From the Departments of Biology and
§ Biochemistry, Queen's University, Kingston, Ontario K7L
3N6, Canada
Received for publication, November 4, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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Phosphoenolpyruvate carboxylase (PEPC) is
believed to play an important role in producing malate as a substrate
for fatty acid synthesis by leucoplasts of the developing castor
oilseed (COS) endosperm. Two kinetically distinct isoforms of COS PEPC were resolved by gel filtration chromatography and purified. PEPC1 is a
typical 410-kDa homotetramer composed of 107-kDa subunits (p107). In
contrast, PEPC2 exists as an unusual 681-kDa hetero-octamer composed of
the same p107 found in PEPC1 and an associated 64-kDa polypeptide (p64)
that is structurally and immunologically unrelated to p107. Relative to
PEPC1, PEPC2 demonstrated significantly enhanced thermal stability and
a much lower sensitivity to allosteric activators (Glc-6-P, Glc-1-P,
Fru-6-P, glycerol-3-P) and inhibitors (Asp, Glu, malate) and pH
changes within the physiological range. Nondenaturing PAGE of clarified
extracts followed by in-gel PEPC activity staining indicated that the
ratio of PEPC1:PEPC2 increases during COS development such that only
PEPC1 is detected in mature COS. Dissimilar developmental profiles and
kinetic properties support the hypotheses that (i) PEPC1 functions to
replenish dicarboxylic acids consumed through transamination reactions
required for storage protein synthesis, whereas (ii) PEPC2 facilitates
PEP flux to malate in support of fatty acid synthesis. Interestingly,
the respective physical and kinetic properties of COS PEPC1 and PEPC2
are remarkably comparable with those of the homotetrameric low
Mr Class 1 and heteromeric high
Mr Class 2 PEPC isoforms of unicellular green algae.
Phosphoenolpyruvate carboxylase
(PEPC)1 is a ubiquitous
cytosolic enzyme in vascular plants that is also widely distributed in
green algae and bacteria (1). It catalyzes the irreversible Relative to C4 and CAM PEPCs, the properties of the enzyme
from non-green plant tissues are less well understood. Although proposed roles for nonphotosynthetic PEPCs are diverse, a crucial PEPC
function is the anaplerotic replenishment of citric acid cycle
intermediates consumed during biosynthesis and nitrogen assimilation
(1). As with C4 and CAM PEPCs, the PEPC of C3 leaves and nonphotosynthetic tissues can be controlled by allosteric effectors and reversible phosphorylation (4-10). However, despite the
probable central role of PEPCs in the metabolism of developing and
germinating seeds (11-16), no seed PEPC has been fully purified and
thoroughly characterized.
Storage lipids account for as much as 65% of the weight of mature
castor oilseeds (COS). Triacylglyceride accumulation depends on the
synthesis of long chain fatty acids, which in developing oilseeds
occurs in specialized plastids termed leucoplasts. This process
requires the transport of both sucrose-derived carbon skeletons and
energetic intermediates across the plastid envelope (17).
L-Malate supports significant rates of fatty acid synthesis by isolated leucoplasts from developing COS (18). Malate imported from
the cytosol into the leucoplast stroma is mediated by a
malate/Pi translocator within the COS leucoplast envelope
(19). Sangwan and co-workers (16) hypothesized that the large increase
in PEPC activity and concentration that accompanies COS development facilitates malate production for fatty acid synthesis. The increased PEP to malate flux would also serve as an anaplerotic source of C-skeletons for transamination reactions associated with COS storage protein synthesis.
The aim of this study was to purify and characterize PEPC from
developing COS. Here we present unexpected evidence for two PEPC
isoforms from developing COS and examine their structural and kinetic
properties. Although one isoform is a typical PEPC homotetramer, the
other represents a unique high Mr PEPC complex unprecedented in vascular plants but remarkably reminiscent of Class 2 PEPC isoforms recently described in unicellular green algae (20-23).
We provide evidence that the association of a common 107-kDa PEPC
catalytic subunit with an unrelated but PEPC-like 64-kDa polypeptide is
responsible for the dramatic differences in the physical and kinetic
properties observed between the PEPC homotetramer and high
Mr PEPC complex of developing COS.
Plant Material--
Castor plants (Ricinus communis
L., cv Baker 296) were cultivated in a greenhouse at 24 °C and 60%
relative humidity under natural light supplemented with 16 h of
artificial light. COS were harvested at stages of development
previously described (24). Dissected endosperm (free of cotyledon) was
frozen in liquid N2 and stored at Enzyme and Protein Assays and Kinetic Studies--
PEPC activity
was assayed at 25 °C using a Molecular Devices microplate reader as
previously described (8). Standard assay conditions were: 100 mM Hepes-KOH (pH 8), 10% (v/v) glycerol, 2.5 mM PEP, 5 mM KHCO3, 5 mM MgCl2, 2 mM dithiothreitol, 0.15 mM NADH, and 5 units/ml desalted malate dehydrogenase. All
assays were corrected for background NADH oxidation and were linear
with respect to time and the concentration of enzyme assayed. One unit of PEPC activity is defined as the amount of enzyme resulting in the
production of 1 µmol of oxaloacetate min
Apparent Vmax (Vmax,app),
Km, and I50 and Ka
values (concentrations of inhibitors and activators producing 50%
inhibition or activation of PEPC activity, respectively) were calculated using Brooks' computer program (27). All kinetic parameters
represent means of at least three separate determinations and are
reproducible to within ± 10% (S.E.) of the mean value. Stock
solutions of metabolites were made equimolar with MgCl2 and
adjusted to pH 7.5.
PEPC Purification--
All procedures were carried out at
0-4 °C, and 10 µg/ml chymostatin, 0.5 µg/ml leupeptin, and 50 nM microcystin-LR were added to all resuspended
pellets and pooled fractions. Malate and 2,2'-dipyridyl disulfide were
omitted during the purification of proteolyzed PEPC. All buffers
contained 1 mM dithiothreitol, 5 mM
MgCl2, 1 mM EDTA, 1 mM EGTA, 20 mM NaF, and 5 mM malate in addition to the
following. Buffer A contained 50 mM Hepes-KOH (pH 7.5),
0.1% (v/v) Triton X-100, 20% (v/v) glycerol, 4% (w/v) PEG 8000, 1% (w/v) insoluble poly(vinylpolypyrrolidone), 5 mM thiourea,
2 mM 2,2'-dipyridyl disulfide, 2 mM
phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 10 µg/ml
chymostatin, and 50 nM microcystin-LR. Buffer B contained
50 mM Hepes-KOH (pH 7.1) and 20% (saturation) (NH4)2SO4. Buffer C was buffer B
lacking (NH4)2SO4 but containing 10% (v/v) glycerol. Buffer D contained 50 mM Hepes-KOH (pH
8) and 15% (v/v) glycerol. Buffer E contained 50 mM
Hepes-KOH (pH 7.5), 15% (v/v) glycerol, 100 mM KCl, and
0.02% (w/v) NaN3.
Stage VII developing COS endosperm (100 g) was homogenized (1:2; w/v)
in buffer A using a Polytron. After centrifugation, the supernatant was
filtered through two layers of Miracloth and recentrifuged. PEG 8000 (50% (w/v) in 50 mM Hepes-KOH, pH 7.5) was added to the
supernatant to a final concentration of 20% (w/v) and stirred for 30 min. After centrifugation, PEG pellets were resuspended in buffer B
(lacking (NH4)2SO4) to a final
protein concentration of about 10 mg/ml. After centrifugation, solid
(NH4)2SO4 was added to the
supernatant to 20% (saturation). The solution was stirred for 20 min,
centrifuged, and loaded at 4 ml/min onto a column (3.2 × 5.7 cm)
of butyl-Sepharose 4 Fast Flow equilibrated with buffer B. The column
was connected to an ÄKTA FPLC system and washed with buffer B
until the A280 approached base line. PEPC
activity was eluted by 50% buffer C (50% buffer B) (9 ml/fraction). Pooled peak fractions were diluted with an equal volume of 50% (w/v)
PEG 8000, stirred for 30 min, and centrifuged. PEG pellets were
dissolved in buffer D to a protein concentration of approximately 10 mg/ml, centrifuged as described above, and loaded at 0.6 ml/min onto a
column (1.1 × 12 cm) of Fractogel EMD DEAE-650 (S) that had been
connected to the FPLC and equilibrated with buffer D. The column was
washed with buffer D, and PEPC activity was eluted with a 0-400
mM KCl gradient (96 ml) in buffer D (3 ml/fraction). Pooled
peak fractions were concentrated to 1 ml using an Amicon YM-30
ultrafilter and applied at 0.3 ml/min onto a Superdex 200 HR 16/50
column equilibrated with buffer E (1 ml/fraction). Two PEPC activity
peaks were resolved (see "Results"). Each PEPC pool was
concentrated to approximately 0.2 ml and applied separately at 0.2 ml/min onto a Superose-6 HR 10/30 column equilibrated with buffer E. Pooled peak fractions were concentrated to <1 ml, divided into 25-µl
aliquots, frozen in liquid N2, and stored at Electrophoresis and Immunoblotting--
SDS and nondenaturing
PAGE, subunit Mr estimates via SDS-PAGE, and
immunoblotting using affinity-purified rabbit anti-(Brassica napus PEPC) IgG were performed as described (8, 28). Gels were
stained for protein with Coomassie Blue R-250 or Sypro Red or for PEPC
activity using the Fast Violet B method (28). Sypro Red-stained gels
were visualized using a Typhoon 8600 fluorescence imager, and the
relative band densities were quantified using software provided by the
manufacturer. For second dimension PAGE, Coomassie Blue-stained PEPC
was excised from a nondenaturing gel and subjected to SDS-PAGE as
described (9).
N-terminal Sequencing and Mass
Spectrometry--
Sequencing was performed by automated Edman
degradation at the Biotechnology Research Institute (Montreal, Quebec,
Canada). Similarity searches were performed using the BLAST program
available on the National Center for Biotechnology Information web site (www.ncbi.nlm.nih.gov/). For MS analyses, Coomassie Blue-stained polypeptides were excised from SDS gels and digested with trypsin using
standard protocols. Samples destined for MALDI-TOF MS were mixed 1:1
(v/v) with 5 mg/ml Purification and Physical, Immunological, and Structural
Characterization
PEPC Purification--
Initial purification trials resulted in a
PEPC preparation (specific activity, 28 units/mg) having a native
molecular mass of approximately 406 kDa as estimated via
analytical gel filtration (Fig. 1).
SDS-PAGE and immunoblot analysis of this preparation revealed an
approximate 1:1 ratio of 107- and 98-kDa protein-staining polypeptides
(p107 and p98, respectively) that cross-reacted with anti-(B.
napus PEPC) IgG with similar intensities (Fig.
2, A and B,
lane 1). BLAST analysis of the N-terminal amino acid
sequence of each polypeptide indicated that p98 probably arose via the action of a COS endopeptidase that hydrolyzed an approximate 120- amino
acid polypeptide from the N terminus of p107 (Fig.
3). Thus, we modified the purification
protocol by adding 2 mM 2,2'-dipyridyl disulfide to the
extraction buffer and 5 mM malate to all purification buffers. 2,2'-Dipyridyl disulfide is an active site-directed covalent affinity label of papain (29) that also suppresses the activity of COS
cysteinyl endopeptidase(s) (30). Malate helps to preserve the integrity
of the N-terminal phosphorylation domain of vascular plant PEPCs during
extraction and subsequent purification (1). With these buffer
additions, partial degradation of p107 was prevented (Fig. 2,
A and B). Moreover, two distinct peaks of PEPC
activity were resolved during Superdex 200 FPLC (Fig. 1B).
Additional gel filtration via Superose 6 FPLC resulted in an
approximately 200-fold purification of PEPC1 and PEPC2 to a final
specific activity of approximately 10 units/mg (Table
I). Although Superose 6 FPLC did not
increase the specific activity of PEPC1 or PEPC2 beyond that achieved
at the Superdex 200 step, it was included to ensure a clean separation
of PEPC1 from PEPC2. With the bicinchoninic acid protein assay (26) the
specific activity of the final PEPC1 and PEPC2 preparations was
increased to 24.2 and 29.2 units/mg, respectively. Calibration of the
Superdex 200 column with molecular mass standards yielded respective
native molecular masses of 410 ± 5 kDa for PEPC1 and 681 ± 9 kDa for PEPC2 (n = 3).
PAGE and Immunoblot Analysis--
A Coomassie Blue- and PEPC
activity-staining polypeptide that cross-reacted with anti-(B.
napus PEPC) IgG was observed after the nondenaturing PAGE of PEPC1
and PEPC2 (Fig. 4,
A-C, lanes 1 and 2). This
analysis was consistent with the respective native Mr estimations by gel filtration, because the
smaller PEPC1 migrated significantly further than the larger PEPC2. The
additional faster migrating protein-staining polypeptide observed
during nondenaturing PAGE of PEPC2 (Fig. 4A, lane
2) is a probable contaminant. It did not stain for PEPC activity
or cross-react with the anti-PEPC IgG (Fig. 4, B and
C, lane 2) and was eliminated when the final PEPC2 preparation was subjected to Mono-Q FPLC (Fig.
1C).
SDS-PAGE of the final PEPC1 and PEPC2 preparations resolved a
protein-staining p107 that cross-reacted with anti-(B. napus PEPC) IgG (Fig. 2, A and B, lanes 2 and
3). However, PEPC2 contained two additional protein-staining
polypeptides of approximately 64-kDa (p64) and 57-kDa (Fig.
2A, lane 3). When the protein- and PEPC
activity-staining band obtained after nondenaturing PAGE of PEPC2 was
excised, equilibrated with SDS, and subjected to SDS-PAGE, p107 and p64
were resolved (Fig. 2A, lane 4), indicating that
the p64 was complexed with p107 in the native PEPC2. This result was
corroborated by SDS-PAGE of fractions collected during analytical
Mono-Q FPLC of PEPC2 in which both the p107 and p64 co-eluted with PEPC
activity and a symmetrical A280 peak (Fig. 1C). The p64 was not recognized by the anti-(B.
napus PEPC) IgG (Fig. 3A). Densitometric analysis of
Sypro Red- stained SDS gels of Mono-Q-purified PEPC2 allowed us to
estimate a p107:p64 molar ratio of 1:1. The native PEPC2 therefore
appears to exist as an unusual hetero-octomeric complex composed of
four p107 and four p64 subunits with a combined theoretical molecular
mass of 684 kDa. This value closely agrees with the molecular mass of
681 kDa estimated for native PEPC2 during Superdex 200 FPLC. PEPC1, by
contrast, is a typical PEPC homotetramer of p107 subunits, which likely
correspond to the same p107 found in PEPC2.
Nondenaturing PAGE of clarified extracts (prepared in buffer A)
followed by in-gel PEPC activity staining indicated that the ratio of
PEPC1:PEPC2 progressively increases during COS development such that
only PEPC1 is detected in stage IX (maturation phase) and dry (mature)
COS (Fig. 4B). Overall PEPC activity was relatively abundant, peaking at approximately 2.6 units·(g fresh
weight) Thermal Stability--
PEPC1 was relatively heat labile, losing 0, 19, 25, 40, 82, and 100% of its original activity when incubated for 3 min at 30, 35, 40, 45, 50, and 55 °C, respectively. By contrast,
PEPC2 was much less heat labile, losing 0, 20, and 100% of its
original activity when incubated for 3 min at 45, 50, and 55 °C, respectively.
N-terminal Sequencing and Mass Spectrometry--
The 20 N-terminal
amino acids of the p107 and p98 of proteolyzed PEPC were sequenced by
Edman degradation (Fig. 3). BLAST analysis revealed significant matches
with the corresponding region of various plant PEPCs and included the
conserved regulatory seryl phosphorylation site found in all plant
PEPCs examined to date (Fig. 3). The sequences of 12 amino acid
residues of the N termini of the p107 of PEPC1 and PEPC2 were
determined and found to be identical to that of the p107 of proteolyzed
PEPC. This result indicates that PEPC1, PEPC2, and proteolyzed PEPC may
share a common p107. This idea was corroborated by MALDI-TOF MS
analysis of PEPC1 and PEPC2 p107 tryptic peptides. Their mass
fingerprints were very similar, with 21 identical peptides (results not
shown). Both mass fingerprints best matched the same tomato
(Lycopersicon esculentum) PEPC (GenBankTM GI
number 6688531).
The p64 of PEPC2 was also subjected to MALDI-TOF MS. However, the p64
mass fingerprint did not produce any significant matches in the NCBI
data base. In addition, none of the 19 tryptic peptides of p64 occurred
in those of PEPC1 or PEPC2 p107 (results not shown). The p64 was
further analyzed by Q-TOF MS/MS. The Q-TOF data best matched two
putative PEPCs from rice (Oryza sativa) and
Arabidopsis genome sequence databases (Fig.
5A). Neither of these PEPCs
was identified by MALDI-TOF MS of p107, further suggesting that p107 and p64 are structurally dissimilar. The most significant hit matched 7 individual peptides of a putative rice PEPC (Fig. 5B). BLAST
analyses of this 102-kDa rice PEPC indicated that its first 70 N-terminal amino acids are unique compared with most plant PEPCs, although the full-length sequence contains all three conserved domains believed to be required for PEPC activity (Fig. 5B)
(1).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-carboxylation of PEP in the presence of Mg2+ and
HCO
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C.
1. Protein
concentration was determined by the Coomassie Blue G-250 (25) or
bicinchoninic acid (26) colorimetric methods using bovine
-globulin
as the protein standard.
80 °C. PEPC activity was stable for at least 3 months when stored frozen. In
some instances, the final PEPC2 preparation was subjected to anion-exchange FPLC on a Mono-Q HR 5/5 column equilibrated in buffer D.
-cyanohydroxycinnamic acid matrix in 50% (v/v)
acetonitrile and 0.1% (v/v) trifluoroacetic acid before
analysis on a Micromass MALDI (positive ion mode). Samples destined for
ESI/Q-TOF MS/MS were applied automatically by capillary liquid
chromatography to a Micromass Q-TOF Ultima GLOBAL using a C18
column (75 µm × 150 mm) running at 200 nl/min. Mass data from
both machines were used to search the NCBInr data base using MASCOT
(www.matrixscience.com).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Superdex 200 and Mono-Q FPLC of COS
PEPC. A and B, Superdex 200 elution
profiles for COS PEPC purified in the absence (A) and
presence (B) of 2,2'-dipyridyl disulfide and malate.
Vo denotes the void volume of the column.
C, Mono-Q FPLC analysis of the co-purification of the
p107 and p64 of COS PEPC2. Inset, aliquots (5-µl) from the
PEPC-active Mono-Q fractions were subjected to SDS-PAGE (9% separating
gel). The gel was stained with Sypro Red and scanned on a fluorescence
imager.
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Fig. 2.
SDS-PAGE and immunoblot analysis of COS PEPC
isoforms. A, SDS-PAGE (9% gel) of 1.5 µg each of the
final preparations of proteolyzed PEPC (lane 1), PEPC1
(lane 2), and PEPC2 (lane 3). Lanes 4 and 5 contain 2 µg of PEPC1 and PEPC2, respectively, that
had been excised from a nondenaturing gel (PEPC active bands; see Fig.
4), equilibrated with SDS, and subjected to SDS-PAGE. Staining was
performed using Coomassie Blue R-250. B, immunoblot analysis
was performed using affinity-purified rabbit anti-(B. napus
PEPC) IgG (8). Lanes 1-3 contain 50 ng each of purified
proteolyzed PEPC, PEPC1, and PEPC2, respectively. The remaining lanes
contain clarified extracts (corresponding to 1.5 mg of fresh tissue)
from various stages of COS development. Lanes (developmental
stage) III, V, VII, and IX
correspond to the heart-shaped embryo, midcotyledon, full cotyledon,
and maturation stages of development, respectively (24).
Lane "Dry" designates a fully mature COS.
Asterisk denotes the stage at which PEPC purification was
conducted.
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Fig. 3.
Comparison of the N-terminal sequences of
COS p107 (A) and p98
(B) with those of other plant PEPCs. The COS
(R. communis) PEPC sequences were determined by Edman
sequencing of p107 and p98 of the "proteolyzed PEPC" preparation
(see Fig. 2, A and B, lane 1). Other
PEPC sequences were derived by translation of the corresponding genes.
Sequence numbering represents amino acid position relative
to the N terminus. Hyphens denote amino acid residues that
are identical to those of the respective COS PEPC sequences. An
asterisk indicates the conserved regulatory seryl
phosphorylation site, and underlined letters indicate the
consensus target sequence for plant PEPC protein kinase (1).
Purification of PEPC isoforms from 100 g of stage VII (full cotyledon)
developing COS endosperm
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Fig. 4.
Nondenaturing PAGE (5% gel) analysis of COS
PEPC isoforms. A, staining was performed with
Coomassie Blue R-250. Lanes 1 and 2, respectively, contain 1 µg each of purified PEPC1 and PEPC2. B, immunoblot
analysis was performed using affinity-purified rabbit anti-(B.
napus PEPC) IgG (8). Lanes 1 and 2,
respectively, contain 50 ng of purified PEPC1 and PEPC2.
C, in-gel PEPC activity staining was performed as
described (9). Lanes 1 and 2, respectively,
contain 1 µg each of purified PEPC1 and PEPC2. The remaining lanes
contain clarified extracts from various stages of COS development, as
described in the legend for Fig. 2. Inset, corresponding
PEPC activity in clarified extracts of COS endosperm (means ± S.E., n = 3).
1 in stage VII developing COS endosperm
(Fig. 4B, inset). Immunoblots of the same
clarified extracts were probed with the anti-(B. napus PEPC)
IgG and demonstrated that p107 was present throughout COS development
but declined after stage VII (Fig. 2B). Immunoreactive polypeptides of 98-102 kDa were detected in the later developmental stages. These polypeptides may represent in vivo proteolytic
degradation products of p107, because the same antigenic polypeptides
were observed on an immunoblot of stage IX COS extract prepared under denaturing conditions in 10% (w/v) trichloroacetic acid (31) (results not shown).
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Fig. 5.
Q-TOF MS/MS analysis of p64 tryptic
peptides. A, Q-TOF data were submitted to the
MASCOT search engine and used to match the nonredundant NCBI data base.
All significant matches (MOWSE score > 34) are shown.
B, predicted primary sequence of a putative PEPC from
rice (O. sativa cv. Japonica) (GenBankTM GI
number 13486638). Peptides found in the tryptic digest of p64 are
underlined. Boldface sequences represent
conserved functional domains (1).
Kinetic Properties
Effect of pH and PEP Saturation Kinetics--
Similar to other
plant PEPCs (1), PEPC1 and PEPC2 activity increased with pH in the
range of 6.5-8.0. However, PEPC1 exhibited optimal activity at pH 8.5, whereas PEPC2 displayed optimal activity at pH 8.0 (Fig.
6). Moreover, PEPC1 displayed a
significantly greater sensitivity to pH changes within the
physiological range. Between pH 8 and 6.5, PEPC1 activity decreased by
more than 30-fold, whereas PEPC2 activity decreased by less than 3-fold
(Fig. 6).
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PEP saturation kinetics and response to metabolite effectors were determined at pH 8 and 7.3. Hyperbolic PEP saturation kinetics was always observed. At pH 8.0 and 7.3 the respective Vmax,app values of PEPC1 were 24.5 and 20.7 units/mg, whereas those of PEPC2 were 29.7 and 28.8 units/mg. No difference was noted in the Km(PEP) values for PEPC1 and PEPC2, which were both approximately 0.06 and 0.12 mM at pH 8 and 7.3, respectively.
Metabolite Effects-- A wide variety of compounds were tested as possible effectors of PEPC1 and PEPC2 at pH 7.3 and pH 8 with subsaturating PEP (0.2 mM). The following compounds exerted little or no influence on the activity of either isoform (± 20% of the control rate): 2-P-glycerate, dihydroxyacetone-P, Fru-1,6-P2, NAD+, Gly, Glu, Arg, Ala, Leu, Asn, Phe, pyruvate, and AMP (2 mM each); CoA, malonyl-CoA, acetyl-CoA, and oleyl-CoA (50 µM each). Table II lists those compounds that significantly influenced PEPC activity. Similar to other plant PEPCs (1), PEPC1 and PEPC2 displayed pH-dependent modulation by several metabolites that were more effective at pH 7.3 than at pH 8. PEPC1, however, was much more sensitive to the various metabolite effectors than PEPC2 (Table II). PEPC1 was potently activated at pH 7.3 by the hexose-mono-Ps and by glycerol-3-P, whereas PEPC2 was only weakly activated by these compounds. Similarly, PEPC1 was far more sensitive to inhibition by malate, Asp, Glu, and ATP, relative to PEPC2. Fig. 7 demonstrates the marked differential response of PEPC1 and PEPC2 activity to increasing concentrations of the most widely recognized allosteric effectors of plant PEPC (1), namely malate and Glc-6-P.
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DISCUSSION |
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When partial in vitro proteolysis of p107 was prevented, two COS PEPC isoforms that significantly differed in their physical and kinetic properties were resolved by Superdex 200 FPLC and highly purified. Tissue- and/or developmentally specific PEPC isozymes are known to occur in vascular plants (1-3), and genetic evidence indicates that developing Glycine max (soybean) and Vicia faba seeds express more than one PEPC gene (15, 32, 33). To our knowledge, this is the first report of the isolation of two PEPC isoforms from the same plant tissue.
COS PEPC1 is a p107 homotetramer, typical of most other plant PEPCs studied to date. By contrast, PEPC2 appears to exist as an unusual 681- kDa hetero-octamer composed of the same p107 found in PEPC1 and an associated p64 that is structurally and immunologically unrelated to p107. Nevertheless, Q-TOF MS/MS analysis of tryptic peptides revealed that p64 is highly similar to two putative PEPCs identified by annotation of the rice and Arabidopsis genomes (Fig. 5). Although they contain conserved regions required for PEPC activity, these putative PEPCs exhibit a unique N-terminal region that lacks the regulatory seryl phosphorylation site thought to be conserved among all plant PEPCs (1) and have predicted molecular masses of 102 (rice) and 110 kDa (Arabidopsis). Although it is feasible that p64 represents an in vivo or in vitro degradation product of a larger polypeptide, the data indicate that p64 is a PEPC-like polypeptide that interacts with p107 to give rise to the COS PEPC2 heteromeric complex. This association may either physically block the allosteric sites of p107 or promote an allosteric transition in p107 such that effectors have limited access to their respective sites.
COS PEPC1 and PEPC2 Emulate Green Algal "Class 1" and "Class 2" PEPC Isoforms-- Interestingly, the respective properties of COS PEPC1 and PEPC2 are remarkably comparable with those of the homotetrameric low Mr Class 1 and heteromeric high Mr Class 2 PEPC isoforms of the green algae Selenastrum minutum and Chlamydomonas reinhardtii (20-22). All algal PEPC isoforms share the same 102-kDa catalytic subunit (p102). Similar to COS PEPC2, the algal Class 2 PEPCs also contain associated polypeptides that are immunologically unrelated to p102. MALDI-TOF MS and microsequencing revealed that like the p64 of COS PEPC2, the 130- kDa subunit (p130) of S. minutum Class 2 PEPC represents a distinct PEPC-like polypeptide that is only distantly related to p102 (22). Moreover, relative to COS PEPC1 and algal Class 1 PEPCs, the COS PEPC2 and algal Class 2 PEPCs demonstrate significantly enhanced thermal stability and a much lower sensitivity to allosteric effectors and pH changes within the physiological range (20, 21). Taken together, the data imply that high and low Mr PEPC isoforms arose in green algae before the evolution of vascular plants, with this feature being conserved as a key structure-function aspect of at least some plant PEPCs.
Possible Functions and Interconversion of COS PEPC1 and
PEPC2--
Similar to PEPCs from other non-green plant tissues (6, 8,
9, 11, 15), COS PEPC1 was activated by hexose-mono-Ps and
potently inhibited by malate, Asp, and Glu at pH 7.3 (Table II). This
result indicates that PEPC1 may fulfill a key anaplerotic role to
replenish dicarboxylic acids consumed through transamination reactions
required to support storage protein synthesis. The inhibition of PEPC1
by Asp and Glu provides a tight feedback control that could closely
balance PEPC1 activity with the production of C-skeletons (i.e. oxaloacetate, 2-oxoglutarate) required for
NH
It remains to be determined whether and how COS PEPC1 and PEPC2
interconvert. However, protein-kinase-mediated phosphorylation of p102
appears to be involved in the control and structural organization of
green algal (S. minutum) Class 2 PEPCs (23). COS p107
contains the N-terminal regulatory seryl phosphorylation site
characteristic of most plant PEPCs (Fig. 3A). It will be of
interest to determine whether COS PEPC1 and PEPC2 are interconverted
via a phosphorylation-dephosphorylation mechanism involving p107 and/or p64.
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ACKNOWLEDGEMENTS |
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We thank Dr. Jean Rivoal (University of Montreal) for helpful discussions. We also gratefully acknowledge Dr. David Hyndman of the Queen's Protein Function Discovery Research and Training Program for his invaluable assistance with the MS analyses of p107 and p64.
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FOOTNOTES |
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* This work was supported by research and equipment grants from the Natural Sciences and Engineering Research Council of Canada (to 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: Dept. of Biology, Queen's University, Kingston, Ontario K7L 3N6, Canada. Tel.: 613-533-6150; Fax: 613-533-6617; E-mail: plaxton@biology.queensu.ca.
Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M211269200
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ABBREVIATIONS |
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The abbreviations used are: PEPC, PEP carboxylase (EC 4.1.1.31); PEP, phosphoenolpyruvate; CAM, crassulacean acid metabolism; COS, castor oilseed; ESI/Q-TOF MS/MS, electrospray quadrupole-time of flight tandem mass spectrometry; MALDI-TOF MS, matrix-assisted laser desorption ionization-time of flight mass spectrometry; FPLC, fast protein liquid chromatography; PEG, polyethylene glycol; MES, 4-morpholineethanesulfonic acid.
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