Co-existence of two regulatory NADP-glyceraldehyde 3-P
dehydrogenase complexes in higher plant chloroplasts
Renate Scheibe
1
, Norbert Wedel
2
, Susanne Vetter
1
, Vera Emmerlich
3
and Sonja-Manuela Sauermann
1
1
Plant Physiology, University of Osnabrueck, Germany;
2
Planton GmbH, Kiel, Germany;
3
Plant Physiology, University of
Kaiserslautern, Kaiserslautern, Germany
Light/dark modulation of the higher plant Calvin-cycle
enzymes phosphoribulokinase (PRK) and NADP-depend-
ent glyceraldehyde 3-phosphate dehydrogenase (NADP-
GAPDH-A
2
B
2
) involves changes of their aggregation state
in addition to redox changes of regulatory cysteines. Here we
demonstrate that plants possess two different complexes
containing the inactive forms (a) of NADP-GAPDH and
PRK and (b) of only NADP-GAPDH, respectively, in

modulation of their activity, brought about by a redox-
modification at specific cysteine residues mediated by the
ferredoxin/thioredoxin system [1]. The activity of each of
these enzymes is adjusted by fine-tuning, the rates of
reduction and/or of oxidation being influenced by specific
metabolites [2]. At constant redox conditions, this allows for
independent changes in the steady-state activities of each of
the enzymes merely by changes in the metabolic state of the
chloroplast [3,4]. In some cases, the reversible changes of
redox and activation states are accompanied by oligomeri-
zation and re-dissociation of transient complexes.
Enzyme aggregations of various compositions have been
described repeatedly, their occurrence under in vivo condi-
tions still being under debate [5,6]. But even the actual
composition of enzyme aggregates containing NAD(P)-
dependent glyceraldehyde-3 P dehydrogenase [NAD(P)-
GAPDH] and phosphoribulokinase (PRK) is controversial.
Both activities appear to occur in high-molecular-mass
forms in darkened chloroplasts, either in homo-oligomers
[7–10] or in hetero-oligomers [11,12], the latter involving a
small chloroplast protein, CP12, with a high sequence
similarity to the C-terminus of subunit B of the unique
chloroplast form of GAPDH [13]. Here we describe the
presence of both types of aggregations in darkened spinach
chloroplasts, one consisting of GAPDH A and B alone, the
other consisting of PRK, GAPDH, and CP12. The
differential stability of both complexes upon reductive
activation, the formation of the A
8
B

glyceraldehyde 3-P dehydrogenase; GSH and GSSG, reduced and
oxidized glutathione; PRK, phosphoribulokinase.
Enzymes: NAD(P)-dependent glyceraldehyde 3-phosphate
dehydrogenase (EC 1.2.1.13); phosphoribulokinase (EC 2.7.1.19).
(Received 3 June 2002, revised 21 August 2002,
accepted 18 September 2002)
Eur. J. Biochem. 269, 5617–5624 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03269.x
either without (dark) or with 20 m
M
dithiothreitol, and
incubated in a darkened vial for 30 min at 25 °C. For light-
activation experiments, intact chloroplasts were isolated and
incubated in bicarbonate-containing medium as described
in [9].
Gel filtration on Superdex S-200
Preincubated samples were filtered through a calibrated
Superdex 200 column (Hiload 16/60) (Pharmacia, Freiburg,
Germany) at 1 mLÆmin
)1
, collecting fractions of 1 mL
which were used for enzyme assays. The column buffer
consisted of 10 m
M
bicine/KOH, 150 m
M
NaCl, 140 l
M
NAD, pH 7.8, for ÔdarkÕ samples, and additionally 2.5 m
M
dithiothreitol for ÔdithiothreitolÕ samples.

Protein purification
PRK was purified from spinach leaves according to [18]
with some modifications. During all purification steps
10 m
M
dithiothreitol was present. The proteins precipitating
between 40 and 55% of the saturation of ammonium sulfate
at 4 °C were resuspended and subjected to acid precipitation
with acetic acid at pH 5.0. The supernatant was adjusted to
pH 6.8 and dialyzed over night. The diluted and clarified
solution in 10 m
M
bicine/KOH, 10 m
M
dithiothreitol,
pH 6.8, was subjected to affinity chromatography on
Reactive Red (Merck, Darmstadt, Germany), washed with
the same buffer. After a washing step with 10 m
M
potassium
phosphate, 10 m
M
dithiothreitol, pH 6.9, the PRK was
eluted in 10 m
M
potassium phosphate, pH 7.2, 10 m
M
dithiothreitol, and 5 m
M
ATP. The fractions with PRK

M
imidazol,
0.5
M
NaCl in 20 m
M
Tris/HCl, pH 7.9. For reconstitution
experiments the His-tag was removed by proteolysis using
thrombin according to the manufacturer’s protocol (Strate-
gene, Heidelberg, Germany). The CP12 clone for the
mature protein was originally described in [13].
Artificial stroma conditions
Reconstitution experiments with the purified proteins were
performed in a solution simulating the high protein
conditions in the stromal sample using a modified Ôartificial
stromaÕ [20]. In more detail, the sample for dark conditions
consisted of 5 m
M
MgSO
4
,10m
M
NaCl, 5 m
M
KNO
3
,
5m
M
KH

electroblotted and immunodecorated as in [12].
Immunoprecipitation
Protein-A Sepharose was preincubated with either preim-
mune serum or the indicated antisera in Tris-buffered saline
for 30 min at room temperature, washed once and then
addedtostromalextractsthathadbeenpreincubatedwith
140 l
M
NAD and 20 m
M
GSSG. After incubation for
30 min, the supernatant was used to assay for GAPDH and
PRK activities after full activation. The antisera against
CP12 and NADP-GAPDH have been obtained from
rabbits using the spinach proteins purified from E. coli
and spinach, respectively. Antiserum against PRK was a
kind gift from Fred Hartman, Oak Ridge, USA.
RESULTS
Reversible aggregation of GAPDH and PRK
in chloroplasts
When the soluble fraction of isolated darkened spinach
chloroplasts was subjected to gel filtration, both GAPDH
and PRK were obtained as high-molecular-mass aggregates
(Fig. 1A). Enzyme activities in the fractions were detected
after full activation. The activity peaks do not coincide
completely, GAPDH eluting somewhat earlier than PRK in
all cases. This tendency was even more pronounced in maize
chloroplasts (Fig. 2), where the GAPDH activity formed a
distinct shoulder at 600 kDa in addition to the peak at
550 kDa that coincided with the PRK activity. The high-

complex
which was still intact after incubation with dithiothreitol
alone [16], and the GADPH/PRK/CP12 complex described
by [12] which, upon reduction, releases GAPDH and PRK
as tetramer and dimer, respectively. CP12 and PRK could
not be detected by the respective antisera in the high-
molecular-mass fractions after dithiothreitol treatment,
while all three proteins were detectable with antisera in the
peak fractions of the untreated sample (Fig. 1C,D). The
high-molecular-mass fraction did not contain other enzyme
activities such as phosphoglycerate kinase [22] or fructose
1,6-bisphosphatase [23] that had been suggested to also
form high-molecular-mass aggregates (data not shown).
Furthermore, we never detected any tetramic GAPDH in
dark stroma which has been suggested to occur as A
4
by
[24].
Differential activation behaviour for GAPDH and PRK
upon dithiothreitol and light treatment
Incubation of chloroplast stroma with increasing concen-
trations of dithiothreitol resulted in 100% activation of
PRK even at low dithiothreitol concentrations at pH 8.0
(Fig.4A).Incontrast,evenupto20m
M
resulted in only
60–70% for the maximal GAPDH activity. Only in the
presence of added ATP and/or 3-phosphoglycerate, thus
increasing the 1,3-bisphosphoglycerate concentration in the
stroma [9], 100% activation of GAPDH was reached. This

to SDS/PAGE. The gel was stained with
Poinceau Red (0.2% in 3% (w/v) trichloro-
acetic acid), destained, and immunodecorated
with the indicated antisera (C,D). Fractions
reacting with the antiserum against CP12 are
highlightedwithanarrow.
Fig. 2. Gel filtration of chloroplast stroma from darkened maize leaves
in the absence of dithiothreitol. PRK (s) and NADP-GAPDH (d)
activities were determined after full activation as described in Materials
and methods.
Ó FEBS 2002 Two regulatory GAPDH complexes in chloroplasts (Eur. J. Biochem. 269) 5619
redox-modification mediated by thioredoxin has been
analyzed in much detail. The redox-active Cys residues
have been identified (Cys15 and Cys55) [25], their redox
potential has been determined [3,26], and the interaction
with thioredoxin f has been studied [21].
In order to analyze the structural changes upon redox
modification, we have reoxidized the purified, reduced
enzyme. Incubation with 50 m
M
dithiothreitol
ox
or with
25 m
M
GSSG at pH 8.0 resulted in an almost complete
inactivation. This inactivation could be reversed by the
addition of reductant (Table 1). However, both the reduced
and the oxidized enzyme forms appeared as dimers upon gel
filtration (Table 1) (Fig. 3C). This is in contrast to the

GSSG or with 50 m
M
oxidized
dithiothreitol, pH 8.0, for 15 min at 20 °C.
Reduced Oxidized
Activity (UÆmg protein
)1
) 437 8
Molecular mass (kDa) 80 80
5620 R. Scheibe et al. (Eur. J. Biochem. 269) Ó FEBS 2002
dark form of PRK appears as high-molecular-mass form.
Such controversial behaviour has been described already
[8,21].
In order to investigate the requirement for a small stromal
protein (i.e. CP12) for reaggregation of dimeric PRK, we
separated the dissociated enzyme (Fraction II: 80–120 kDa
in Fig. 5A) from the higher-molecular-mass fraction at
600 kDa (Fraction I) by gel filtration. Then a concentrated
fraction containing the smaller stromal proteins (Fraction
III: 20–60 kDa) and the enzyme fraction II were incubated
together with GSSG. After another step of gel filtration, a
new high-molecular-mass fraction became apparent con-
taining PRK activity (after reductive activation of the
fractions) (Fig. 5B). This peak contained almost equal
activities of GAPDH and PRK.
Reconstitution of GAPDH (A
8
B
8
) and PRK/GAPDH/CP12

proteins were incubated in Ôartificial stromaÕ as described under
Materials and methods. GAPDH (400 lg) pretreated with 380 l
M
1,3-bisphosphoglycerate and desalted in order to generate the tetra-
meric A
2
B
2
form, was present in addition to 200 lg purified PRK and
200 lg recombinant CP12. (C) Recombinant GAPDH consisting of
the subunits A and B in the soluble extract from IPTG-induced E. coli
cells was directly applied to a Superdex-200 gel filtration column. All
proteins were kept in their oxidized form by incubation with 20 m
M
GSSG.
Ó FEBS 2002 Two regulatory GAPDH complexes in chloroplasts (Eur. J. Biochem. 269) 5621
the PRK and all of the GAPDH (Fig. 6B). The low yield of
PRK reaggregation was probably due to the rather artificial
conditions. Some of the aggregated GAPDH was most
likely in its A
8
B
8
form.
Finally, the expression of both GAPDH subunits in
E. coli was performed. A high-molecular-mass complex
formed immediately in E. coli upon expression of the
combined construct for GapA and GapB and eluted as a
600-kDa form (Fig. 6C). The activation characteristics of
the recombinant GAPDH are typical for the A

together with 96.1% of the PRK. This again indicates the
presence of an independent GAPDH-A
8
B
8
complex apart
from the PRK/GAPDH/CP12-mixed complex. The fact
that the antiserum raised against recombinant CP12 only
removed a small proportion of the enzymes from the
solution, is most likely due to inaccessibility of the CP12 in
the native complex, because the serum recognized native
soluble CP12 in reduced stromal extracts (data not shown).
DISCUSSION
In general, evidence is increasing that cellular contents are
well organized in microcompartments due to protein–
protein interactions between partners of a metabolic path-
way or of a signal transduction cascade. In particular for
chloroplasts, there have been various attempts to show the
presence of bi- or multi-enzyme complexes of Calvin-cycle
enzymes (reviewed in [27]); however, there are intrinsic
technical problems when trying to confirm any of the
interactions unambiguously. The critical step is always
the breakage of the cell or the organelle, since changes of
the protein concentration and of the low-molecular-mass
components of the soluble medium will occur.
In order to avoid any new formation of complexes,
ammonium sulfate precipitation has been omitted in our
procedure and stromal fractions were applied directly to the
gel filtration column. This leads to reproducable elution
profiles on the Superdex 200 column in the presence of

GAPDH form present in darkened chloroplasts [9,16].
Later, we showed by limited proteolysis [29] and using
truncated constructs expressed in E. coli [30] that the unique
C-terminal sequence extension of GapB is responsible for
Fig. 7. Activation properties of A
8
B
8
-GAPDH in stromal extract and in
E. coli. GAPDH activites in the 600-kDa peak fractions of dithio-
threitol-treated stroma and of the recombinant GapA/GapB prepar-
ation were determined. The pooled fractions of peak I from Fig. 5A
(grey bars) and from Fig. 6C (hatched bars) were either assayed
directly. (– DTT), after incubation with 10 m
M
dithiothreitol
(+ DTT), or with 10 m
M
dithiothreitol and 21 lg 1,3-bisphospho-
glycerate (+ DTT/1,3bisPGA). DTT, dithiothreitol.
Table 2. Percentage coprecipitation of PRK-, GAPDH- and CP12-
containing complexes from oxidized stroma. The activities remaining in
the supernatant were determined after full activation.
Antiserum NADP-GAPDH PRK
Preimmune 100 100
Anti-GAPDH 8.5 17.5
Anti-PRK 57.1 3.9
5622 R. Scheibe et al. (Eur. J. Biochem. 269) Ó FEBS 2002
aggregation and inactivation of GAPDH in the dark.
Likewise, expression of the complete subunit B in E. coli

due to sterical hindrance. Taken together, it could be
established that GAPDH activity is present in two different
complexes reacting with distinct sensibility towards dithio-
threitol and the effector 1,3-bisphosphoglycerate leading to
dissociation and activation. On the other hand, all PRK
activity was present in a mixed complex forming only in the
presence of both GAPDH and CP12.
In order to understand the differential regulation of
Calvin-cycle enzymes, their midpoint redox potentials have
been determined and compared to physiological data
obtained with the intact system [34]. The apparent discrep-
ancies with respect to GAPDH could be easily explained
with the presence of two differently responding GAPDH-
containing aggregates: (a) the early evolved system, namely
PRK/GAPDH/CP12, which is activated merely by reduc-
tion and is already present in cyanobacteria [33]; and (b) the
A
8
B
8
form that evolved with the appearance of multicellular
green organisms when GapB emerged in Characeae for the
first time (R. Cerff and J. Peterson, Institut fu
¨
r Genetik, Tu
Braunschweig, Germany, personal communication).
The occurrence of both aggregates in plants indicates their
special role for optimal photosynthesis under all conditions.
A fast-responding system for complete PRK activation and
activation of a portion of GAPDH is required for CO

activity in both complexes.
From our experience, even very dim light during chloro-
plast isolation or preparation of extract leads to a significant
level of PRK activity and to some basic GAPDH activity
that is often described as Ôdark activityÕ. In contrast, strict
darkness during these steps will result in complete inactiva-
tion. The residual GAPDH activity obtained in vitro is
rather the result of the high 1,3-bisphosphoglycerate con-
centration (21 l
M
) in the standard assay leading to some
activity of the inactivated enzyme (low affinity for substrate
1,3-bisphosphoglycerate) as opposed to the in vivo situation
with a stromal concentration of 1–2 l
M
1,3-bisphospho-
glycerate [9,16].
Regulation of the activity at the PRK step is exclusively
achieved by noncovalently acting inhibitors, with ribulose
1,5-P
2
(K
i
¼ 0.7 m
M
), 3-phosphoglycerate (K
i
¼ 2m
M
),

common principle for individual control. Plant Physiol. 96,1–3.
3. Faske, M., Holtgrefe, S., Ocheretina, O., Meister, M.,
Backhausen, J.E. & Scheibe, R. (1995) Redox equilibria between
the regulatory thiols of light/dark-modulated chloroplast enzymes
and dithiothreitol: fine-tuning by metabolites. Biochim. Biophys.
Acta 1247, 135–142.
4. Holtgrefe, S., Backhausen, J.E., Kitzmann, C. & Scheibe, R.
(1997) Regulation of steady-state photosynthesis in isolated intact
chloroplasts under constant light: responses of carbon fluxes,
metabolite pools and enzyme-activation states of changes of
electron pressure. Plant Cell Physiol. 38, 1207–1216.
5. Su
¨
ss, K H. & Sainis, J.K. (1997) Supramolecular organization of
water-soluble photosynthetic enzymes in chloroplasts. In Hand-
book of Photosynthesis (Pessarakli, M., ed.), pp. 305–314. Marcel
Dekker Inc., New York – Basel – Hong Kong.
6.Rault,M.,Giudici-Orticoni,M T.,Gontero,B.&Ricard,J.
(1993) Structural and functional properties of a multi-enzyme
complex from spinach chloroplasts. 1. Stoichiometry of the
polypeptide chains. Eur. J. Biochem. 217, 1065–1073.
7. Wara-Aswapati, O., Kemble, R.J. & Bradbeer, J.W. (1980) Acti-
vation of glyceraldehyde-phosphate dehydrogenase (NADP) and
phosphoribulokinase in Phaseolus vulgaris leaf extracts involves
the dissociation of oligomers. Plant Physiol. 66, 34–39.
Ó FEBS 2002 Two regulatory GAPDH complexes in chloroplasts (Eur. J. Biochem. 269) 5623
8. Porter, M.A. (1990) The aggregation states of spinach phos-
phoribulokinase. Planta 181, 349–357.
9. Baalmann, E., Backhausen, J.E., Kitzmann, C. & Scheibe, R.
(1994) Regulation of NADP-dependent glyceraldehyde 3-phos-

18. Krieger, T.J., Mende-Mueller, L. & Miziorko, H.M. (1987)
Phosphoribulokinase: isolation and sequence determination of the
cysteine-containing active-site modified by 5¢-p-fluorosulfonyl-
benzoyladenosine. Biochim. Biophys. Acta 915, 112–119.
19. Pupillo, P. & Faggiani, R. (1979) Subunit structure of three gly-
ceraldehyde 3-phosphate dehydrogenases of some flowering
plants. Arch. Biochem. Biophys. 194, 581–592.
20. Kaiser, W.M., Schro
¨
ppel-Meier, G. & Wirth, E. (1986) Enzyme
activities in an artificial stroma medium. Planta 167, 292–299.
21. Geck, M.K. & Hartman, F.C. (2000) Kinetic and mutational
analysis of the regulation of phosphoribulokinase by thioredoxins.
J. Biol. Chem. 275, 18034–18039.
22. Macioszek, J., Anderson, J.B. & Anderson, L.E. (1990) Isolation
of chloroplastic phosphoglycerate kinase. Kinetics of the two-
enzyme phosphoglycerate kinase/glyceraldehyde-3-phosphate
dehydrogenase couple. Plant Physiol. 94, 291–296.
23. Grotjohann, N. (1997) Activation of chloroplast fructose-1,6-bis-
phosphatase of Pisum sativum by mole mass change. Bot. Acta
110, 323–327.
24. Scagliarini, S., Trost, P. & Pupillo, P. (1998) The non-regulatory
isoform of NAD(P)-glyceraldehyde-3-phosphate dehydrogenase
from spinach chloroplasts. J. Exp. Bot. 49, 1307–1315.
25. Porter, M.A., Stringer, C.D. & Hartman, F.C. (1988) Character-
ization of the regulatory thioredoxin site of phosphoribulokinase.
J. Biol. Chem. 263, 123–129.
26. Hirasawa, M., Brandes, H.K., Hartman, F.C. & Knaff, D.B.
(1998) Oxidation-reduction properties of the regulatory site
of spinach phosphoribulokinase. Arch. Biochem. Biophys. 350,

tion of spinach chloroplast glyceraldehyde 3-phosphate dehy-
drogenase: effect of glycerate 1,3-bisphosphate. Planta 190,
320–326.
33. Wedel, N. & Soll, J. (1998) Evolutionary conserved light regula-
tion of Calvin cycle activity by NADPH-mediated reversible
phosphoribulkinase/CP12/glyceraldehyde-3-phosphate dehydrog-
enase complex dissociation. Proc.NatlAcad.Sci.USA95, 9699–
9704.
34. Hutchison, R.S., Groom, Q. & Ort, D.R. (2000) Differential ef-
fects of chilling-induced photooxidation on the redox regulation of
photosynthetic enzymes. Biochemistry 39, 6679–6688.
35. Gardemann, A., Stitt, M. & Heldt, H.W. (1983) Regulation of
spinach ribulose-5-phosphate kinase by stromal metabolite levels.
Biochim. Biophys. Acta 722, 51–60.
5624 R. Scheibe et al. (Eur. J. Biochem. 269) Ó FEBS 2002