Two Unrelated Phosphoenolpyruvate Carboxylase Polypeptides Physically Interact in the High Molecular Mass Isoforms of This Enzyme in the Unicellular Green Alga Selenastrum minutum*

Jean RivoalDagger §, Stacy Trzos, Douglas A. Gage, William C. Plaxton||, and David H. Turpin**

From the Dagger  Department of Plant Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada, the  Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, the || Departments of Biology and Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada, and the ** Department of Biology, University of Victoria, Victoria, British Columbia V8W 3N5, Canada

Received for publication, November 7, 2000, and in revised form, January 17, 2001



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the chlorophyte Selenastrum minutum, phosphoenolpyruvate carboxylase (PEPC) exists as two kinetically distinct classes of isoforms sharing the same 102-kDa catalytic subunit (p102). Class 1 PEPC is homotetrameric, whereas Class 2 PEPCs consist of three large protein complexes. The different Class 2 PEPCs contain p102 and 130-, 73-, and 65-kDa polypeptides in different stoichiometric combinations. Immunoblot, immunoprecipitation, and chemical cross-linking studies indicated that p102 physically interacts with the 130-kDa polypeptide (p130) in Class 2 PEPCs. Immunological data and mass spectrometric and sequence analyses revealed that p102 and p130 are not closely related even if a p130 tryptic peptide had significant similarity to a conserved PEPC C-terminal domain from several sources. Evidence supporting the hypothesis that p130 has PEPC activity includes the following. (i) Specific activity expressed relative to the amount of p102 was lower in Class 1 than in Class 2 PEPCs; (ii) reductive pyridoxylation of both p102 and p130 was inhibited by magnesium-phosphoenolpyruvate; and (iii) biphasic phosphoenolpyruvate binding kinetics were observed with Class 2 PEPCs. These data support the view that unicellular green algae uniquely express, regulate, and assemble divergent PEPC polypeptides. This probably serves an adaptive purpose by poising these organisms for survival in different environments varying in nutrient content.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP>. 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 gamma -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,
V=<FR><NU>V<SUB><UP>max1</UP></SUB></NU><DE>1+K<SUB>m1</SUB>/[<UP>S</UP>]</DE></FR>+<FR><NU>V<SUB><UP>max2</UP></SUB></NU><DE>1+K<SUB>m2</SUB>/[<UP>S</UP>]</DE></FR> (Eq. 1)
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), beta -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), beta -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-alpha -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (22K):
[in this window]
[in a new window]
 
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").


View larger version (23K):
[in this window]
[in a new window]
 
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.

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).

                              
View this table:
[in this window]
[in a new window]
 
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.

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 alpha -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.


View larger version (49K):
[in this window]
[in a new window]
 
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.

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.


View larger version (30K):
[in this window]
[in a new window]
 
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.


View larger version (26K):
[in this window]
[in a new window]
 
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.

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.

                              
View this table:
[in this window]
[in a new window]
 
Table II
MALDI-MS analysis of p102 and p130 tryptic peptides

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.


View larger version (21K):
[in this window]
[in a new window]
 
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).

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).


View larger version (26K):
[in this window]
[in a new window]
 
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.

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).


View larger version (21K):
[in this window]
[in a new window]
 
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.

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).


View larger version (27K):
[in this window]
[in a new window]
 
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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    ACKNOWLEDGEMENT

We are grateful to Dr. Florencio Podestá for stimulating discussions.

    FOOTNOTES

* This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (to D. H. T. and W. C. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 204-474-6098; Fax: 204-474-7528; E-mail: rivoalj@ms.umanitoba.ca.

Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M010150200

    ABBREVIATIONS

The abbreviations used are: PEPC, phosphoenolpyruvate carboxylase; PEP, phosphoenolpyruvate; DMS, dimethyl suberimidate; PAGE, polyacrylamide gel electrophoresis; CAPS, 3-(cyclohexylamino)propanesulfonic acid; MALDI-MS, matrix-assisted laser desorption ionization mass spectrometry; HPLC, high pressure liquid chromatography; PLP, pyridoxal phosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Chollet, R., Vidal, J., and O'Leary, M. H. (1996) Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 273-298[CrossRef]
2. Vidal, J., and Chollet, R. (1997) Trends Plant Sci. 2, 230-237[CrossRef]
3. Huppe, H. C., and Turpin, D. H. (1994) Annu. Rev. Plant Physiol. Plant Mol. Biol. 45, 577-607[CrossRef]
4. Law, R. D., and Plaxton, W. C. (1995) Biochem. J. 307, 807-816[Medline] [Order article via Infotrieve]
5. Schuller, K. A., Plaxton, W. C., and Turpin, D. H. (1990) Plant Physiol. 93, 1303-1311
6. Rivoal, J., Dunford, R., Plaxton, W. C., and Turpin, D. H. (1996) Arch. Biochem. Biophys. 332, 47-57[CrossRef][Medline] [Order article via Infotrieve]
7. Falkowski, P. G., Barber, R. T., and Smetacek, V. (1998) Science 281, 200-207[Abstract/Free Full Text]
8. Field, C. B., Behrenfeld, M. J., Randerson, J. T., and Falkowski, P. (1998) Science 281, 237-240[Abstract/Free Full Text]
9. Raven, J. A., Wollenweber, B., and Handley, L. L. (1992) New Phytol. 121, 5-18
10. Turpin, D. H. (1990) J. Phycol. 27, 14-20
11. Vanlerberghe, G. C., Schuller, K. A., Smith, R. G., Feil, R., Plaxton, W. C., and Turpin, D. H. (1990) Plant Physiol. 94, 284-290
12. Rivoal, J., Plaxton, W. C., and Turpin, D. H. (1998) Biochem. J. 331, 201-209[Medline] [Order article via Infotrieve]
13. Allen, M. M. (1968) J. Phycol. 4, 1-4
14. Brooks, S. P. G. (1994) BioTechniques 17, 1154-1161[Medline] [Order article via Infotrieve]
15. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
16. Brooks, S. P. G. (1992) BioTechniques 13, 906-911[Medline] [Order article via Infotrieve]
17. Tipton, K. F. (1993) in Enzyme Assays, a Practical Approach (Eisenthal, R. , and Danson, M. J., eds) , pp. 1-58, Oxford University Press, Oxford
18. Laemmli, U. K. (1970) Nature 277, 680-685
19. Wray, W., Boulikas, T., Wray, V. P., and Hancock, R. (1981) Anal. Biochem. 118, 197-203[Medline] [Order article via Infotrieve]
20. Doucet, J. P., and Trifaró, J. M. (1988) Anal. Biochem. 168, 265-271[CrossRef][Medline] [Order article via Infotrieve]
21. Jiao, J. A., Podestà, F. E., Chollet, R., O'Leary, M. H., and Andreo, C. S. (1990) Biochim. Biophys. Acta 1041, 291-295[Medline] [Order article via Infotrieve]
22. Podestà, F. E., Iglesias, A. A., and Andreo, C. S. (1986) Arch. Biochem. Biophys. 246, 546-553[Medline] [Order article via Infotrieve]
23. Plaxton, W. C., and Moorhead, G. B. G. (1989) Anal. Biochem. 178, 391-393[Medline] [Order article via Infotrieve]
24. Toh, H., Kawamura, T., and Izui, K. (1994) Plant Cell Environ. 17, 31-43
25. Dong, L., Patil, S., Condon, S. A., Haas, E. J., and Chollet, R. (1999) Arch. Biochem. Biophys. 371, 124-128[CrossRef][Medline] [Order article via Infotrieve]
26. Kai, Y., Matsumura, H., Inoue, T., Terada, K., Nagara, Y., Yoshinaga, T., Kihara, A., Tsumura, K., and Izui, K. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 823-828[Abstract/Free Full Text]
27. Rajagopalan, A. V., Tirumala Devi, M., and Raghavendra, A. S (1994) Photosynth. Res. 39, 115-135
28. Law, D. L., and Plaxton, W. C. (1997) Eur. J. Biochem. 247, 642-651[Abstract]
29. Nhiri, M., Bakrim, N., Pacquit, V., El Hachimi-Messouak, Z., Osuna, L., and Vidal, J. (1998) Plant Cell Physiol. 39, 241-246
30. Baret, P., Cesari, M., Queiroz, C., Rouch, C., Meunier, J. C., and Cadet, F. (1999) C. R. Acad. Sci. Paris Ser. III 322, 29-34[Medline] [Order article via Infotrieve]
31. Srere, P. A., and Mathews, C. K. (1990) Methods Enzymol. 182, 539-551[Medline] [Order article via Infotrieve]
32. Turpin, D. H. (1988) in Growth and Reproductive Strategies of Freshwater Phytoplankton (Sandgren, G. D., ed) , pp. 316-368, Cambridge University Press, Cambridge, United Kingdom
33. Falkowski, P. G., and Raven, J. A. (1997) Aquatic Photosynthesis , pp. 300-335, Blackwell Publishers, Oxford
34. Smith, R. G., Vanlerberghe, G. C., Stitt, M., and Turpin, D. H. (1989) Plant Physiol. 91, 749-755
35. Barber, R. T. (1992) in Primary Productivity and Biogeochemical Cycles in the Sea (Falkowski, P. G. , and Woodhead, A. D., eds) , pp. 89-106, Plenum Press, New York
36. Fleischmann, R. D., Adams, M. D., White, O., Clayton, R. A., Kirkness, E. F., Kerlavage, A. R., Bult, C. J., Tomb, J.-F., Dougherty, B. A., Merrick, J. M., McKenney, K., Sutton, G., Fitzhugh, W., Fields, C. A., Gocayne, J. D., Scott, J. D., Shirley, R., Liu, L. I., Glodek, A., Kelley, J. M., Weidman, J. F., Phillips, C. A., Spriggs, T., Hedblom, E., Cotton, M. D., Utterback, T., Hanna, M. C., Nguyen, D. T., Saudek, D. M., Brandon, R. C., Fine, L. D., Fritchman, J. L., Fuhrmann, J. L., Geoghagen, N. S., Gnehm, C. L., McDonald, L. A., Small, K. V., Fraser, C. M., Smith, H. O., and Venter, J. C. (1995) Science 269, 496-512[Medline] [Order article via Infotrieve]
37. Fujita, N., Miwa, T., Ishijima, S., Izui, K., and Katsuki, H. (1984) J. Biochem. (Tokyo) 95, 909-916[Abstract]
38. Nakamura, T., Yoshioka, I., Takahashi, M., and Izui, K. (1995) J. Biochem. (Tokyo) 118, 319-324[Abstract]
39. Luinenburg, I., and Coleman, J. R. (1992) J. Gen. Microbiol. 138, 685-691[Medline] [Order article via Infotrieve]
40. O'Regan, M., Tierbach, G., Bachmann, B., Villeval, D., Lepage, P., Viret, J. F., and Lemoine, Y. (1989) Gene (Amst.) 77, 237-251[Medline] [Order article via Infotrieve]
41. Koizumi, N., Sato, F., Terano, Y., and Yamada, Y. (1991) Plant Mol. Biol. 17, 535-539[Medline] [Order article via Infotrieve]
42. Cushman, J. C., and Bonhert, H. J. (1989) Nucleic Acids Res. 17, 6743-6744[Medline] [Order article via Infotrieve]
43. Crétin, C., Keryer, E., Tagu, D., Lepiniec, L., Vidal, J., and Gadal, P. (1991) Gene (Amst.) 99, 87-94[CrossRef][Medline] [Order article via Infotrieve]
44. Izui, K., Ishijima, S., Yamaguchi, Y., Katagiri, F., Murata, T., Shigesada, K., Sugiyama, T., and Katsuki, H. (1986) Nucleic Acids Res. 14, 1615-1628[Abstract]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.