
carries mutations in both the TCATTCATTT and AAAATTGCGAG elements and
was constructed using primers MV24 and MV30 with pMV54 as template. The
polymerase chain reaction products were digested with SphI
and SalI and cloned into the native SphI,
SalI sites of pMV46 (Fig. 2).
The mutated TGGGCTAAT sequence at nt
186 (pMZ86) was introduced with
primers MV40 and MV2, with direct cloning into the SalI,
PstI sites of pMZ1. Plasmid pMZ89 contains mutations in the
TCATTCATTT, AAAATTGCGAG, and TGGGCTAAT elements and was constructed by
subcloning the SalI, PstI fragment from pMZ86
(contains mutated TGGGCTAAT element) into the SalI,
PstI sites of pMZ61 (mutated TCATTCATTT and AAAATTGCGAG elements).

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 1.
DNA sequence of the 544 bp upstream of the
mae2 gene. Putative regulatory elements indicated
by shaded blocks include the recognition sites for the
TGGCA-binding protein at nt 394 and 354, CCAAT-binding protein at
nt 397 and 294, ADR1 at nt 358 and 250, AP-2 at nt 358, Sp1
at nt 186 ATF/CREB at nt 175 and 102, and TATA element at nt
88. The annealing positions of primers used for mutagenesis of
putative elements are indicated with arrows, and the altered
sequences are shown above or underneath the nucleotide sequence.
Restriction enzyme sites are indicated in bold letters as
are transcription initiation (tsp) and translation
initiation (ATG) sites.
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 2.
Introduction of the mutated CCAAT-like
element in pMZ53. Plasmid pMV46 contains the mae2 gene
cloned in the SacI, HindIII sites of pRS315
(SalI-XhoI sites destroyed with restriction
digest). Primers MV24 and MV26 were used to yield fragment A (nt 483
to 282) and primers MV25 and MV27 for fragment B (nt 305 to 180).
Both fragments were digested with BstEII and ligated, and
the product was amplified with primers MV24 and MV27. The product was
digested with SphI and SalI and cloned in the
native SphI, SalI sites of pMV46. The
mae2 ORF was replaced by the lacZ ORF from YEp356
and flanking regions as a PstI-ApaI
fragment.
|
|
In pMV88, the TCCCCTGGCA sequence at nt
297 was mutated with primers
MV24 and MV42, and MV41 and MV2, and the putative TATA element at nt
88 in pMV59 with primers MV24 and MV31, and MV32 and MV2. Both were
subcloned in the SphI, PstI sites of pMV46. Primers MV24 and MV44 were used for mutation of AGGGGGA at nt
251 in
pMV292 (subcloned in the SphI, DraI sites of
pMV46); primers MV43 and MV2 for mutation of TGACGT at nt
175 in
pMZ291 (subcloned in the SalI, PstI sites of
pMZ1); and primers MV45 and MV2 for mutation of TGACG at nt
102 in
pMV293 (subcloned in the AvaII, PstI sites of
pMV46). For all the pMV constructs, the mae2 ORF was
replaced with the lacZ ORF as described above. All deletions and mutations were verified with restriction enzyme digestions and
sequence analysis (13).
 |
RESULTS |
Malic Enzyme Activity and Levels of mae2 Transcription--
The
mae2 gene carried on the episomal plasmid pMV652
complemented the S. pombe mae2
mutant in its
ability to reduce NAD+ to NADH via the malic enzyme
reaction (Table III). There was no difference in the growth rate of S. pombe wild type,
mae2
or transformed strains (data not shown)
that would indicate that the malic enzyme is essential for growth. When
cells of S. cerevisiae were transformed with pMV652, no
increase in malic enzyme activity was observed relative to the
untransformed strain.
-Galactosidase assays on S. cerevisiae cells transformed with pMZ1 showed only 4% activity
relative to S. pombe (data not shown), suggesting that the
mae2 promoter is not recognized by S. cerevisiae.
View this table:
[in this window]
[in a new window]
|
Table III
Malic enzyme activity in wild type and transformed yeast strains
Yeast cells were cultured overnight in SC leu medium with or
without 0.2% L-malate, and crude cell extracts were used
for malic enzyme assays (18). Activities are given as µmol of NADH
produced/min/mg of protein. ND, not determined.
|
|
No evidence for an increase in either the malic enzyme activity or
mae2 transcription was observed for S. pombe
cells grown in the presence of 0.2% L-malate (Fig.
3). However,
-galactosidase assays
done with pMZ1 (Table IV) showed
transcription levels of 122 and 187% for cells grown in 2% glucose
and 8% glucose, respectively, relative to the 100% for cells grown in
0.2% glucose and 105% in 2% raffinose. Furthermore, the levels of
transcription were increased more than 5-fold when grown under
anaerobic conditions in 2% glucose. This was confirmed with Northern
analysis (Fig. 4) on the wild type
strain; a small increase was observed when cells were shifted to 2%
glucose, with a stronger response under anaerobic conditions.

View larger version (55K):
[in this window]
[in a new window]
|
Fig. 3.
Deletion and mutational analysis of the
S. pombe mae2 promoter region. Yeast
transformants were cultured in SC leu medium with or
without 0.2% malate. Assays were done in duplicate on permeabilized
cells, and results are given in percentage relative to that obtained
for pMZ1.
|
|
View this table:
[in this window]
[in a new window]
|
Table IV
-Galactosidase values for S. pombe 603 transformed with pMZ1 and
cultured in various carbon sources
Yeast transformants were cultured in SC leu medium with the
carbon sources as indicated. Anaerobic conditions were obtained with
mineral oil. Assays were done in duplicate on permeabilized cells (13),
and results are given in percentage relative to that obtained for
growth in 0.2% glucose.
|
|

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 4.
. Northern analysis of
mae2 expression. Cells were grown overnight in
1% glucose and shifted to fresh 2% glucose, either aerobic or
anaerobic, or 2% glycerol, ethanol medium. Total RNA was probed with
the 510-bp BglII-EcoRV fragment of the
mae2 gene or the 1.2-kilobase ClaI fragment of
the S. cerevisiae ACT1 gene.
|
|
Sequence Analysis of the mae2 Promoter--
The DNA sequence of
the region upstream of the mae2 ORF is available in the
GenBankTM and EMBL data bases (accession number SPCC794). Computerized
analysis (16) of the mae2 promoter indicated a putative TATA
element (TTATTTAAAA) at nt
88 in the mae2 promoter (Fig.
1). A predicted CAP signal (ACAGTAAT) corresponding to the putative TATA element was identified at nt
51, and primer extension reactions (data not shown) confirmed that it serves as the
transcription initiation site. Comparative analysis of the 5'-flanking
regions of the S. pombe mae2 and malate transporter
(mae1) genes,2
revealed three conserved elements: GTTGATT at nt
298 in
mae2 (GTTTGATT at nt
292 in mae1), a direct
repeat TCATTCATTT at nt
244 (TCATTCATTT at nt
227 in
mae1), and AAATTGCGAG at nt
202 (AAATTGCTAG at nt
152 in
mae1).
Putative binding sites for several eukaryotic activators were
identified in the mae2 promoter (Fig. 1). The conserved
CCAAT sequence that serves as binding site for the CCAAT-binding
protein (20) is present in the reverse orientation at nt
397 and
294. Recognition sites for the TGGCA-binding protein (21), a member of the ubiquitous eukaryotic family of NF1-like nuclear proteins that
are involved in transcriptional regulation (22), are present at nt
395 and nt
354. Furthermore, the TGGGCTAAT sequence at nt
186
shows strong homology with the conserved
(T/G)(G/A)GGCG(T/G)(A/G)(A/G)(T/C) sequence reported for the binding of
the mammalian transcription activator Sp1 (23).
The putative cis-acting elements that were identified based
on homology with the recognition sites of cAMP-dependent
transcription factors include the conserved GG(A/G)G core sequence for
the yeast transcription factor ADR1 (24) at nt
251 and inverted at nt
359. The ADR1 binding site was first identified in the
upstream-activating sequence UAS1 of the S. cerevisiae ADH2
gene, where it functions as a cis-acting element involved in
glucose repression (24). The ADR1 protein (adr1p) is inactivated
through phosphorylation by a cAMP-dependent kinase (25),
therefore rendering the activity of adr1p sensitive to the levels of
glucose. More recently, adr1p was also reported to be involved in
derepression of the S. cerevisiae acetyl-CoA synthetase gene
in response to sugar limitation (26).
The putative ADR1 binding site at nt
359 overlaps with the CCCMNSSS
recognition sequence reported for the mammalian transcription factor
AP-2 that is involved in cAMP-induced expression of the human
metallothionein IIA protein (27). Furthermore, the TGACGT sequence at
nt
175 and the TGACGA sequence at nt
102 resemble the binding site
for the mammalian activating transcription factor ATF/CREB (cAMP
response element binding) (28). ATF/CREB is activated through
phosphorylation by the cAMP-dependent kinase A (29) and was
reported to bind as a dimer to the GTGACGTACAG consensus sequence
(30).
Deletion and Mutation Analysis of Putative Regulatory Elements in
mae2 Promoter--
Eighteen unidirectional deletion fragments of the
mae2 promoter were fused in-frame with the lacZ
ORF (pMZ1 to pMZ18 in Table I) to evaluate their ability to support
transcription. Transcription from plasmids pMZ1 to pMZ12 were
unaffected as determined by
-galactosidase assays (only the results
for pMZ12 are shown in Fig. 3). Removal of the sequences upstream of nt
218 decreased the activity by more than 50% (pMZ15), whereas
deletion of the adjacent 36 nucleotides in pMZ16 restored the wild type
levels of activity. Deletion of the sequences upstream of nt
73 in
pMZ17 removed the putative TATA element at nt
88 and reduced
transcription to only 4%. Smaller deletions within the
SphI-AvaII region (nt
472 to
122) showed decreased transcription, especially for pMZ48 and pMZ71. In contrast, deletions within the XhoI-AvaII region increased
transcription 4-fold for pMZ47, 7- to 8-fold for pMZ77, and 8- to
9-fold for pMZ79. This suggested the presence of positive regulatory
sequences between nt
362 and
284 and negative regulatory sequences
between nt
218 and
182.
Site-directed mutations of nine putative cis-acting elements
were introduced with polymerase chain reaction primers (Fig. 1). Three
of these elements were identified as conserved elements in the
mae1 and mae2 promoters, whereas the other
elements were identified based on homology with recognition binding
sites for eukaryotic regulatory proteins. Mutation of the putative TATA element at nt
88 (pMZ59) decreased transcription to only 10%, confirming an essential function in transcription of the
mae2 gene. Mutation of the CCAAT-like element at nt
298
(pMZ53), ADR1 at nt
251 (pMZ292), or ATF/CREB at nt
102 (pMZ293)
did not affect transcription significantly (Fig. 3). However, mutation
of TCCCCTGGCA at nt
359 (homology with the ADR1, AP-2, and TGGCA
binding sites) in pMZ88 and TGACGT at nt
175 (homology with ATF/CREB
binding site) in pMZ291 resulted in only 53 and 20% of wild type
levels, respectively. These elements therefore seem to serve as
upstream activating sequences that regulate the expression of the
mae2 gene and are designated UAS1 and UAS2, respectively, in
further discussions (Fig. 5).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 5.
Proposed model for the regulated expression
of the S. pombe malic enzyme gene. UAS1 and UAS2
serve as activator sites and have homology with the binding sites for
eukaryotic transcription-activating factors as indicated. The novel
URS1, URS2, and URS3 elements act cooperatively to repress
transcription of the mae2 gene. The putative TATA element
indicated is required for wild type levels of expression.
|
|
Mutation of TCATTCATT at nt
244 (pMZ54), AAATTGCGAG at nt
202
(pMZ55), and TGGGCTAAT at nt
186 (pMZ86) increased transcription to
154, 170, and 224%, respectively (Fig. 3). These elements seem to
function as upstream repressor sequences and were designated URS1,
URS2, and URS3, respectively (Fig. 5). Furthermore, pMZ61 (mutated URS1
and URS2) and pMZ89 (mutated URS1, URS2, and URS3) showed increased
levels of transcription of approximately 3- and 4-fold, respectively,
suggesting that URS1, URS2, and URS3 may confer a cooperative
repression of mae2 expression.
 |
DISCUSSION |
Transcription of S. pombe class II promoters is typical
of higher eukaryotes where TFIID is the general transcription factor that binds to the TATA box and initiates the assembly of the
transcription complex (31). However, the promoter structure of S. pombe genes in general differ from that of S. cerevisiae and in some cases more closely resembles that of
mammalian promoters (32). Our results showed that mutation of the
putative TATA element 37 bp upstream of the transcription initiation
site reduced transcription to only 10% (pMZ59, Fig. 3). The distance
between the putative TATA box and the initiation start site is typical
for S. pombe (32) and within the 35 to 180 nt reported for
S. cerevisiae (33). However, the S. pombe mae2
promoter was not functional when expressed in S. cerevisiae
as measured by malic enzyme activity and
-galactosidase assays. This
lack of promoter function could be because of the inability of the
S. cerevisiae transcription complex to recognize the
mae2 promoter structure or the absence of essential
transcription factors.
The S. pombe malic enzyme has a high substrate affinity
(Km of 3.2 mM) (34) and decarboxylates
L-malate to pyruvate and CO2 with the
concurrent reduction of NAD+ to NADH. Under anaerobic
conditions, the pyruvate is further metabolized to ethanol and
CO2 with the re-oxidation of NADH to NAD+. The
degradation of L-malate in S. pombe was reported
not to be subject to substrate induction or glucose repression (35). Our results confirmed that expression of mae2 was not
affected by 0.2% L-malate (Fig. 3), but induced levels of
mae2 transcription were obtained when grown in 8% glucose
or under anaerobic conditions (Table IV). A 2- to 3-fold increase in
transcription under anaerobic conditions was reported for the S. cerevisiae malic enzyme gene (12), and a role in the provision of
intramitochondrial NADPH or pyruvate under anaerobic conditions was
proposed for the NADP-dependent enzyme. Unlike the S. cerevisiae malic enzyme, the DNA sequence of the mae2
gene (6) did not indicate the presence of a mitochondrial signal
sequence, suggesting that the enzyme is located in the cytosol.
Little information is available on the regulation of enzymes involved
in carbon metabolism in S. pombe cells. Expression of the
S. pombe fbp1 gene, encoding fructose 1,6-bisphosphatase, was reported to be repressed in 8% glucose (36) because of regulation by a glucose-induced cAMP signal (37). The increased expression of
mae2 observed in high concentrations of glucose (and
therefore high cAMP levels) suggested that cAMP induction may be
involved in this response. We have found two positive
cis-acting elements, UAS1 and UAS2, that show homology with
binding sites for cAMP-dependent regulatory proteins. UAS1
contains overlapping recognition sites for the transcriptional
activator proteins ADR1, AP-2, and TGGCA-binding protein (Fig. 5),
whereas UAS2 has homology with the transcriptional activator ATF/CREB.
ADR1 is inactivated by a cAMP-dependent kinase, whereas the
activity of AP-2 and ATF/CREB are induced in the presence of cAMP
(24-30). Mutation of UAS1 (pMZ88) and UAS2 (pMZ291) decreased the
levels of transcription to 53 and 20%, respectively (Fig. 3). It would
be of interest to determine whether these mutations also affect the
induced expression of mae2 under fermentative conditions.
Deletion and mutation analysis of the mae2 promoter
indicated the presence of three negative-acting elements, URS1, URS2, and URS3. Mutation of URS3 (pMZ86) had a stronger effect than that of
URS1 (pMZ54) or URS2 (pMZ55). However, mutation of either URS1 plus
URS2 (pMZ61) or all three elements (pMZ89) resulted in higher levels of
transcription than for any one of the individual elements, suggesting
that they function cooperatively to repress transcription of the
mae2 gene (Fig. 5). Homologous copies of the URS1 and URS2
elements are also present in the promoter of the S. pombe
malate transporter gene, mae1, indicating possible co-regulation of enzymes involved in malate degradation in S. pombe. Further analysis of these elements and the regulatory
proteins that bind them is essential to elucidate the transcriptional
regulation of the mae2 gene and perhaps also that of the
mae1 gene.
Because cells of S. pombe do not utilize
L-malate as the only source of carbon or energy, the
physiological importance of the active transport and strong
intracellular degradation is intriguing. The oxidative decarboxylation
of malate coincides with the reduction of NAD+ to NADH,
suggesting that the malic enzyme may play an important role in
maintaining the redox balance under aerobic conditions. Under anaerobic
conditions, however, the pyruvate produced during this reaction is
further metabolized to ethanol with the concomitant oxidation of NADH
to NAD+. Because there is no net gain or loss in NADH
during the conversion of malate to ethanol, the increased expression of
the malic enzyme under fermentative conditions should not affect the
redox balance.
The S. pombe cells may use the malic enzyme to provide
pyruvate for essential anaplerotic reactions under fermentative
conditions. Pyruvate plays an essential role in the provision of
-ketoglutarate and oxaloacetate for the biosynthesis of amino acids
and nucleotides. Both these precursors are synthesized in the
mitochondria and transported to the cytosol for biosynthetic reactions;
therefore alternative pathways have to be utilized for the synthesis of these precursors when the mitochondria are not functional (38). These
anaplerotic reactions include the carboxylation of pyruvate to
oxaloacetate via pyruvate carboxylase, the oxidation of malate to
pyruvate via the malic enzyme, and the production of succinate via the
glyoxylate cycle. The induced expression of the S. pombe malic enzyme under fermentative conditions may therefore serve as an
important auxiliary pathway for the production of pyruvate for other
metabolic requirements.
To our knowledge, this is the first report on the glucose-induced
expression of a malic enzyme gene in yeast. Results presented here
indicate the differential expression of the S. pombe mae2 gene under fermentative conditions, i.e. 8% glucose and
anaerobic conditions. We therefore propose that the
NAD-dependent malic enzyme from S. pombe may
provide cytosolic pyruvate for anaplerotic pathways under fermentative
conditions. A function in the provision of NADH for reductive
biosynthesis and maintenance of the redox balance is, however, not
excluded. Further research is required to elucidate the physiological
role of the malic enzyme in S. pombe and the importance of
the various UASs and URSs in the regulated expression of the
mae2 gene.