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INTRODUCTION |
All plants and a wide range of prokaryotes contain
phosphoenolpyruvate carboxylase
(PEPC1; EC 4.1.1.31) (1),
which catalyzes the irreversible carboxylation of the glycolytic
intermediate phosphoenolpyruvate (PEP) to yield oxaloacetate and
Pi. In C4 and CAM (crassulacean
acid metabolism) plants, PEPC catalyzes the
first step in photosynthetic carbon fixation. Because of its
central role in the metabolism and primary productivity of economically
important C4 crops, PEPC has been extensively characterized at the
structural and regulatory levels in organisms such as maize and sorghum
(1, 2). C4 PEPCs are homotetramers with a subunit size of 100-110 kDa.
(2). C4 PEPC is subject to regulatory phosphorylation by a
Ca2+-insensitive PEPC kinase that phosphorylates a Ser
residue near the N terminus (e.g. Ser-15 in maize), thus
attenuating PEPC sensitivity to inhibition by malate (1). In C3
photosynthetic and non-photosynthetic tissues and in many prokaryotes,
PEPC also fulfills an important metabolic function, which is the
anaplerotic supply of carbon to the tricarboxylic acid cycle for
biosynthetic purposes (1-6). Despite this fact, C3 PEPCs have received
a lot less attention than their C4 counterparts.
This situation is particularly obvious in the case of photoautotrophs.
Estimates indicate that 50% of global photosynthesis is carried out by
phytoplankton in aquatic ecosystems (7, 8). Moreover, these organisms
are also responsible for >70% of global nitrogen assimilation
(9). Indeed, up to 50% of algal carbon metabolism is coupled to
nitrogen metabolism (10). However, despite the importance of algae in
the global carbon and nitrogen economy, little is known at the
molecular level about the enzymes involved in the interactions between
carbon and nitrogen metabolism in these organisms.
Previous studies using the chlorophyte Selenastrum minutum
as a model system have demonstrated that PEPC catalyzes a key
regulatory step in the interactions between carbon and nitrogen
metabolism (3). The addition of a nitrogen source to nitrogen-limited S. minutum culture induces a rapid activation of PEPC
activity (3, 5), and the in vivo carbon flux through PEPC is
tightly coupled to nitrogen assimilation rates (11). Moreover, S. minutum PEPC is activated by Gln and inhibited by Glu (3, 6).
These data demonstrate that PEPC has an anaplerotic function in green algae in supplying the tricarboxylic acid cycle with carbon skeletons that are used for nitrogen assimilation through the synthesis of amino
acids (3).
We have undertaken a characterization of the structure and regulatory
features of PEPC in green algae. Our results indicate that algal PEPCs
are quite different from the enzyme present in higher plants.
Unicellular green algae such as S. minutum and Chlamydomonas reinhardtii typically contain two classes of
PEPC isoforms with strikingly different structural, kinetic, and
regulatory properties (6, 12). The kinetic and regulatory properties of
the two classes of green algal PEPCs are now well established (6, 12).
Class 1 PEPCs (termed PEPC1) are homotetrameric and have a relatively
high Km for their substrate PEP. Class 2 PEPCs,
which represent the bulk of extractable PEPC activity in green algae,
correspond in S. minutum to three large protein complexes
(PEPC2, PEPC3, and PEPC4) with a relatively low Km for PEP. In addition, the catalytic subunit of ~100 kDa present in
green algae is phylogenetically distinct from that of higher plant
PEPCs (12). Class 2 PEPCs are quite unusual and so far have been
described only in green algae. Neither the structure of Class 2 PEPCs
nor their subunit composition is known in any detail. It has
nevertheless been established that both algal PEPC classes contain the
same catalytic subunit (6, 12) with, in the case of S. minutum PEPC, a molecular mass of 102 kDa. Immunological data
indicate that the other subunits of the Class 2 PEPC complexes are not
related to this catalytic subunit (6, 12). The possibility that these
complexes may represent purified metabolons has been investigated, but
so far, no enzyme activity other than PEPC has been detected in Class 2 PEPCs (6). It is also possible that the polypeptides associated with
the PEPC catalytic subunits in Class 2 PEPCs represent non-catalytic
regulatory subunits of the enzyme (6). In this study, we undertook an
investigation of S. minutum Class 2 PEPCs. Taking PEPC2, the
most abundant S. minutum PEPC isoform, as a representative
of this class, we provide evidence that distantly related PEPC
polypeptides physically interact in these protein complexes. This
interaction between two types of PEPC subunits may add an additional
level of complexity to the metabolic regulation of this important enzyme.
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EXPERIMENTAL PROCEDURES |
Materials--
All chemicals were of analytical grade from Sigma
or BDH Chemicals (Toronto, Ontario, Canada). Butyl-Sepharose Fast Flow, protein A-Sepharose CL-4B, prepacked Superose 6 HR 10/30, and MonoQ 5/5 columns were from Amersham Pharmacia Biotech (Baie
d'Urfé, Province of Québec, Canada).
NaB3H4 and the fluorographic reagent
EN3HANCE was from PerkinElmer Life Sciences (Mississauga,
Ontario). The cross-linker DMS was from Pierce. Anti-banana fruit PEPC
and anti-S. minutum PEPC1 antisera were obtained as
described (4, 6). The latter antiserum is designated anti-p102
hereafter. Ribi adjuvant was obtained from Ribi Immunochemical Research
(Hamilton, MT), and alkaline phosphatase-tagged goat anti-rabbit IgG
was from Promega (Madison, WI).
Growth Conditions--
S. minutum (Naeg.)
Collins (UTEX 2459) was grown in chemostat cultures maintained at
20 °C under constant light (120 microeinsteins·m
2·s
1)
in Allen's medium (13) containing 5 mM
NO
. The steady-state growth of the
culture was 1 day
1. The cells were
harvested from the outflow of the chemostats by centrifugation (10 min
at 1100 × g), and the pellets were resuspended in
harvesting buffer (25 mM Hepes-KOH (pH 7.5), 20% (v/v)
glycerol, 1 mM EDTA, 1 mM EGTA, 10 mM MgCl2, 0.1% (v/v) Triton X-100, and 5 mM malate) using 1 ml of harvesting buffer/liter of
culture. The resuspended pellets were frozen in liquid N2
and kept at
80 °C.
PEPC Extraction, Assay, and Purification--
Cells were thawed,
and the slurry was adjusted to 1 mM EGTA, 2 mM
dithiothreitol, 2 mM phenylmethylsulfonyl fluoride, 2 mM 2,2'-dipyridyl, 10 µg/ml pepstatin, 10 µg/ml
chymostatin, and 10 µg/ml leupeptin (complete extraction buffer).
Cells were lysed using a French pressure cell (three passages at 18,000 p.s.i.). PEPC activity was assayed as described before (6) on Dynatech or Molecular Device microplate readers (14). The purification of PEPC
isoforms was carried out exactly as described (6), except that NaF was
omitted from the resuspension buffer after the PEG precipitation step
and that EGTA, phenylmethylsulfonyl fluoride, 2,2'-dipyridyl,
pepstatin, chymostatin, and leupeptin were added to the buffers used to
store the enzyme between chromatographic steps at the concentrations
given above. Protein was determined using bovine
-globulin as a
standard (15).
p130 Proteolytic Degradation Studies--
In the experiment
described for Fig. 1, all procedures were carried out on ice. Pellets
of cells corresponding to 2 liters of culture were resuspended in 2 ml
of (i) 10% (w/v) trichloroacetic acid; (ii) harvesting buffer
lacking EGTA but containing 2 mM dithiothreitol, 2 mM phenylmethylsulfonyl fluoride, and 10 µg/ml chymostatin (incomplete extraction buffer); or (iii) complete extraction buffer. Proteins were extracted by cell disruption by a
single passage through a 5-ml French pressure cell (16,000 p.s.i.)
followed by a 5-min centrifugation at 16,000 × g. The trichloroacetic acid precipitate was neutralized by resuspension in SDS
sample buffer adjusted to pH 8.2 and centrifuged at 16,000 × g to remove cellular debris. The supernatant was designated the trichloroacetic acid extract and was boiled before analysis. Supernatants made with incomplete and complete extraction buffers were
incubated on ice for various times before being denatured by boiling in
the above SDS sample buffer. Samples were analyzed by SDS-PAGE on an
8.5% acrylamide gel followed by immunoblotting. Incubation times
indicated in the legend to Fig. 1 refer to time after cell disruption.
Kinetic Analysis--
For kinetic analysis, MonoQ-purified PEPC2
was used. V values were routinely calculated using a
nonlinear least-square regression computer program (16). For PEPC2 PEP
saturation kinetics presented in Fig. 9, data were examined using
nonlinear regression analysis software (SigmaPlot Version 6.0 (SPSS
Inc., Chicago, IL) and DataFit Version 7.0 (Oakdale Engineering,
Oakdale, PA)). Experimental points were fitted to two possible models
(17): (i) a unique catalytic site model responding to the
Michaelis-Menten equation and (ii) a two-catalytic site (S1 and S2)
michaelian model responding to Equation 1,
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(Eq. 1)
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where V is the observed reaction velocity
corresponding to the sum of two rectangular hyperbolas.
Vmax1 and Vmax2 refer to the maximum velocities of S1 and S2, respectively, whereas
Km1 and
Km2 refer to the respective apparent
Michaelis constants of S1 and S2 for PEP. Kinetic parameters are the
mean of four determinations.
SDS-PAGE, Immunoblotting, and Fluorography--
Unless otherwise
stated, SDS-PAGE analysis was performed according to Laemmli (18) on
7.5% acrylamide gels. Subunit molecular masses were estimated using
the following protein standards: carbonic anhydrase (29 kDa), ovalbumin
(45 kDa), bovine serum albumin (66 kDa), phosphorylase b
(97.4 kDa),
-galactosidase (116 kDa), and myosin (205 kDa). Proteins
were stained with silver nitrate (19). Densitometric analysis was
performed on an Amersham Pharmacia Biotech Ultroscan XL laser
densitometer using Gelscan XL Version 2.1 software. Immunoblotting of
acrylamide gels was performed as described earlier (6). Gels were
routinely electroblotted onto polyvinylidene difluoride membranes for
80 min at a constant voltage of 125 V. For the analysis of cross-linked
products, proteins were electroblotted for 16 h at a constant
voltage of 30 V. Transfer efficiency was followed using the following
Bio-Rad high molecular mass prestained standards: ovalbumin (49.5 kDa),
bovine serum albumin (80 kDa),
-galactosidase (120 kDa), and myosin
(205 kDa). For protein cross-linking experiments, thyroglobulin monomer
(335 kDa) and cross-linked thyroglobulin (670 kDa) were also used. For
fluorography, the gels were first stained with Coomassie Blue to
visualize molecular mass standards. After destaining, they were treated
with 50 ml of EN3HANCE according to the manufacturer's
directions. After drying, the gels were exposed for 35 days at
80 °C on Kodak X-Omat film.
Preparation of Antisera and Affinity Purification of
IgGs--
Purified PEPC2 was subjected to preparative SDS-PAGE.
Polypeptides were stained with Coomassie Blue for 15 min, and the band corresponding to the 130-kDa polypeptide (p130) was excised. The p130
protein was electroeluted using a Bio-Rad electroeluter according to
the directions of the manufacturer. The protein solution (1 ml) was
then dialyzed for 18 h against 20 mM potassium
Pi (pH 7.4) and 150 mM NaCl. The dialysate was
stored at
20 °C until used for antiserum production. Antibodies
were raised using a 2-kg New Zealand White rabbit. After
collection of the preimmune serum, p130 (100 µg, emulsified in Ribi
adjuvant) was injected subcutaneously into the back of the rabbit.
Booster injections were performed at days 30 and 45 with 50 µg of
p130 freshly emulsified in Ribi adjuvant. Fifteen days after the final
injection, blood was collected by cardiac puncture. The serum was
collected after centrifugation at 1500 × g, frozen in
liquid N2, and kept at
80 °C. For immunoblot analysis,
anti-p130 and anti-p102 IgGs were affinity-purified against 15-20 µg
of purified p130 and PEPC1, respectively, as described (6).
Affinity-purified IgGs were diluted 10-fold in 50 mM
Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% (v/v) Tween 20, and
0.04% (w/v) NaN3 containing 0.03% (w/v) bovine serum
albumin and stored at 4 °C.
Immunoprecipitation--
PEPC2 was subjected to
immunoprecipitation with anti-p102 and anti-p130 antisera. Aliquots of
purified PEPC2 (1.5 milliunits) were incubated for 3 h with 20 µl of serum or with the corresponding preimmune serum in 100 µl of
immunoprecipitation buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.03% (w/v) bovine serum albumin, 20% (v/v)
glycerol, and 1% (v/v) Triton X-100). Antigen-antibody complexes were
pelleted with 20 µl of protein A-Sepharose beads (60 µg of protein
A) previously equilibrated in immunoprecipitation buffer. The beads
were pelleted by centrifugation for 1 min at 16,000 × g. Pellets were washed five times with 500 µl of
immunoprecipitation buffer. Immunoprecipitates were solubilized by
boiling in SDS sample buffer and analyzed by SDS-PAGE followed by immunoblotting.
N-terminal Sequencing--
Proteins were separated by SDS-PAGE
as described above, except that 0.1 mM sodium thioglycolate
was added to the upper buffer chamber. Following electrophoresis, the
gel was soaked for 10 min in transfer buffer (10 mM CAPS
(pH 11) and 10% (v/v) methanol) and then transferred onto a Bio-Rad
Sequiblot membrane for 90 min at 250 mA. The membrane was washed in
MilliQ H2O, stained for 5 min with 0.1% (w/v) Coomassie
Blue R-250 in 50% (v/v) methanol, and destained for 7 min in
methanol/acetic acid/water (50:10:40, v/v/v). After rinsing (5 × 2 min) with 20 ml of MilliQ H2O and air-drying, the p130
band on the membrane (5 µg of protein) was subjected to automated
Edman degradation.
Preparation and Purification of Tryptic Peptides for MALDI-MS and
Edman Sequencing--
For mass spectrometric analysis and
microsequencing, purified PEPC2 was subjected to preparative
electrophoresis on a 1.5-mm-thick SDS-7.5% acrylamide gel. The
separated polypeptides were electroblotted onto a polyvinylidene
difluoride membrane as described above. The membrane was then stained
for 1 min with 0.2% (w/v) Ponceau S in 1% (v/v) acetic acid. The
areas of the membranes corresponding to p102 and p130 were cut out. The
membrane strips were then washed (6 × 5 min) with MilliQ
H2O and dried. A blank piece of membrane with no protein
was processed as a control at the same time. For in situ
digestion with trypsin, membrane pieces were cut into 1-mm squares and
placed in a microcentrifuge tube. HPLC-grade methanol (20 µl) was
added to wet the membrane thoroughly. The tube was vortexed and
centrifuged and excess liquid was removed. Digestion buffer (75 µl;
100 mM Tris-HCl (pH 8.15), 1% (v/v) Triton X-100 (reduced,
Sigma X-100-RS), and 10% (v/v) acetonitrile) was added to the tube.
This solution was incubated with the membrane pieces at 37 °C for 15 min. Modified trypsin (1.5 µg; Promega) was then added to the tube,
and incubation was continued for 12 h. The tube was then sonicated
for 10 min, and the supernatant was transferred to a new tube. A
100-µl aliquot of 1% (v/v) trifluoroacetic acid solution was added
to wash the membrane pieces; and after another 10-min sonication, the
wash was removed and pooled with the supernatant collected initially.
The combined solution was then separated by microbore reverse-phase
HPLC on a Micchrom BioResources UMA instrument using a Reliasil
C18 column (1.0 × 150 mm, 5 µm, 300-A pore
size). Elution was with a linear solvent gradient from 100% solvent A
(H2O/acetonitrile/trifluoroacetic acid, 94.9:5:0.1, v/v/v)
to 100% solvent B (acetonitrile/H2O/trifluoroacetic
acid, 90:9.9:0.1, v/v/v). The elution profile was monitored at 214 nm. Peaks were collected in minicentrifuge tubes as they eluted. For automated Edman degradation, tryptic peptides were further purified using C18 reverse-phase HPLC as described above, but
substituting trifluoroacetic acid with heptafluorobutyric acid in all solvents.
MALDI-MS--
The collected fractions were used directly to
prepare samples for MALDI-MS analysis. A 1-µl aliquot of an HPLC
fraction was added to 1 µl of a saturated 4-hydroxy-
-cyanocinnamic
acid matrix solution containing 0.1% (v/v) trifluoroacetic acid on a
sample plate containing a 10 × 10 array of wells for the
individual samples. The solution was allowed to air-dry before
introduction into the mass spectrometer. MALDI spectra were acquired on
a Voyager Elite MALDI-TOF instrument (PerSeptive Biosystems) operated
in the linear mode with positive ion detection. A nitrogen laser (337 nm, 3-ns pulses) was used to irradiate the target, and the acceleration potential was 23 kV. Sixty-four laser shots were averaged to produce one spectrum. Only the peaks at m/z >948 were retained for
analysis to avoid confusion with background noise in the low
m/z value region of the spectra.
Protein Cross-linking--
PEPC2 was cross-linked with 2 mM DMS in buffer containing 100 mM Hepes-KOH
(pH 8) for 60 min at 20 °C. The reaction was then quenched by adding
50 mM Tris-HCl (pH 9.5) for 15 min. The samples were
denatured by boiling in SDS sample buffer and analyzed by SDS-PAGE
using 5% final acrylamide monomer separating gels (20) without
stacking gel. The gels were run at a constant voltage of 100 V until
the prestained 80-kDa marker eluted from the bottom of the gel.
Electrophoresis was followed by immunoblotting as described above.
PLP Labeling--
PEPC1 and PEPC2 (1 µg) were subjected to
reductive pyridoxylation with 2.5 mM PLP. The reaction was
allowed to proceed for 30 min at 25 °C. PLP forms a Schiff base with
an essential Lys residue present at the active site of the enzyme (21,
22). NaB3H4 (17.5 Ci/mol) was then added to the
reaction mixture at a 1 mM final concentration (35 µCi).
The mixture was further incubated for 15 min on ice. This procedure
allows the reduction of the Schiff base and thereby introduces a
radioactive label at the active site of the enzyme. As a control for
the specificity of the PLP labeling, 10 mM Mg-PEP was added
to the incubation mixture. Binding of Mg-PEP to the active site
protects from inactivation by PLP (22). The polypeptides were then
analyzed by SDS-PAGE and fluorography as described above.
Peptide Mapping by CNBr Fragmentation--
Polypeptides were
subjected to preparative SDS-PAGE, excised from the gel, and cleaved
in situ as described (23).
 |
RESULTS |
Polypeptide Composition of Undegraded Purified PEPCs--
Previous
results indicated that, when purified in the presence of
phenylmethylsulfonyl fluoride and chymostatin as the only protease
inhibitors, a faintly detectable polypeptide of ~130 kDa (p130) is
present in purified Class 2 PEPCs together with major polypeptides of
102, 73, 70, 65, and 61 kDa (6). Preliminary results indicated that
CNBr cleavage of p130 and the 70- and 61-kDa polypeptides generated
common fragments (data not shown), suggesting that these lower
molecular mass polypeptides were possibly derived by proteolytic
cleavage of p130. Indeed, using affinity-purified anti-p130 IgGs, we
were able to determine that in the absence of protease inhibitors,
there was almost complete clipping of p130 after 60 min at 4 °C,
yielding mainly immunoreactive polypeptides of 70 and 61 kDa in a 1:1
ratio (Fig. 1). Interestingly, these proteolytic fragments appeared to be quite stable after the initial extraction step. They were not subject to further degradation during
purification and were found associated with purified Class 2 PEPCs when
isolated under the conditions described previously (6). This suggested
that p130 was sensitive to endogenous protease activity. Separate
additions of the metalloprotease inhibitors 2,2'-dipyridyl, EGTA,
pepstatin, and leupeptin to the extraction buffer described in Ref. 6
significantly increased the stability of p130. Maximum stability was
achieved when all these inhibitors were present (<5% degradation
occurring over 6 h at 4 °C as judged from immunoblot
quantification) (Fig. 1). S. minutum PEPC was purified from
50 g of cells using these improved extraction conditions. As
previously reported (6), the purification yielded four isoforms. The
specific activities of PEPC1, PEPC2, PEPC3, and PEPC4 were 29.7, 34.3, 36.1, and 27.5 units/mg of protein, respectively. Thus, the specific
activities of non-proteolyzed Class 2 PEPCs are almost 2-fold higher
those reported when these isoforms contain degraded p130 (6). When
analyzed by SDS-PAGE (Fig.
2A), purified Class 2 PEPCs
all contained polypeptides with molecular masses of 130 kDa (p130) and
102 kDa (p102). p102 corresponds to the same catalytic subunit present
in PEPC1 (6). These complexes also contained two other polypeptides
with molecular masses of 73 and 65 kDa. These latter polypeptides were
already identified as part of the Class 2 PEPC complexes (6), whereas
the purification and high recovery of p130 with Class 2 PEPCs appear to
be due to the protease inhibitors used in the present study. Antisera raised against p130 and p102 (6) were used to analyze the purified isoforms by immunoblotting (Fig. 2, B and C).
Both antisera recognized a single band in the complexes, indicating
that p130 and p102 are immunologically distinct. Because these
polyclonal antisera were raised against SDS-denatured polypeptides, it
is unlikely that clipping would totally eliminate antigenicity. This
supports the view that the 73- and 65-kDa polypeptides are
immunologically distinct from p102 and p130.

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Fig. 1.
Degradation of p130 in clarified S. minutum extracts is prevented by protease inhibitors.
Clarified S. minutum extracts (85 µg of protein) were
prepared under various extraction conditions, incubated on ice for
various times, and subjected to immunoblot analysis with
affinity-purified anti-p130 IgGs. The numbers below each
lane indicate the length of the incubation on ice in minutes before
denaturation in SDS sample buffer. The molecular mass running positions
are indicated on the left. Lane 1, 10% (w/v)
trichloroacetic acid; lanes 2 and 3, incomplete
extraction buffer; lanes 4 and 5, complete
extraction buffer (as described under "Experimental
Procedures").
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Fig. 2.
Subunit analysis of purified S. minutum PEPC isoforms. Pooled active fractions from the
last step of the purification were analyzed by SDS-PAGE (A)
and immunoblotting (B and C). For SDS-PAGE, 1.5 µg of protein was loaded onto each lane, and the gel was stained with
silver nitrate. For immunoblotting, proteins (250 ng) were transferred
to polyvinylidene difluoride membrane, and immunodetection was carried
out with affinity-purified anti-p130 (B) and anti-p102
(C) IgGs. The molecular mass running positions are indicated
on the right. Lanes 1, PEPC1; lanes 2, PEPC2,
lanes 3, PEPC3; lanes 4, PEPC4.
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Subunit Stoichiometry in Class 2 PEPCs--
To examine the subunit
stoichiometry in Class 2 PEPCs, the purified isoforms were subjected to
SDS-PAGE, and the gels were scanned with a laser densitometer after
silver staining. Assuming that all polypeptides were equally sensitive
to the staining technique, this method allows the estimation of subunit
stoichiometry in the complexes (Table I).
Results are consistent with the hypothesis that PEPC2, PEPC3, and PEPC4
arise from different stoichiometric combinations of p130, p102, and the
other polypeptides. When the size of the complexes increased, the
representation of p102 and p130 followed opposite trends, with p102
being more abundant in PEPC2 and p130 becoming more predominant in
PEPC4. The relative stoichiometry of p130 and p102 in Class 2 PEPC
isoforms was confirmed by quantitative immunoblot analysis using
affinity-purified anti-p130 and anti-p102 IgGs (data not shown).
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Table I
Determination of relative subunit stoichiometry in purified PEPC
isoforms
Gels similar to those in Fig. 2A were scanned with a laser
densitometer. Intensities of the scanned bands (area counts) were
proportional to protein loading (100-400 ng/lane). Results are
expressed as percent representation on a protein basis. Data correspond
to a mean of nine estimations from three different preparations. S.D.
values were <15% of the mean between replicates. To determine
relative subunit stoichiometry (in parentheses), results have been
normalized to the subunit molecular masses, and the values were rounded
to the closest integral number. The measured molecular masses
correspond to experimental determination using gel filtration on two
Superose 6 columns arranged in tandem (Ref. 6 and this study). The
calculated molecular mass values are based upon the relative subunit
stoichiometry.
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p130 Is Tightly Associated with Class 2 PEPCs and Is in Physical
Contact with p102--
To investigate whether p130 is merely a
contaminant or an integral part of Class 2 PEPCs, several experiments
were conducted. First, an aliquot of each chromatographic fraction
obtained during the various steps of Class 2 PEPC purification was
subjected to immunoblot analysis using anti-p102 and anti-p130 IgGs
(Fig. 3). During all the chromatographic
steps of the purification, p130 coeluted with the p102 polypeptide
present in Class 2 PEPCs. The coelution of the two polypeptides was
associated with PEPC activity peaks (Fig. 3, A-D). On the
butyl column (Fig. 3A), the elution maxima of p102 and p130
are displaced by a few fractions. This probably reflects the fact that
the various PEPC isoforms present at this early stage of the
purification eluted slightly differently from the hydrophobic medium.
The 102-kDa polypeptide eluting in the early part of the KCl gradient
on the Fractogel EMD DEAE-650 column (Fig. 3B) represents
the homotetrameric PEPC1 isoform. It is noteworthy that throughout the
purification, p130 was never found eluting without p102. In addition,
attempts to dissociate p102 from p130 were conducted. Incubations (1 h
at 4 °C) of tandem Superose 6-purified PEPC2 with 1 M
NaCl, 50 mM dithiothreitol, 10 mM EDTA, 10 mM EGTA, 25 mM MgCl2, 10 mM CaCl2, 5 mM ATP, 5 mM NH4Cl, 5 mM NaNO3, 5 mM Gln, 5 mM Glu, 5 mM Asp, 5 mM dihydroxyacetone phosphate, 5 mM
-ketoglutarate, 25 mM malate, or 50% (v/v) ethylene glycol failed to dissociate the PEPC2 complex as judged from the fact
that the coelution of the p102 and p130 polypeptides from a Superose 6 column remained unchanged. Similar results were obtained when PEPC2 was
chromatographed on a Superose 6 column pre-equilibrated in buffer
containing 0.3 M NaCl, 50 mM dithiothreitol, 10 mM EDTA, 10 mM EGTA, 10 mM
CaCl2, 5 mM Gln, 5 mM Glu, or 5 mM Asp. Denaturation of the complex with 6 M
guanidinium Cl or 8 M urea allowed a subsequent partial
separation of p102 and p130 by Superose 6 gel-filtration fast protein
liquid chromatography. However, efforts aimed at the renaturation of
both peptides by slow dialysis were not successful. The above results
suggested a tight association between p102 and p130 and that both
polypeptides are part of the native Class 2 PEPCs.

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Fig. 3.
Immunoblot analysis of the copurification of
p130 and p102 with Class 2 PEPCs. A 10-µl (A) or
5-µl (B-D) aliquot of each fraction obtained at the
different steps in the purification of Class 2 PEPCs was subjected to
immunoblot analysis using affinity-purified anti-p102 (p102)
and anti-p130 (p130) IgGs. For each chromatographic step,
the blots are superimposed onto the elution profile. The fractions
pooled and carried to the next step are bracketed.
Numbers above the blots refer to the fractions. PEPC
activity ( ), the A280 trace from the UV
monitor (- - -), the (NH4)2SO4
gradient (------) (A), and the KCl gradient (------)
(B and D) are plotted. A, hydrophobic
interaction chromatography on butyl-Sepharose; B,
anion-exchange chromatography on Fractogel DEAE (peak 1,
PEPC1; peak 2, Class 2 PEPCs); C, size-exclusion
fast protein liquid chromatography on tandem Superose 6 (P2,
PEPC2; P3, PEPC3; P4, PEPC4); D,
anion-exchange fast protein liquid chromatography of PEPC2 on
MonoQ.
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These finding were supported by the following experiments. First, PEPC2
was subjected to immunoprecipitation with anti-p102 and anti-p130
antisera. The immunoprecipitates were analyzed for the presence of p102
and p130 by immunoblotting (Fig. 4). The anti-p102 antiserum was able to immunoprecipitate p102 and p130. Conversely, the anti-p130 antiserum was able to immunoprecipitate p102
and p130. Immunoprecipitation with the respective preimmune sera was
negative. Since the anti-p102 and anti-p130 antisera are highly
specific for their respective antigens (Fig. 2), these results provide
very good evidence that both polypeptides are physically associated
with and part of the Class 2 PEPCs. Second, PEPC2 was subjected to
chemical cross-linking with DMS. Cross-linked protein was analyzed
using a highly porous SDS-PAGE system (20) in conjunction with
immunoblotting (Fig. 5). Analysis of
cross-linked proteins blots with anti-p102 and anti-p130 IgGs revealed
the presence of immunoreactive polypeptides migrating at the same molecular masses on both blots. This indicates the presence of chemically cross-linked p102 and p130. Specifically, polypeptides potentially corresponding to a p102/p130 heterodimer (230 kDa) and a
(p102)2/p130 heterotrimer (330 kDa) were observed. Under the experimental conditions used, monomeric p102 and p130 were the main
detectable bands, indicating that only a fraction of the complex had
reacted with the cross-linking agent and thereby ensuring the
specificity of the cross-linking reaction. A number of other bands were
detected on the blot corresponding to cross-linked p130. They probably
correspond to cross-linking with the 65- and 73-kDa polypeptides. Using
various experimental conditions, we did not observe the formation of
cross-linked protein corresponding to the entire complex. We attribute
this to the fact that under the conditions used, the probabilities of
cross-linking all the subunits within a single complex and then
transferring the 106-kDa protein onto polyvinylidene
difluoride are relatively low. An alternative possibility is
preferential cross-linking between subunits due to optimum
subunit-subunit distance.

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Fig. 4.
Analysis of PEPC2 by
immunoprecipitation. PEPC2 (1.5 milliunits) was subjected to
immunoprecipitation using anti-p102 preimmune serum (lane
1), anti-p102 immune serum (lane 2), anti-p130
preimmune serum (lane 3), or anti-p130 immune serum
(lane 4). Immunoprecipitates were pelleted with protein
A-Sepharose beads and analyzed by immunoblotting using anti-p130
(A) and anti-p102 (B) IgGs. Lane 5 corresponds to 250 ng of purified PEPC2 serving as a positive control
for immunoblot analysis.
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Fig. 5.
Analysis of PEPC2 by chemical cross-linking
followed by immunoblotting. PEPC2 (5 µg) was incubated in the
absence (lanes 1) or presence (lanes 2) of 2 mM DMS. Cross-linked protein was analyzed by SDS-PAGE on a
5% acrylamide separating gel and by immunoblotting. Immunoblot
analysis was carried out with anti-p130 (A) and anti-p102
(B) IgGs.
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N-terminal Sequencing of p130--
The N-terminal sequence of p130
was determined by Edman degradation. A 19-amino acid sequence was
obtained: VARTPR(P)LDPAALAKL(L)D(D). No significant sequence similarity
was found between this sequence and that previously determined for
C. reinhardtii PEPC1 (12) or any of the protein sequences
present in GenBankTM/EBI Data Bank.
Comparative Peptide Mapping of p130 and p102 Tryptic Fragments by
MALDI-MS--
The p130 and p102 polypeptides of PEPC2 were isolated
and subjected to digestion with trypsin, and the tryptic fragments were separated by reverse-phase chromatography. Fractions eluting from the
column were collected and subjected to MALDI-MS analysis. Due to the
complex background peaks from the matrix in the low mass region,
interpretation of the spectra focused on the region at m/z
>940. The m/z values corresponding to the tryptic peptide present in each fraction are reported in Table
II. Only a single match was found between
p130 and p102 (fraction 5, m/z ~2174). Related
polypeptides analyzed by this technique are expected to share a
significantly higher number of tryptic fragments. Hence, the data
demonstrate that p102 and p130 are not closely related, confirming the
immunological data presented in Fig. 2. A computer search of the Swiss
Protein Database for tryptic fragments matching those listed in Table
II was done with the MS-FIT program (Protein Prospector
package). This search did not reveal any similarity between
proteins already present in the data base and p130 or p102.
Microsequencing of a p130 Tryptic Peptide Reveals Similarity to C
Termini from Various PEPCs--
Two p130 tryptic fragments (peptides A
and B) were further purified using a second reverse-phase HPLC step and
sequenced using automated Edman degradation. Peptides A and B yielded
the following respective amino acids sequences: AYIDPNILQVELLR and
(L)(I)TEGMDP(V/A)ELFTE. A FASTA3 search revealed no similarity for
peptide B. However, peptide A had significant similarity to the C
termini of several PEPCs. An alignment of p130 peptide A with the C
termini of PEPCs from various origins is presented in Fig.
6. Peptide A exhibited 50-78% and
43-50% identities to the C-terminal regions of a number of
prokaryotic and higher plant PEPCs, respectively. It is interesting to
note that the C-terminal region is highly conserved in PEPCs and that
p130 peptide A contains two species-invariant amino acids (Gln and Arg)
at positions 9 and 14, respectively (24). This result suggested that
p130 could be a PEPC polypeptide only distantly related to p102 and
other PEPCs.

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Fig. 6.
Alignment of p130 tryptic peptide A with the
C-terminal amino acid sequences of prokaryotic and higher plant
PEPCs. The sequence of peptide A was obtained by automated Edman
degradation. Other sequences were derived from the translation of PEPC
nucleotide sequences. Swiss-Prot accession numbers are shown in
parentheses. Numbering is relative to the first amino acid
for each PEPC clone. Gaps (dashes) have been introduced to
maximize alignment, and colons indicate an amino acid
residue in the same position in the p130 peptide A sequence.
Underlining indicates a previously identified
species-invariant amino acid residue (24). The sources for the
sequences are as indicated: Hemophilus influenzae (36),
Mycobacterium leprae (D. R. Smith and K. Robison,
direct submission), E. coli (37), Thermus
sp. strain 71 (38), Anabaena sp. strain PCC7120 (39),
Corynebacterium glutamicum (40), Nicotiana
tobacum (41), Mesembryanthemum crystallinum (42),
Sorghum vulgare (43), and Zea mays (44).
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p130 Cross-reacts with an Anti-banana Fruit PEPC Antiserum--
To
further test the possibility that p130 could be related to PEPC, we
carried out an immunoblot analysis of PEPC2 with an antiserum raised
against banana fruit PEPC (4). This antiserum has been shown to
cross-react strongly with various higher plant PEPCs (12) and weakly
with S. minutum p102 (Fig. 7).
Fig. 7 shows that the anti-banana fruit PEPC antiserum cross-reacted weakly not only with p102, but also with p130, whereas the anti-p130 antiserum did not recognize purified banana fruit PEPC. Likewise, the
anti-p102 antiserum did not cross-react with higher plant PEPCs
(12).

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Fig. 7.
Immunological relationship between p130 and
banana fruit PEPC. Purified PEPC1, PEPC2, and banana fruit PEPC
were subjected to immunoblot analysis with anti-p130 IgG (A)
and anti-banana fruit PEPC immune serum (B). Lanes
1 and 5, 125 ng of PEPC1; lanes 2 and
6, 250 ng of PEPC2; lanes 3 and 7, 25 ng of banana fruit PEPC; lanes 4 and 8, 50 ng of
banana fruit PEPC. MW, molecular mass markers.
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Evidence That p130 Has PEPC Activity Based on Specific Activity
Calculations--
The data presented in Fig. 2 and Table I clearly
indicate that, on a protein basis, Class 2 PEPCs contain far less p102
compared with PEPC1. From the specific activities and the
quantification of the p102 content of each purified isoform, PEPC
specific activities relative to the amount of p102 in each isoform can
be calculated. Such calculations indicate that Class 2 PEPCs have a
much higher specific activity than PEPC1. The specific activities
relative to the p102 content alone are 56.4, 129.4, and 184.5 units/mg for PEPC2, PEPC3, and PEPC4, respectively. These results indicate that
(i) there is another polypeptide with PEPC activity in Class 2 PEPCs,
or (ii) there is a polypeptide activating p102 in Class 2 PEPCs. We
further characterized PEPC2 to provide support for one of these possibilities.
PLP Labeling of p130 Can Be Alleviated by Mg-PEP--
PLP
is known to form a Schiff base with an essential Lys residue on the
active site of PEPC (e.g. Lys-606 in maize PEPC) (21). This
property has been used to identify the species-invariant active-site
residue in this enzyme (21). The reaction between PEPC polypeptides and
the covalently bound PLP moiety can be visualized by reduction with
NaB3H4 (22). Mg-PEP, the substrate of the
enzyme, protects it from PLP binding. A 30-min incubation of PEPC2 with
2.5 mM PLP resulted in an 85% reduction of PEPC activity.
This was almost completely negated by the inclusion of 10 mM Mg-PEP in the incubation mixture: only a 5% reduction
of PEPC2 activity was observed in this case. We therefore used this
approach to label PEPC2 with PLP and to visualize the labeled
polypeptides by fluorography (Fig. 8).
These data show that p130 and p102 were both labeled in the absence of
Mg-PEP and that the presence of the substrate protected both polypeptides from reductive pyridoxylation. This suggests that Mg-PEP
binding affects one or several Lys residues on both subunits, indicating the possible presence of a PEPC catalytic site on both polypeptides. The 65-kDa polypeptide associated with PEPC2 also appeared to be heavily labeled, but labeling was not sensitive to the
presence of Mg-PEP. This may indicate the reduction of amino acid
residues by NaB3H4 in this polypeptide
(22).

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Fig. 8.
SDS-PAGE and fluorographic analysis of
3H-labeled PLP-modified PEPC2. Purified PEPC1
(lanes 1 and 2) and PEPC2 (lanes 3 and
4) were incubated with PLP in the absence (lanes
1 and 3) or presence (lanes 2 and
4) of 10 mM Mg-PEP. After 30 min, the PLP-enzyme
adduct was reduced using NaB3H4. The reaction
mixture was denatured in SDS sample buffer, and the polypeptides were
analyzed by SDS-PAGE followed by fluorography. The gel was exposed to
x-ray film for 35 days at 80 °C.
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PEPC2 Shows Biphasic PEP Binding Kinetics--
Since
chemical modification-protection studies suggested that p130 and p102
are both able to bind PEP, we investigated the possibility that a
kinetic analysis for PEP binding could provide further support that
p102 and p130 are both catalytically active. Analysis of PEP binding to
PEPC1 is cooperative and gives an S0.5 value of 1.56 mM and a Hill coefficient of 1.3 (6). In contrast, the
saturation of PEPC2 with Mg-PEP was hyperbolic (Fig.
9, inset). These data were
analyzed for best fitting to a single catalytic site Michaelis-Menten
model or to Equation 1 using two nonlinear fitting programs (SigmaPlot
Version 6.0 and DataFit Version 7.0). With both programs, the fit to
Equation 1 had a higher probability (p < 0.001). Data
analyzed with SigmaPlot Version 6.0 (Fig. 9, inset) indicate
that PEPC2 has two PEP-binding sites with significantly different
apparent Km values for PEP (1.23 and 0.13 mM) and Vmax values (7.28 and 28.27 units/mg of protein). For a visual confirmation of this result,
the values were introduced into Equation 1 represented in the
Eadie-Hofstee plot (Fig. 9). The plot is clearly biphasic. The
theoretical plot of the two-site model (solid line) fitted
the experimental points with a correlation coefficient of 0.97. On the
other hand, the plot of the equation obtained assuming a single
catalytic site (dashed line) has a lower correlation coefficient to experimental points (0.94). The ratio of
Vmax values between the low and high apparent
Michaelis constant sites (0.25) follows the same trend as the
distribution of p102 and p130 in PEPC2 obtained from Table I
(0.16).

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Fig. 9.
Analysis of PEP saturation kinetics for
PEPC2. Enzyme velocities (V) were measured on a
Dynatech microplate reader at 25 °C and pH 8.5. Inset,
direct representation of experimental data. Experimental points ( )
were fitted to Equation 1 using the SigmaPlot Version 6.0 nonlinear
regression analysis package. The derived kinetic parameters of the low
and high Km sites are given in the inset.
In the main panel, the Eadie-Hofstee plot of these data is
shown. Experimental points ( ) were fitted to Equation 1 (solid
line; r = 0.97) or to the Michaelis-Menten
equation assuming a unique PEP-binding site (dashed line;
r = 0.94).
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DISCUSSION |
In unicellular green algae, two classes of PEPC isoforms can be
distinguished based on their physical and kinetic properties (6, 12).
Class 1 PEPC is similar in molecular structure to higher plant and
prokaryotic PEPCs. Class 2 PEPCs have, to date, been reported only for
green algae. The work was undertaken to provide a better understanding
of the structure and composition of these large protein complexes.
Purification of Intact PEPC Protein Complexes and Characterization
of Their Subunit Composition--
We previously reported the
purification and characterization of four PEPC isoforms from the green
alga S. minutum (6). All isoforms contain the same
p102 catalytic subunit, as evidenced by CNBr cleavage patterns. High
molecular mass (Class 2) PEPC isoforms, however, contain a set of
associated polypeptides with no immunological relationship to p102 (6),
including a protease-susceptible 130-kDa polypeptide (p130). The
present work provides unequivocal evidence that p130 is an integral
part of Class 2 PEPCs. First, immunoblot analysis was used to
demonstrate the copurification of p130 with Class 2 PEPCs (Fig. 3).
These data were supported by immunoprecipitation experiments in which
an antiserum specific for p102 immunoprecipitated p130 present in the
native Class 2 complex, and conversely, specific anti-p130 antiserum
was able to immunoprecipitate p102 under the same conditions (Fig. 4). Finally, chemical cross-linking studies carried out with DMS support the direct interaction between p102 and p130 in PEPC2 through the
detection of cross-linked p102 and p130 in particular bands (Fig. 5).
These data strongly suggest that p102 and p130 physically interact
in vivo.
p130 Is a Novel PEPC Polypeptide in Green Algae--
This study
provides evidence that p130 represents a novel PEPC polypeptide in
S. minutum and is only very distantly related to p102, the
already known PEPC catalytic subunit in this organism. Furthermore, our
results suggest that p130 has PEPC activity. The evidence that p130 has
limited homology to p102 is supported by the fact that they do not
share common immunogenic epitopes (Fig. 2, B and
C). Evidence can also be found in the MALDI-MS analysis of
p130 and p102 tryptic peptides (Table II). Only one common tryptic
fragment was found between p102 and p130. Proteins sharing a high
degree of similarity are expected to have a significantly higher number
of common tryptic fragments. For example, comparison of the tryptic
digests of the C4 and C3 forms of maize PEPC (Swiss-Prot accession numbers P04711 and P51059, respectively) using the MS-Digest
program available in the Protein Prospector package produced seven
identical masses at m/z 940. Our results support the view
that p102 and p130 are less related than are the C4 and C3 maize PEPCs.
It is clear, on the other hand, that a tryptic peptide derived from
p130 has primary sequence identity to the C termini of other PEPCs
(Fig. 6). The C terminus is conserved in prokaryotic and eukaryotic
PEPCs (24, 25). Preliminary data indicate that this domain is involved
in enzyme maximal catalytic activity in higher plant PEPCs (25).
Interestingly, alignment of p130 peptide A with other PEPCs also
positions it very closely to a conserved Arg residue involved in the
binding of the inhibitor Asp residue (e.g. Arg-832 in
Escherichia coli PEPC) (26). The presence of such a
conserved domain in p130 indicates that it potentially plays a similar
role in the Class 2 PEPC protein complexes. An immunological analysis
carried out with the anti-banana fruit PEPC antiserum showed that p130
shares a few common epitopes with higher plant PEPCs. Similarly, p102
was shown to have limited immunological relatedness to the higher plant
enzymes (Fig. 7) (12).
These results raise the question of whether the sequence similarity of
p130 to other PEPCs is limited (e.g. to the C-terminal domain) or whether it extends to other regions of the protein. The
N-terminal sequence of p130 showed no homology to the corresponding highly conserved regulatory phosphorylation N-terminal domain of higher
plant PEPCs or to the C. reinhardtii PEPC N terminus (12),
further supporting the view that p130 is divergent from known PEPC
sequences. We next investigated whether or not p130 has PEPC activity.
Despite our efforts, the Class 2 complexes were resistant to treatments
aiming at their dissociation, and we were not able to separate and
hence assay p130 and p102 for PEPC activity. We therefore used several
independent indirect methods to investigate if p130 has PEPC activity.
Our results provide strong circumstantial evidence that there are two
catalytic sites in Class 2 PEPCs and that these sites are probably
carried by p102 (high Km) and p130 (low
Km). The data presented in Table I clearly indicate
that Class 2 PEPCs contain less p102 compared with PEPC1, yet
comparable PEPC-specific activities are found in Class 1 and 2 PEPCs.
These results can be explained if (i) Class 2 PEPCs have polypeptides
with PEPC activity other than p102; or (ii) p102 is activated in Class
2 PEPCs, and this activation raises its specific activity. A review of
our experimental data provides support for the former hypothesis.
First, reductive pyridoxylation resulted in an 85% reduction in
S. minutum PEPC2 activity. Moreover, the results in Fig. 8
suggest that both p102 and p130 bind Mg-PEP, consistent with the view
that both polypeptides are catalytically active. Second, PEP saturation
kinetics for PEPC2 fit a two-binding site model such as the one
described by Equation 1 (Fig. 9). The apparent
Km(PEP) for the two sites is different by an order
of magnitude. On the other hand, PEPC1 contains only one site with high
Km(PEP) (6). The high Km value
for PEPC2 is close to the Km(PEP) for PEPC1 (1.23 and 1.56 mM, respectively). Similarly, the
Vmax value for the PEPC2 high
Km site and that for PEPC1 are very close (28.27 and
29.7 units/mg of protein, respectively). This suggests that these
properties belong to p102. Moreover, the ratio of low to high
Km sites based on their Vmax values (0.25) is reasonably comparable to the calculated ratio of p130
to p102 present in PEPC2 (0.16; Table I). Together, these results
strongly suggest, but does not prove, that p130 carries the low
Km(PEP) site. Molecular cloning of p130 will ultimately help to determine whether p130 has PEPC activity. Also of
interest will be to determine the respective identities and roles of
the 73- and 65-kDa polypeptides that appear to be part of the Class 2 PEPC complexes.
Taken as a whole, our results support the conclusion that
S. minutum is able to coexpress and assemble very divergent
PEPC polypeptides into large complexes, thereby producing the PEPC isoenzyme system present in all green algae studied so far. This ability to assemble PEPC polypeptides that are probably the products of
divergent genes has not been reported so far for vascular plants in
which PEPC is a homotetrameric enzyme (2). The only well documented
exception to this rule is the case of banana fruit PEPC (C3 form),
which is a heterotetramer containing two very similar PEPC subunits
(4). PEPC isoforms do exist in higher plants, but are expressed in
different tissues or cell types and/or are differently regulated at the
developmental level (27). However, it is important to reemphasize that
PEPC protein characterization has been overwhelmingly focused on the C4
leaf form of the enzyme, which is metabolically specialized in the
photosynthetic fixation of atmospheric CO2 (1, 2, 27) and
fairly novel, evolutionarily speaking (150-300 million years) (24).
Interestingly, the notion that PEPC is able to form complexes with
unrelated polypeptides is currently emerging in higher plants (28-30).
In particular, in different studies, PEPC was shown to be associated
with its regulatory Ca2+-independent protein kinase (28,
29). However, no PEPC protein kinase activity has been detected in
purified S. minutum Class 2 PEPCs (6). A critical aspect of
the work involving the characterization of protein complexes is the
preservation of a relatively high protein concentration (31). Green
algae are very rich in proteins (e.g. 17% of the fresh
weight is composed of proteins in S. minutum). They can be
extracted in a minimal volume of medium and therefore constitute the
material of choice for the purification and characterization of protein
complexes. It is possible that, with the development and use of
specialized protein purification techniques aiming at the preservation
of protein complexes (e.g. inclusion of glycerol or
polyethylene glycol in all purification buffers) (31), more PEPC
protein complexes will be characterized from higher plants.
Function of Different PEPC Polypeptides in Unicellular Green
Algae--
An important question raised by our findings concerns the
adaptive value conferred by the simultaneous expression of different PEPC polypeptides in S. minutum and their assembly into two
classes of strikingly different isoforms. In contrast to higher plants, green algal cells are all in direct contact with their environment, and
their survival ultimately depends upon their capacity to adapt rapidly
and efficiently to this environment (32).
Nitrogen and phosphorus availabilities are the two main factors
limiting primary production in aquatic ecosystems (7, 8, 33). This
study provides support for our hypothesis that Class 2 PEPCs may have a
housekeeping function (6). From the detailed characterization of PEPC1
and the fact that Class 2 PEPCs are largely insensitive to metabolic
effectors, it was concluded that PEPC1 is specialized in the
anaplerotic fixation of CO2 when a nitrogen source is
available (6). It is also clear that p102 (PEPC1) is not metabolically
significant if nitrogen is not available (i.e. if its
activator Gln is not present). Under physiological pH conditions and in
the absence of the activators Gln and dihydroxyacetone phosphate, PEPC1
has an S0.5(PEP) of >9 mM (6). This value is
more than an order of magnitude higher than the S. minutum physiological PEP concentration, which can be estimated below 0.2 mM, taking an average chlorophyll concentration of 10 mg/g (fresh weight) (34). It is nevertheless necessary for the cell to have
a low level of anaplerotic carbon flux for maintenance purposes when
nitrogen is limited. Our results indicate that this role may be carried
out by p130. In addition, algal ecophysiology may offer a rationale for
the coexpression of p102 and p130. Nutrient distribution in aquatic
ecosystems is not homogeneous either temporally or spatially; and
several times a day, an algal cell may encounter or be driven away from
a nutrient source (32, 35). The metabolic cost of synthesizing and
degrading the enzymatic machinery necessary for nitrogen assimilation
could be enormous. We therefore hypothesize that the simultaneous
expression of different PEPCs such as p102 and p130 poises green algae
for any eventuality. Additional regulatory mechanisms such as
reversible protein phosphorylation and isoform interconversion may also
be involved.