From the Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900
Received for publication, December 27, 2002 , and in revised form, April 1, 2003.
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ABSTRACT |
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INTRODUCTION |
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The yeast MDH isozymes are all homodimers with similar subunit
Mr values (33,500 for MDH1; 40,700 for MDH2; and 37,200
for MDH3), and they share primary sequence identities of 43 to 50%. Distinct
differences among the enzymes include a 17-residue mitochondrial targeting
sequence for MDH1 that is removed upon import
(2), and a carboxyl-terminal
tripeptide targeting sequence (Ser-Lys-Leu) required for peroxisomal import of
MDH3 (11). In addition, MDH2
has a 12-residue extension on the amino terminus that is not present on mature
MDH1 or on MDH3 (Fig. 1). We
have previously shown that removal of this 12-residue extension, generating a
truncated enzyme designated nMDH2, appears to have no effect on
cellular levels of activity of the enzyme
(12). However, the
nMDH2 enzyme is resistant to catabolite inactivation, a regulatory
phenomenon in yeast involving glucose-induced inactivation and degradation of
gluconeogenic and glyoxylate cycle enzymes
(13,
14). In addition, when glucose
is added to a culture of a strain expressing
nMDH2, adaptation to
utilization of glucose as a carbon source is slowed
(12), suggesting that
catabolite inactivation may provide a physiological advantage by rapidly
removing enzymes that interfere with optimal rates of glycolysis. In this
report, we also provide evidence that the catalytically active
nMDH2
enzyme may not function efficiently in gluconeogenesis in vivo.
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Because of the unfavorable equilibrium for formation of oxaloacetate from
malate (G0'
+7 kcal/mol), it has been
proposed that physical interactions between malate dehydrogenase and the next
enzyme in the same metabolic pathway may be necessary to ensure direct
transfer of oxaloacetate (15),
a metabolite present in very low cellular concentrations. In support of this
proposal, substantial experimental evidence has been presented for
interactions between the mitochondrial tricarboxylic acid cycle enzymes,
malate dehydrogenase and citrate synthase, in yeast and other organisms
(1619).
In the current study, we present evidence for physical interactions of MDH2
with PCK1 and FBP1. These results suggest an association of these cytosolic
enzymes in vivo that may facilitate metabolic flux through
gluconeogenesis.
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EXPERIMENTAL PROCEDURES |
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Plasmid Constructs and Yeast Two-hybrid AssaysFor growth
phenotype analyses, the mdh2 strain was transformed
(22) with pRS314 plasmids
(23) carrying authentic
(MDH2) or truncated (
nMDH2) coding regions of
MDH2. The pRS314MDH2 plasmid was previously described
(24), and was used for
mutagenesis to remove 12 codons following the AUG translation initiation codon
(12) to produce the
pRS314
nMDH2 plasmid.
For subcloning into two-hybrid vectors (Clontech, Inc.), PCR was used to
introduce BamHI restriction sites onto the 5' and 3' ends
of the coding region of MDH2 for in-frame fusions with the 3'
end of the GAL4 DNA-binding domain sequence in pAS2-1 (carrying
GAL4(1147) and TRP1
for selection) and with the 3' end of the GAL4
transcription-activation domain sequence in pACT2 (carrying
GAL4(768881) and LEU2
for selection). This generated plasmids designated pASMDH2 and
pACTMDH2. A truncated form of the MDH2 gene
(nMDH2) lacking codons for the first 12 amino acids was
obtained by oligonucleotide-directed mutagenesis of pACTMDH2, and the
truncated coding region was transferred to pAS2, generating both
pACT
nMDH2 and pAS
nMDH2.
To construct "reverse" two-hybrid vectors to express fusion
proteins with GAL4 domains on the carboxyl-terminal side, the EcoRI
and BamHI sites in the multicloning sites of pACT and pAS2-1 were
eliminated by oligonucleotide-directed mutagenesis. Subsequent mutagenesis was
used to reinsert EcoRI and BamHI sites between the
ADH1 promoter and the GAL4 domain coding sequences in these
plasmids. The resulting reverse two-hybrid vectors permit in-frame cloning of
coding sequences (carrying a 5' translation-initiator AUG codon) onto
the 5' end of the GAL4 domain sequences. PCR fragments
containing MDH2 and nMDH2 coding sequences with
EcoRI restriction sites on the 5' and 3' ends were cloned
into the reverse pACT and pAS2-1 plasmids as illustrated in
Fig. 4.
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To construct conventional two-hybrid vectors (pASPCK1 and pACT-PCK1) containing PCK1 sequences, PCR was conducted with a YEp352 plasmid carrying PCK1 (provided by Dr. E. Cardemil) (25) to generate the PCK1 coding region flanked by EcoRI restriction sites for subcloning. Similar vectors (pASFBP1 and pACTFBP1) containing FBP1 sequences were generated using PCR with yeast genomic DNA as the template to generate the FBP1 coding region with 5' and 3' XhoI restriction sites for ligation into plasmid SalI or XhoI sites. Coding sequences in two-hybrid plasmids were verified by DNA sequence analysis.
The two-hybrid plasmids were transformed singly and in pairwise pAS/pACT
combinations into Y190, and transformants were selected by growth on YNB
plates lacking tryptophan and/or leucine. A positive control strain was
obtained by transformation with plasmid pCL1, a centromere-based plasmid
carrying the full GAL4 gene and LEU2 for selection
(26). Transformants were
restreaked for colony filter assays of lacZ reporter gene expression
as described in protocols provided by Clontech. The time required for colony
color development was monitored over a 24-h period. Enzyme assays for
-galactosidase activity were conducted using extracts prepared by
freeze-thaw cycles from transformants harvested from YP glucose cultures at
measured densities (A600 nm = 12).
-Galactosidase activity was measured spectrophotometrically
(A578 nm) at times (min) following color
development with chlorophenol red-
-D-galactopyranoside as the
substrate (27). Units are
expressed as A578 nm x 103/min
ml of A600 nm.
Affinity Purification and Kinetic AnalysesTo insert tags
for affinity purification, MDH2 and nMDH2 genes on
pRS426 plasmids, and the PCK1 gene on a YEp352 plasmid, were used for
oligonucleotide-directed mutagenesis to add codons for six histidine residues
between the final codon and the stop codon of each gene. Each plasmid
contained
500 bp of the authentic 5' promoter sequence for
expression of each gene. A 2.0-kb DNA fragment containing the FBP1
coding region and adjacent promoter sequences was cloned using PCR from yeast
genomic DNA and inserted into pRS426. Oligonucleotide-directed mutagenesis was
subsequently used to insert nine codons for the residues in a streptavidin tag
(28) into the 3' ends of
PCK1 and FBP1 genes. Each plasmid was transformed into the
yeast strain containing a disruption in the corresponding MDH2, PCK1,
or FBP1 chromosomal locus.
For affinity purification, transformants were grown in 10-ml cultures of
YNB glucose medium to maintain plasmid selection, then transferred to
2501000-ml cultures of YP ethanol medium to induce expression of
gluconeogenic enzymes (6). The
cells were harvested at A600 nm = 45,
and cell pellets were stored at 20 °C. For purification of
histidine-tagged enzymes, the cell pellets were lysed with glass beads, and
the lysate was diluted 1:1 with a buffer containing 100 mM sodium
phosphate (pH 8.0), 600 mM sodium chloride, and 40 mM
imidazole. The diluted lysate was incubated with 2 ml of
Ni2+-NTA superflow resin (Qiagen) for 1 h at 4 °C
and loaded into a 1-cm diameter column. The column flow-through fraction was
collected, and the column was washed with three column volumes of a buffer
containing 50 mM sodium phosphate (pH 8.0), 300 mM
sodium chloride, and 20 mM imidazole. Bound protein was eluted with
1.0 ml of the same buffer containing 250 mM imidazole. For
purification of streptavidin-tagged enzymes, 2 µg of avidin was added to
the cell lysates, and the lysates were loaded onto 1.0-ml streptavidin-agarose
columns (immunopure affinity Pak immobilized streptavidin, Pierce). The
columns were washed with 5 ml of phosphate buffer (100 mM sodium
phosphate, 50 mM sodium chloride, and 1 mM
phenylmethylsulfonyl chloride). We found that the streptavidin-tagged enzymes
bind weakly to these columns and are eluted with a second 5-ml phosphate
buffer wash. Yields for the affinity purified enzymes were 0.5 mg of
histidine-tagged enzymes and 0.10.2 mg of streptavidin-tagged enzymes
per 1.21.7 g of cells harvested from 250-ml cultures. Protein
concentrations were determined using absorbance at A280
nm and calculated extinction coefficients
(29). Samples obtained during
affinity purification were electrophoresed on 10% polyacrylamide/sodium
dodecyl sulfate gels for Coomassie Blue staining or for immunoblotting.
Enzyme activities for malate dehydrogenase
(1), phosphoenolpyruvate
carboxykinase (30), and
fructose-1,6-bisphosphatase
(31) were measured as
previously described. Activities measured for affinity purified enzymes were:
200250 units/mg for MDH2His and nMDH2His,
8 units/mg for PCK1His and PCK1Strep, and
10
units/mg for FBP1Strep. These values are 23-fold lower than
those reported for conventionally purified MDH2
(6), PCK1
(30), and FBP1
(31).
For determination of kinetic parameters, 0.15 µg of MDH2His
or nMDH2His were used in 1.0-ml assays containing 50
mM potassium phosphate (pH 7.5). NADH concentrations were varied
from 0 to 0.4 mM in the presence of 0.35 mM
oxaloacetate, or oxaloacetate concentrations were varied from 0 to 1.0
mM in the presence of 0.4 mM NADH. Units are defined as
micromole of NAD+ produced per minute.
Immunoblots and Co-immunoprecipitationImmunoblot analysis
of MDH2 or nMDH2 was conducted using a polyclonal antiserum prepared
against MDH2 (6). The antiserum
was preadsorbed with extracts from a
mdh2 strain and used at
dilutions of 1:250. A polyclonal antiserum against FBP1 was obtained from Dr.
K. Eschrich (32) and was used
at dilutions of 1:500. Histidine-tagged enzymes were also detected using a
monoclonal penta-histidine antiserum obtained from Qiagen. Immunocomplexes
were detected with the enhanced chemiluminescence method (Amersham
Bioscience).
For co-immunoprecipitation experiments, cells were pelleted from YP ethanol cultures of the parental strain or, for controls, of gene disruption strains. Pellets of cells from 20 ml of the cultures (A600 nm = 1.52.0) were lysed in 400 µl of 10 mM sodium phosphate (pH 7.5) containing a protease mixture (20 µg/ml chymostatin, 10 µg/ml pepstatin A, 0.5 µg/ml leupeptin, 5 mM benzamidine, 20 µg/ml E-64, and 2 mM phenylmethylsulfonyl chloride) and sodium chloride at concentrations ranging from 10 to 150 mM. The cleared lysates were incubated for 1 h at 4 °C with 25 µl of antiserum against MDH2 or FBP1. Protein A-Sepharose beads (50 µl; Sigma) were added and the incubation continued for 30 min. The Sepharose beads were pelleted and washed three times with 400 µlof10mM sodium phosphate buffer (pH 7.5) containing 50 mM sodium chloride, then resuspended and boiled in 50 µl of SDS electrophoresis sample buffer. After centrifugation to remove the beads, the supernatants were used for gel electrophoresis and immunoblot analysis.
Hummel-Dreyer AnalysesGel filtration chromatography was conducted with a Superdex 200 HR 10/30 column (24 ml bed volume) attached to an Applied Biosystems HPLC model 120A. The chromatography buffer contained 50 mM sodium phosphate (pH 7.4) and 50 mM sodium chloride. Samples and standard proteins (at concentrations of 4 µM for calibration and otherwise as indicated in the text) were injected with a 50-µl loop and chromatographed at a flow rate of 0.25 ml/min. For some experiments, 4.0 µM MDH2 was included in the chromatography buffer.
Surface Plasmon Resonance AnalysesExperiments were
performed on a BIAcore 2000 surface plasmon resonance (SPR) instrument using
steptavidin and carboxymethyl dextran (CM5) research grade chips (BIAcore,
Piscataway, NJ). Runs were conducted at 25 °C with HBS buffer (10
mM HEPES, pH 7.0, 150 mM sodium chloride, 3
mM EDTA, and 0.005% surfactant P20) at a flow rate of 5 µl/min.
Proteins were immobilized on steptavidin chips by injection in 50
mM HEPES (pH 8.0) containing 300 mM sodium chloride. CM5
chips were activated by a 6-min injection of 50% N-hydroxysuccinimide
and 50% N-ethyl-N'-(dimethylaminopropyl)carbodiimide.
Proteins (ligands) were immobilized on activated CM5 chips at 10003500
response units (RU; 1 RU = a change of 1 ng/mm2 in surface
protein concentration) at pH 7.5. After immobilization, the surfaces were
blocked by a 6-min injection of 1.0 M ethanolamine (pH 8.5).
Soluble analyte proteins or bovine serum albumin as the control were passed
over the surfaces at the indicated concentrations for 3 min. The surfaces were
regenerated between binding runs by a 3-min injection of 1 M sodium
chloride or 4 M guanidine HCl.
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RESULTS |
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We additionally found that transformation of the mdh2
strain with a centromere-based plasmid containing the MDH2 gene
restores the ability to grow on minimal medium plates with ethanol or acetate
as carbon sources. In contrast, transformation of the same strain with a
similar plasmid bearing the
nMDH2 gene fails to restore growth
under these conditions. These results were extended by measuring culture
doubling times in minimal medium. When shifted from minimal glucose to minimal
ethanol medium, the parental strain and the
mdh2 strain
expressing MDH2 exhibit a lag of
24 h before attaining doubling times of
5.9 and 6.9 h, respectively (Table
I). In contrast, all three gluconeogenic gene disruption strains
and the
mdh2 strain expressing
nMDH2 fail to grow under
these conditions. Similar results were obtained when strains and transformants
were shifted to minimal medium with acetate as the carbon source
(Table I), although doubling
times for the parental strain and for the
mdh2 strain
expressing MDH2 were significantly longer on this medium. These results
suggest that
nMDH2 does not compensate for the normal gluconeogenic
function of MDH2.
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Similar levels of malate dehydrogenase activity (78 units/mg of
protein) were measurable with cellular extracts from the mdh2
strains expressing MDH2 or
nMDH2. However, because of expression of
other MDH isozymes in those strains, immunoblot analysis was conducted to
specifically examine levels of expression of MDH2 and
nMDH2. As
illustrated in Fig. 2, this
analysis indicates no significant difference between levels of MDH2 (lane
2) and levels of
nMDH2 (lane 3) in extracts from
respective transformants. Levels of the plasmid-expressed enzymes exceed that
of chromosomally expressed MDH2 in extracts from the parental strain (lane
1) by 23-fold, and MDH2 is clearly absent in the
mdh2 strain (lane 4).
Fig. 2 also illustrates the
differences in electrophoretic mobilities of authentic MDH2 (lanes 1
and 2) relative to
nMDH2 (lane 3) and relative to
purified MDH2 carrying a histidine tag (lane 5), as described
below.
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To examine activity and kinetic properties of the MDH2 and nMDH2
enzymes in more detail, we constructed plasmids for production of
carboxyl-terminal histidine-tagged versions of both enzymes. The enzymes were
affinity purified with similar yields using Ni2+-NTA
chromatography. Basic kinetic properties of the purified enzymes were compared
using standard assay conditions. In assays with increasing concentrations
(0300 µM) of oxaloacetate, both enzymes exhibit
hyperbolic kinetic curves, as illustrated in
Fig. 3A. Higher
concentrations of oxaloacetate produce an inhibition of activity
(inset in Fig.
3A), resulting in velocities lower than the apparent
Vmax as also reported by others
(33). Using data from the
hyperbolic regions of the curves, similar apparent Vmax
and Km values for oxaloacetate were obtained for
MDH2 and
nMDH2 (Table
II). With NADH as the varied assay component, the wild-type and
mutant enzymes exhibit similar kinetic curves
(Fig. 3B), and both
enzymes are inhibited at high concentrations of NADH (inset in
Fig. 3B). On average,
the
nMDH2 enzyme exhibits slightly higher apparent
Km and Vmax values with NADH
than the MDH2 enzyme (Table
II). The kinetic parameters obtained for the affinity purified
wild-type enzyme are similar to those previously reported for the
conventionally purified enzyme
(9), indicating that the
histidine tag has little effect on catalytic properties of the enzyme.
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We also examined kinetic parameters for the reverse reaction as would be
required for function in gluconeogenesis. With NAD+ as the variable
assay component (01.0 mM) and a malate concentration of 50
mM, MDH2 and nMDH2 enzymes exhibit similar apparent
Vmax values (460 and 490 units/mg, respectively) and
Km values for NAD+ (83 and 120
µM, respectively). For malate (concentrations varied from 0 to
200 µM with an NAD+ concentration of 1.0
mM), similar apparent Km values were
obtained (59 µM for MDH2 and 55 µM for
nMDH2), and the mutant enzyme exhibits a higher
Vmax value (130 units/mg) than the wild-type enzyme (44
units/mg). Collectively, these data suggest that the authentic and truncated
MDH2 enzymes are kinetically quite similar, and that any differences in
function in vivo cannot be attributed to inherent catalytic
differences or to differences in steady state levels of expression in
ethanol-grown cultures.
Two-hybrid Analyses of MDH2 and nMDH2Among
possible explanations for differential function of the authentic and truncated
forms of MDH2 in vivo despite their kinetic similarities are
differences in interactions with other cellular components. Because of the
dominant effect of MDH2 truncation on apparent glucose-induced turnover of
other gluconeogenic enzymes
(12), and the inability of the
nMDH2 enzyme to support gluconeogenesis as described above, we
hypothesized that co-localization and/or interactions among gluconeogenic
enzymes in the cytosol might facilitate pathway function and/or regulated
turnover, and that the amino-terminal extension of MDH2 might be essential for
these processes. To investigate these possibilities, we initially tested
possible interactions among gluconeogenic enzymes using the yeast two-hybrid
system because this provides a sensitive in vivo assay.
For two-hybrid assays, MDH2 and nMDH2 were expressed as fusion
proteins with DNA-binding and transcriptional activation domains of GAL4 using
conventional two-hybrid plasmids. These constructs result in GAL4 domains
fused to the amino termini of MDH2 and
nMDH2. However, anticipating the
possibility that such fusions might block potential interactions of the
amino-terminal extension of MDH2
(34), we also constructed
reverse two-hybrid plasmids that permit expression of MDH2 and
nMDH2
with free amino termini and GAL4 domains fused to the carboxyl termini
(Fig. 4).
For initial tests of fusion proteins in two-hybrid assays, we determined
the ability to detect MDH2 and nMDH2 homodimeric interactions. As shown
in Table III, positive results
are obtained with colony filter assays with both conventional and reverse MDH2
and
nMDH2 fusion proteins. Transformants expressing the conventional
pairs of MDH2 and
nMDH2 fusion proteins give less robust results (2 h
for development of blue color indicating
-galactosidase activity) than
does a transformant expressing full-length GAL4 (40 min for development of
blue colony color). Enzyme activity measurements were generally consistent
with colony color except for extracts from the
nMDH2 pair, which gave
measurable but much lower levels of activity for
-galactosidase. In
contrast, both colony filter assays and enzyme assays indicate approximately
equivalent
-galactosidase expression in transformants expressing the
reverse pairs of MDH2 and
nMDH2 fusion proteins, and these levels are
similar to those obtained with full-length GAL4. We also tested and found that
MDH2 and
nMDH2 fusion proteins can be paired together in various
combinations to form "heterodimers" in these two-hybrid assays
(data not shown). These results suggest that (a) both the authentic
and truncated MDH2 polypeptides expressed with these plasmids are
appropriately folded in vivo for homodimeric interactions,
(b) that truncation of MDH2 does not alter the strength of
polypeptide interactions in the homodimer, and (c) that addition of
the GAL4 domains onto the amino termini may slightly retard homodimeric
interactions.
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To test possible heteromeric interactions of MDH2, we constructed
conventional two-hybrid plasmids containing PCK1 and FBP1
coding regions fused to the 3'-end of GAL4 domain sequences.
These plasmids were co-transformed in all possible pairwise combinations with
the conventional and reverse two-hybrid plasmids containing MDH2 and
nMDH2 gene fusions. As illustrated in
Table IV, most of the assays
were negative. However, positive results were observed with two pairs of
constructs. Both contain the GAL4 DNA-binding domain fused to the amino
terminus of MDH2, which appears to interact with the GAL4
transcription-activation domain fused to either PCK1 or FBP1. With both types
of assays, the levels of
-galactosidase activity in these transformants
are lower than those detected in a transformant expressing full-length GAL4 or
the (
n)MDH2 homodimers (Table
III), but they are substantially higher than background levels
detected with negative controls obtained with pairs of empty two-hybrid
plasmids or with pairs expressing only one fusion protein. The MDH2/FBP1 pair
exhibits lower levels of
-galactosidase activity in enzyme assays than
does the MDH2/PCK1 pair. We also tested and found no evidence with this method
for an interaction between PCK1 and FBP1 (data not shown).
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Because putative interactions for MDH2 with PCK1 or with FBP1 can be
detected only when MDH2 is fused to the carboxyl terminus of the GAL4
DNA-binding domain (Table IV,
section A), this suggests that the GAL4 transcription-activation domain in
this position may block the interactions. With similar conventional two-hybrid
constructs, no interactions are detectable for nMDH2 with PCK1 or FBP1
(Table IV, section B),
suggesting that the amino-terminal extension of authentic MDH2 is essential
for such interactions. Neither MDH2 nor
nMDH2 in reverse two-hybrid
fusion proteins exhibits interactions with PCK1 or FBP1 in these assays
(Table IV, sections C and D),
suggesting for MDH2 that relatively large GAL4 domains fused to the carboxyl
terminus may block interactions observed when the GAL4 DNA-binding domain is
fused to the amino terminus of MDH2. The latter suggestion seems reasonable in
light of the proximity of amino and carboxyl termini in three-dimensional
structures of related malate dehydrogenases
(35,
36).
In summary, these data from two-hybrid analyses suggest that free amino
termini enhance homodimer formation for both MDH2 and nMDH2. The
apparent interactions between MDH2 and PCK1 or FBP1 are observed only in
certain pairs of possible domain combinations and orientations. These
interactions appear to require the amino-terminal 12-residue extension of
MDH2.
Physical Assays for Protein Interactions Involving MDH2 To initiate physical approaches to examine possible interactions among yeast gluconeogenic enzymes, we generated constructs to permit affinity purification of PCK1 and FBP1. Plasmids were constructed for expression in yeast of PCK1 carrying a carboxyl-terminal pentahistidine tag, and for expression of PCK1 and FBP1 carrying nine-residue streptavidin tags on their carboxyl termini. Electrophoretic analyses of fractions obtained during affinity purification of MDH2His, FBP1Strep, PCK1His, and PCK1Strep are illustrated in Fig. 5 (panels AD, respectively). We found that the histidine-tagged enzymes are efficiently purified with good yields, whereas the steptavidin-tagged enzymes are purified with significantly lower yields. All purified enzymes are active in standard enzyme assays, confirming retention of the native states during purification.
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Our first test for physical associations among the gluconeogenic enzymes was to assess the possibility of co-purification. As shown in Fig. 5, denaturing electrophoretic analysis of each of the affinity purified enzymes produced no evidence for copurification of significant amounts of other gluconeogenic enzymes. For more sensitive assays, we assayed wash and elution fractions from each affinity column for activity of the other gluconeogenic enzymes. Results of these assays, as well as results of immunoblot analyses of the same fractions with antisera recognizing the other gluconeogenic enzymes, provided no evidence for co-purification (data not shown). We conclude that interactions between MDH2 and PCK1 or FBP1 that are detectable with two-hybrid assays are insufficient to withstand the relatively stringent conditions of affinity chromatography.
Co-immunoprecipitation techniques were also tested as described under
"Experimental Procedures" using polyclonal antisera against MDH2
and FBP1. We found some evidence for co-immunoprecipitation of MDH2 with the
anti-FBP1 antiserum from extracts of the parental strain, and MDH2 was not
precipitated with the same antiserum when extracts were prepared from the
FBP1 strain (data not shown). However, these results were
difficult to routinely reproduce, and the converse co-immunoprecipitation of
FBP1 from parental cell extracts with anti-MDH2 serum was not detected. We
therefore focused on using the affinity purified enzymes for other physical
assays for protein interactions.
With histidine-tagged forms of MDH2 and PCK1, it was possible to purify
sufficient quantities of both enzymes to apply the classical Hummel-Dreyer
chromatography method for binding analyses
(3739).
For this, we developed a gel filtration system for resolution of MDH2 and PCK1
and determined their elution patterns relative to a set of standard proteins
(inset, Fig. 6). MDH2
eluted with a calculated Mr of 71,000 and PCK1 eluted with
a calculated Mr of 212,000, values, respectively,
consistent with the native dimeric and tetrameric forms of these enzymes. The
gel filtration column was then equilibrated with buffer containing 4.0
µM MDH2His and loaded with a sample containing 1.0
µM PCK1His. The elution profile showed, in addition
to the peak for PCK1His, a trough in the basal absorbance (data not
shown), indicating binding of MDH2His in the buffer to
PCK1His (39).
Similar chromatography runs were conducted with the same buffer and samples
containing 1.0 µM PCK1His plus MDH2His at
concentrations ranging from 0 to 14 µM. The size of the PCK1
peak eluting in each column run increased with increasing concentrations of
MDH2 in the sample. The increases in the size of the PCK1 peak were attributed
to binding of MDH2 by PCK1 and were used to calculate the concentration of
bound MDH2 (Fig. 6). The slope
of the plot indicates a binding to PCK1 of 0.14 µmol of MDH2/µmol
of total MDH2 within these concentration ranges. Because of limitations with
absorbance measurements, it was not possible to achieve saturation
concentrations. However, an interaction between these two gluconeogenic
enzymes is clearly measurable using this technique.
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We also tested a second sensitive method for detection of protein-protein interactions, SPR, which requires relatively small amounts of purified proteins. The affinity tags of PCK1Strep and FBP1Strep were used initially to couple the proteins (ligands) to the surfaces of strepavidin chips. MDH2His as the soluble analyte was found to bind to both chips and not to a control chip carrying biotinylated bovine serum albumin. However, the kinetics of binding were slow, precluding accurate measurements of saturation. Therefore, PCK1Strep and FBP1Strep were covalently immobilized on carboxymethyl dextran (CM5) chips, which significantly increased the kinetic response to MDH2 binding.
The binding of MDH2His and nMDH2His to CM5
chips carrying PCK1Strep was measured at equilibrium using a range
of concentrations of each analyte protein. The binding of MDH2 by PCK1 was
found to be dose-dependent and saturable ( in
Fig. 7A), whereas no
binding of bovine serum albumin by PCK1 was observed in parallel experiments
(data not shown). A calculated maximum of 10,590 RU of MDH2 could be bound to
a chip loaded with 1,560 RU of PCK1. Saturation was achieved with ≥8
µM MDH2His as ligand. Binding to PCK1 was also
observed using
nMDH2His as the analyte (
in
Fig. 7A). Whereas the
calculated maximum binding (10,640 RU of
nMDH2His by a chip
loaded with 996 RU of PCK1) was similar to that obtained with the authentic
enzyme, the saturation curve suggests a significantly lower affinity. These
binding data were used for Scatchard plot analysis
(Fig. 7B) to calculate
Kd values of 0.63 µM for the
MDH2/PCK1 interaction and of 7.69 µM for the
nMDH2/PCK1
interaction. Thus, affinity of PCK1 for MDH2 is dramatically reduced by
amino-terminal truncation. The n value obtained from the Scatchard
plot, corrected for relative Mr values for the holoenzymes
(81,400 for dimeric MDH2 and 243,920 for tetrameric PCK1), gives a maximum of
20 mol of authentic MDH2 bound per mol of PCK1.
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Similarly, binding of MDH2His and nMDH2His to
CM5 chips carrying covalently bound FBP1Strep was measured at
equilibrium for a range of concentrations of each analyte protein. The binding
of MDH2 by FBP1 was also found to be dose-dependent and saturable
(Fig. 8A), whereas no
binding of bovine serum albumin by FBP1 was observed in parallel experiments
(data not shown). A maximum of 10,410 RU of MDH2 could be bound to a chip
loaded with 8,710 RU of FBP1. Saturation was achieved with
8
µM MDH2. In contrast, we found that concentrations as high as 20
µM
nMDH2His produced no detectable binding to
chips loaded with equivalent RU of FBP1. Thus, truncation of MDH2 appears to
eliminate measurable interaction with FBP1. Scatchard analysis of the binding
data for authentic MDH2 to immobilized FBP1
(Fig. 8B) was used to
calculate a Kd value of 0.58 µM and
an n value of 0.56 mol of dimeric MDH2 bound per mol of monomeric
FBP1 (Mr = 38,260). The latter value thus implies an
equimolar association of MDH2 and FBP1 polypeptides.
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Collectively, these SPR results provide further evidence for physical
interactions among yeast gluconeogenic enzymes. PCK1 and FBP1 exhibit similar
affinities but different holoenzyme stoichiometries for binding MDH2. The
affinity of PCK1 for nMDH2 appears to be substantially lower than that
for MDH2, and no interaction between FBP1 and
nMDH2 was detectable.
Thus, these results from SPR experiments are generally consistent with those
obtained with yeast two-hybrid assays using the authentic and truncated forms
of MDH2.
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DISCUSSION |
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Positive results from yeast two-hybrid assays are potentially the most
convincing evidence for interactions of MDH2 (but not of nMDH2) with
PCK1 and with FBP1, because these assays are conducted in vivo. Based
on colony filter and enzyme assays for reporter gene expression, these
interactions are weak relative to interactions between identical polypeptides
to form homodimers of MDH2 (and of
nMDH2). The weakness of heteromeric
interactions among gluconeogenic enzymes may in part be because of
localization in the nucleus in these assays. The heteromeric gluconeogenic
enzyme interactions were also found to be highly dependent on orientation and
the type of GAL4 domain fused to MDH2, because positive results are obtained
only when the GAL4 DNA-binding domain is fused to the amino terminus of MDH2.
This orientation dependence is contrary to our initial prediction that
interactions might be stronger with a free amino terminus of MDH2. However,
because of the proximity of amino- and carboxyl-terminal ends of malate
dehydrogenase polypeptides in crystallographic analyses
(35,
36), we assume that fusion of
GAL4 domains onto the carboxyl terminus of MDH2 blocks heteromeric two-hybrid
interactions with PCK1 and FBP1. This is not the case for homodimeric
interactions, which appear to be enhanced by the free amino termini on MDH2
(and
nMDH2) subunits.
We used several methods to study interactions of gluconeogenic enzymes
in vitro. Whereas MDH2 and FBP1 could be co-immunoprecipitated, this
technique was not sufficiently reliable for further analyses. A physical
interaction between MDH2 and PCK1 was demonstrated with Hummel-Dreyer
chromatography. Physical interactions between MDH2 and PCK1 and between MDH2
and FBP1 were also observed using SPR. In contrast, SPR results indicated a
significant reduction in affinity for binding of nMDH2 by PCK1 and no
binding of
nMDH2 by FBP1. Consistent with two-hybrid results, SPR data
indicate that interactions of PCK1 and FBP1 with MDH2 are relatively weak,
with Kd values in the low micromolar range. In
fact, these Kd values are near the limit for
detection with two-hybrid assays
(38,
40). This limit would explain
the inability of two-hybrid assays to detect even weaker
nMDH2/PCK1
interactions. The stoichiometries obtained for MDH2/PCK1 and MDH2/FBP1
interactions by SPR are very different and imply differences in individual
subunit interactions (10:1 for MDH2 and PCK1 subunits and 1:1 for MDH2 and
FBP1 subunits). The stoichiometry for the MDH2/FBP1 interaction is clearly
closer to that expected for a physiologically relevant interaction. However,
it will be of interest to determine how these values correlate with relative
cellular concentrations of the enzymes, because one prediction for association
of enzymes in a multienzyme complex is the maintenance of specific
stoichiometries within the complex.
Our evidence for physical associations, particularly between MDH2 and PCK1, is consistent with a prediction by Srere (15) that close proximity with the next enzyme within a metabolic pathway may be important to ensure direct delivery of oxaloacetate generated in a reaction catalyzed by malate dehydrogenase. An interaction between sequential enzymes in a given metabolic pathway is frequently cited as evidence for the possibility of "channeling" of intermediates within the pathway (41). To our knowledge, ours are the first data demonstrating such interactions involving the cytosolic gluconeogenic isozyme of malate dehydrogenase. However, there is abundant experimental evidence for interactions between the mitochondrial tricarboxylic acid cycle isozyme of malate dehydrogenase and citrate synthase. This evidence has been obtained using a variety of methods including gel filtration chromatography and polyethylene glycol precipitation (17). In addition, kinetic models, based on yeast or mammalian fusion proteins containing citrate synthase fused to the amino terminus of malate dehydrogenase, have been presented to support the potential for channeling of oxaloacetate between the soluble enzymes (17, 18, 42). Vélot and Srere (19) also used a peptide "purtubagen," an in vivo competitor, to identify a region (residues 353366) of yeast citrate synthase (CIT1) that appears to be important in functional interactions with MDH1 in vivo. In preliminary experiments, we have been unable to detect interactions between MDH1 and CIT1 using yeast two-hybrid assays, but results cited above obtained for the fused CIT/MDH enzymes suggest that such interactions may be highly dependent on orientation and on linker spacing between MDH1 or CIT1 and GAL4 domains.
At present, we have no evidence for a kinetic advantage of physical associations among the gluconeogenic enzymes, but their co-localization would theoretically reduce diffusion of intermediates (e.g. oxaloacetate) away from the pathway. In the current study, we have focused upon the enzymes unique to gluconeogenesis. Clearly, however, this pathway also requires several enzymes that also function in glycolysis. Therefore, it will be of interest to examine potential associations with some of the glycolytic enzymes.
A particular reason for focus on the enzymes unique to gluconeogenesis in
yeast is the dramatic coordinate degradation of these enzymes during
catabolite inactivation. Within minutes after addition of glucose to cells
growing on non-fermentable carbon sources, the unique gluconeogenic enzymes
are catalytically inactivated by a process that involves phosphorylation
(43,
44), then degraded by a
mechanism that occurs at a much faster rate than normal cellular protein
turnover (13,
14). The mechanism for such
rapid and specific glucose-induced degradation is of significant interest, but
is currently somewhat controversial. Evidence exists both for a mechanism
involving a vacuolar protease
(45) and/or for a
ubiquitin-dependent mechanism involving the cytosolic proteosome
(46,
47). The amino termini of MDH2
(i.e. the residues missing in nMDH2) and FBP1 share features
including several hydroxylated and charged amino acids. Phosphorylation of
Ser-11 of FBP1 appears to be involved in glucose-induced catalytic
inactivation but not degradation
(43). MDH2, but not
nMDH2, is also phosphorylated in response to glucose
(44). However, an aspartate
replacement of Ser-12 of MDH2 produced an enzyme still subject to
phosphorylation and inactivation but not to rapid degradation. Thus, although
details and requirements for catabolite inactivation are not identical for the
two enzymes, amino termini of both MDH2 and FBP1 are clearly involved in the
process. According to a recent report
(48), the most important
shared feature in this region may be the presence of an amino-terminal proline
residue that is essential for glucose-induced degradation of FBP1. Structural
features important for catabolite inactivation of PCK1, which lacks an
amino-terminal proline residue, have not been analyzed. However, it will
obviously be of interest in the future to examine the importance of the
amino-terminal proline residue of MDH2 in interactions with PCK1 and FBP1
using methods described in this report.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 210-567-3782; Fax:
210-567-6595; E-mail:
henn{at}uthscsa.edu.
1 The abbreviations used are: MDH, malate dehydrogenase; PCK1,
phosphoenolpyruvate carboxykinase; FBP1, fructose-1,6-bisphosphatase;
Ni2+-NTA, nickel-nitrilotriacetic acid; SPR, surface
plasmon resonance; RU, response units.
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ACKNOWLEDGMENTS |
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REFERENCES |
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