Báo cáo khoa học: Isolation and structural characterization of the Ndh complex from mesophyll and bundle sheath chloroplasts of Zea mays pptx

Isolation and structural characterization of the Ndh
complex from mesophyll and bundle sheath chloroplasts
of Zea mays
Costel C. Darie
1
, Martin L. Biniossek
2
, Veronika Winter
3
, Bettina Mutschler
4
and
Wolfgang Haehnel
4
1 Brookdale Department Molecular, Cell and Developmental Biology, Mount Sinai School of Medicine, New York, USA
2 Institut fuer Molekulare Medizin und Zellforschung, Albert-Ludwigs Universitaet, Freiburg, Germany
3De
´
partement de Biologie Mole
´
culaire, Universite
´
de Gene
`
ve, Switzerland
4 Institut fuer Biologie II ⁄ Biochemie der Pflanzen, Albert-Ludwigs Universitaet, Freiburg, Germany
Complex I is a proton-pumping multisubunit-complex
involved in the respiratory electron transport chain,
which provides the proton motive force essential for
the synthesis of ATP. Homologs of this complex exist
in bacteria, the mitochondria of eukaryotes, and the

chloroplasts of plants. The bacterial and mitochondrial
Keywords
chloroplast; maize; mass spectrometry;
native electrophoresis; Ndh complex
Correspondence
C. C. Darie, Brookdale Department of
Molecular, Cell and Developmental Biology,
Annenberg Building, Box1020, Mount Sinai
School of Medicine, One Gustave L. Levy
Place, New York, NY 10029-6574, USA
Fax: +1 718 246 2616
Tel: +1 212 241 8620
E-mail:
(Received 31 January 2005, revised 23
March 2005, accepted 24 March 2005)
doi:10.1111/j.1742-4658.2005.04685.x
Complex I (NADH: ubiquinone oxidoreductase) is the first complex in the
respiratory electron transport chain. Homologs of this complex exist in
bacteria, mitochondria and chloroplasts. The minimal complex I from
mitochondria and bacteria contains 14 different subunits grouped into
three modules: membrane, connecting, and soluble subcomplexes. The com-
plex I homolog (NADH dehydrogenase or Ndh complex) from chloroplasts
from higher plants contains genes for two out of three modules: the mem-
brane and connecting subcomplexes. However, there is not much informa-
tion about the existence of the soluble subcomplex (which is the electron
input device in bacterial complex I) in the composition of the Ndh com-
plex. Furthermore, there are contrasting reports regarding the subunit
composition of the Ndh complex and its molecular mass. By using blue
native (BN) ⁄ PAGE and Tricine ⁄ PAGE or colorless-native (CN) ⁄ PAGE,
BN ⁄ PAGE and Tricine ⁄ PAGE, combined with mass spectrometry, we

attempted to obtain more information about the plastidal Ndh complex
from maize (Zea mays). Using antibodies, we detected the expression of a
new ndh gene (ndhE) in mesophyll (MS) and bundle sheath (BS) chloro-
plasts and in ethioplasts (ET). We determined the molecular mass of the
Ndh complex (550 kDa) and observed that it splits into a 300 kDa mem-
brane subcomplex (containing NdhE) and a 250 kDa subcomplex (contain-
ing NdhH, -J and -K). The Ndh complex forms dimers at 1000–1100 kDa
in both MS and BS chloroplasts. Native ⁄ PAGE of the MS and BS chloro-
plasts allowed us to determine that the Ndh complex contains at least 14
different subunits. The native gel electrophoresis, western blotting and mass
spectrometry allowed us to identify five of the Ndh subunits. We also pro-
vide a method that allows the purification of large amounts of Ndh com-
plex for further structural, as well as functional studies.
Abbreviations
BN ⁄ PAGE, blue native ⁄ PAGE; BS, bundle sheath; CN ⁄ PAGE, colorless native ⁄ PAGE; ET, ethioplasts; MS, mesophyll; Ndh complex,
NADH-dehydrogenase or NADH plastoquinone oxidoreductase; PS, photosystem.
FEBS Journal 272 (2005) 2705–2716 ª 2005 FEBS 2705
complexes function as NADH dehydrogenase (NADH:
ubiquinone oxidoreductase) [1].
The minimal complex I from mitochondria and
bacteria contains 14 polypeptides. The mitochondrial
complex I contains additional subunits with no
counterparts in the bacterial complex. In bacteria, the
14 subunits of the complex I are grouped into three
modules: seven subunits form the membrane subcom-
plex, four subunits form the connecting subcomplex
and the last three subunits form the soluble subcom-
plex. The soluble subcomplex contains the NADH-
binding and -oxidizing site [1].
Genes for 11 of the 14 minimal subunits were also

found in the plastid genome of plants. The 11 ndh
genes on the plastid genome that encode subunits
homologous to those of the NADH dehydrogenase or
complex I of mitochondria and bacteria are highly
conserved in most plants. Their function as a proton-
pumping NADH, plastoquinone oxidoreductase
(NADH dehydrogenase or Ndh complex), has been
suggested [1].
A structural model indicates that the plastid ndhA–
ndhG gene products form the membrane subcomplex,
and the ndhH–ndhK gene products form a connecting
subcomplex that probably mediates the electron trans-
fer from NAD(P)H. Subunits homologous to the three
peripheral subunits (from the soluble subcomplex) of
the NADH-oxidizing domain are likely encoded in the
nucleus, but have not been identified so far.
Although most groups studying the Ndh complex
agree upon its molecular mass as 550–580 kDa [2–7],
other groups have reported detection of the Ndh com-
plex with a molecular mass between 800 and 1000 kDa
[8,9].
To date, not all 11 Ndh polypeptides encoded by
ndh genes have been identified in plastids. Only the
polypeptides corresponding to seven out of the 11
genes have been identified in chloroplasts. Four of
them are components of the connecting subcomplex:
NdhJ [6], NdhH [10], NdhK [11] and NdhI [12]. From
the other seven polypeptides that form the membrane
subcomplex, only three have been identified: NdhA
[13], NdhB [6] and NdhF [14]. Nothing is known

about the expression of ndhC, -D, -E and -G in chloro-
plast, nor in any plastid type.
The C4 plants Sorghum bicolor and Zea mays have
mesophyll (MS) and bundle sheath (BS) chloroplasts.
The chloroplasts of the MS cells contain grana, but
those in the BS cells have a variable degree of grana
development, depending on the species [15]. The grana
from MS and BS chloroplasts exhibit normal photosys-
tem (PS) I activity, but the agranal BS thylakoids have
almost no PS II activity [16,17]. However, transcription
of the ndh genes is much higher in BS chloroplasts, and
elevated amounts of the Ndh complex have been found
in these plastids [18].
The function of the Ndh complex is still a matter of
debate. Some authors have proposed that in chloro-
plasts the Ndh complex is involved in cyclic electron
transport around PS I [18–22]. Other authors have
suggested a second role for this complex in chlorores-
piration [3–5,23,24]. However, controversial reports
about the viability of ndh mutants [2,23,25] have
clearly restarted the debate about the real function(s)
of the Ndh complex.
To contribute to the structural and functional char-
acterization of this large complex in chloroplasts, we
produced antibodies against Ndh subunits from the
membrane (NdhE) and connecting (NdhH, -J and -K)
subcomplexes. NdhE antibodies were used as markers
for the presence of the membrane subcomplex, while
NdhH, -J and -K antibodies were used to identify the
connecting subcomplex.

Using these antibodies, we detected the expression of
a new Ndh subunit (NdhE from the membrane subcom-
plex) in maize MS, BS and ethioplast (ET) plastids. By
using (1) blue-native (BN) ⁄ PAGE and (2) colorless-
native (CN) ⁄ PAGE and BN ⁄ PAGE, we separated the
Ndh complex from both MS and BS chloroplasts and
determined its monomeric and dimeric state. We also
demonstrated that the Ndh complex splits into a
300-kDa membrane subcomplex (containing NdhE) and
a 250-kDa subcomplex (containing NdhH, -J and -K).
By separating the Ndh complex that resulted from
native electrophoresis in denaturing Tricine ⁄ PAGE, we
determined that the Ndh complex contains at least 14
different subunits, five of which were identified by Ndh
antibodies and mass spectrometry.
Results
Detection of a new expressed Ndh protein
In higher plants it has been demonstrated that seven
(ndhA, -B, -F, -H, -I, -J, -K) of 11 ndh genes are
expressed in plastids, but there is no information about
the expression of ndhC, -D, -E and -G. Here we report
that a new Ndh protein (NdhE) is expressed in three
different plastid types. In Fig. 1A, the protein pattern
of the MS, ET and BS plastids is shown. In Fig. 1B,
the three plastid types were separated on SDS ⁄ PAGE,
electroblotted and immunodecorated with Ndh anti-
bodies. The Ndh antibodies detected polypeptides
with a molecular mass of 46 (NdhH), 28 (NdhK),
18 (NdhJ) and 12 (NdhE) kDa, in agreement with
their theoretical mass, calculated from their DNA

Maize Ndh complex C. C. Darie et al.
2706 FEBS Journal 272 (2005) 2705–2716 ª 2005 FEBS
sequences. The NdhE antibodies recognized their cor-
responding antigens in three different plastid types:
MS and BS chloroplasts, as well as in ET. Because of
high homology between NdhE from maize and rice,
this polypeptide was also identified by cross-reaction
of NdhE antibodies in rice chloroplasts, a C
3
type
plant (data not shown). This is the first demonstration
that the ndhE gene produces a stable NdhE polypep-
tide in different types of plastids.
The Ndh complex associates in homodimers
and dissociates in membrane and soluble
subcomplexes; isolation of the Ndh complex
by BN
/
PAGE and Tricine
/
PAGE
Intact MS and BS chloroplasts were used as starting
material for the separation of the Ndh complex.
BN ⁄ PAGE (which separates the protein complexes
based on their molecular mass), of the BS chloroplasts
revealed eight (five dominant and three minor) bands
with molecular masses in the range of 750–200 kDa
(Fig. 2A left, the horizontal gel lane). To identify the
Ndh complex, the gel strips that resulted from the first
native dimension (1D) were separated in a second

dimension (2D) under denaturing and reducing condi-
tions (Tricine ⁄ PAGE), electroblotted and immunodeco-
rated with Ndh antibodies (Fig. 2A). NdhH, -K and -J
antibodies were markers for the Ndh connecting sub-
complex, while NdhE antibodies were markers for
the Ndh membrane subcomplex. The Ndh antibodies
recognized the 46, 28, 18 and 12 kDa polypeptides, cor-
responding to the NdhH, -K, -J and -E subunits of the
Ndh complex.
The intact Ndh complex, which corresponded to the
third band in BN ⁄ PAGE, showed a molecular mass of
520–550 kDa (Fig. 2A, left) and contained at least 14
visible subunits with a molecular mass in a range of
10–80 kDa (Fig. 2B, left). Ndh antibodies also reacted
with their antigens which were part of a 300–320-
(NdhE) or 250-kDa complexes (NdhH, -K, and -J),
MS ET BS MS ET BS
kDa
97-
66-
45-
30-
20-
14-
Nd
h
-H
-K
-J
-E

AB
Fig. 1. SDS ⁄ PAGE of the MS and BS chloroplasts and ET of maize.
After electrophoresis, the gels were stained with Coomassie blue
(A) or transferred onto membrane and probed with Ndh antibodies
(B). The polypeptide pattern of the maize plastids and the molecular
mass markers are shown in (A). The polypeptides detected by Ndh
antibodies are shown in (B) (right).
A
B
Fig. 2. Separation of the MS and BS chloro-
plasts by BN ⁄ PAGE. The horizontal gel lane
represents the first BN ⁄ PAGE dimension
(1D). After reduction and denaturation, the
gel lane was separated in the second dimen-
sion Tricine ⁄ PAGE (2D). The molecular
mass standards and direction of migration
are indicated for both the first (1D) and sec-
ond (2D) dimension. (A) Tricine ⁄ PAGE gels
that resulted in 2D were electroblotted and
immunodecorated with Ndh antibodies. The
position of the immune reaction for the Ndh
antibodies is indicated on the side of the
blots. The experiments were performed in
identical conditions using BS chloroplasts,
except that the running time in the 1D right
was longer. (B) Tricine ⁄ PAGE gels that resu-
lted in 2D were silver stained for the protein
pattern of the protein complexes. The mate-
rial used was BS (left) and MS (right) chloro-
plasts. The position of the Ndh complex in

both gels is indicated.
C. C. Darie et al. Maize Ndh complex
FEBS Journal 272 (2005) 2705–2716 ª 2005 FEBS 2707
suggesting that the Ndh complex splits into a mem-
brane (300 kDa) and a soluble (250 kDa) subcomplex
(Fig. 2A, left).
Similar results were also obtained when MS chloro-
plasts were used as starting material. The intact Ndh
complex had the same molecular mass (520–550 kDa)
(Fig. 2A, right), and split into membrane (300 kDa)
and soluble (250 kDa) subcomplexes (data not shown).
In addition, Ndh antibodies detected the Ndh complex
with a molecular mass of 1000–1100 kDa, suggesting
that it exists in a dimeric form (Fig. 2A, right). How-
ever, due to the low amounts of the Ndh complex in
MS chloroplasts, its polypeptide pattern could not be
observed in the second dimension of the BN ⁄ PAGE
(Fig. 2B, right).
The Ndh complex monomer (520–550 kDa), Ndh com-
plex dimer (1000–1100 kDa) and the 300- and 250-kDa
subcomplexes were also observed in sucrose gradient
and BN ⁄ PAGE, as well as anion exchange and gel fil-
tration experiments (data not shown). Taken together,
these data suggest that the Ndh complex exists as a
monomer and dimer and splits into a membrane and
soluble subcomplexes.
Separation of the Ndh complex by CN
/
PAGE
(1D) and Tricine

/
PAGE (2D) and CN
/
PAGE (1D)
and BN
/
PAGE (2D)
When the subunit composition of a protein complex is
investigated, one problem that can occur in BN ⁄ PAGE
is that two protein complexes with identical molecular
mass may migrate together. To further confirm the
molecular mass and the number of subunits of the
Ndh complex obtained by BN ⁄ PAGE (at least 14 sub-
units), colorless native PAGE (CN ⁄ PAGE; in which
separation of the protein complexes is based on their
internal charge) was used as a prepurification step.
For location and isolation of the Ndh complex, BS
chloroplasts were first separated on CN ⁄ PAGE (1D)
and Tricine ⁄ PAGE (2D) (Fig. 3A). The polypeptide
pattern of the protein complexes from BS chloroplasts
is shown in Fig. 3A (left).
To detect the Ndh complex, the gel that resulted in
2D was electroblotted and incubated with Ndh anti-
bodies (Fig. 3A, right). The polypeptides that reacted
with Ndh antibodies were part of a protein complex,
and corresponded to the second intense band in the
1D CN ⁄ PAGE (Fig. 3A, right). Similar results were
obtained using MS chloroplasts (data not shown).
To further localize, isolate, and characterize the Ndh
complex, the MS and BS chloroplasts were separated

on CN ⁄ PAGE (1D, based on the internal charge of
the protein complexes) and BN ⁄ PAGE (2D, based on
the external charge of the protein complexes and
according to their molecular mass) (Fig. 3B). Based on
previous results, in BN ⁄ PAGE (as a second dimension,
2D), the Ndh complex should correspond with the
second intense band from CN ⁄ PAGE; based on
BN ⁄ PAGE (as a first dimension, 1D) results, it should
have a molecular mass of 520–550 kDa. It should be
located in a square containing the three protein com-
plexes marked a, b and c (Fig. 3B). Indeed, western
1D
-Ndh H
-Ndh K
-Ndh J
-Ndh E
kDa
97-
66-
45-
30-
20-
14-
1D
kDa
2D
1D
1D
-669
-440

-232
232-
440-
669-
kDa
a
a
b
c
b
c
MS
BS
2D
A
B
Fig. 3. Separation of the MS and BS chloro-
plasts by two-dimensional CN ⁄ PAGE.
(A) The BS chloroplasts were separated in
CN ⁄ PAGE (1D, the horizontal bands) and
then in denaturing and reducing conditions
by Tricine ⁄ PAGE (2D). The gel that resulted
from 2D was silver stained (left) or electro-
blotted and immunodecorated with Ndh
antibodies (right). The polypeptides detected
by Ndh antibodies are shown on the right.
(B) The MS (left) and BS (right) chloroplasts
were separated in CN ⁄ PAGE (1D, the hori-
zontal bands) and then in nondenaturing
conditions by BN ⁄ PAGE (2D). The molecular

mass standards and direction of migration
are indicated for both the first (1D) and
second (2D) dimension. The position of the
Ndh complex was detected in the square
containing the protein complexes marked (a)
(b) and (c).
Maize Ndh complex C. C. Darie et al.
2708 FEBS Journal 272 (2005) 2705–2716 ª 2005 FEBS
blotting and silver staining of the in BN ⁄ PAGE (as a
second dimension, 2D) gels confirmed again that the
molecular mass of the Ndh complex is 520–550 kDa
(data not shown). In order to increase the amount of
the Ndh complex for further analysis of its polypeptide
pattern, a three-dimensional preparative isolation of
the Ndh complex was performed.
Three-dimensional preparative isolation of the
Ndh complex from MS chloroplasts: CN
/
PAGE
(1D), BN
/
PAGE (2D) and Tricine
/
PAGE (3D)
Based on the results provided by CN ⁄ PAGE,
BN ⁄ PAGE and western blotting experiments, the thyl-
akoid membranes were separated on CN ⁄ PAGE (1D)
and the second intense band containing the Ndh com-
plex was excised and further separated on BN ⁄ PAGE
(2D). The BN ⁄ PAGE band containing the Ndh com-

plex was excised, reduced, denatured and further separ-
ated on Tricine ⁄ PAGE (3D). The resulting Tricine ⁄
PAGE (3D) gel was further divided into three pieces;
two of them were stained with silver or Coomassie
blue. The third piece was electroblotted and
immunodecorated with Ndh antibodies. A computer-
assisted reconstitution of the initial gel is shown in
Fig. 4A. Both the silver and CBB stained gel pieces
revealed at least 14 visible polypeptides with molecular
masses between 10 and 80 kDa, confirming the results
obtained by BN ⁄ PAGE (1D) and Tricine ⁄ PAGE (2D)
of the BS chloroplasts (Fig. 2B). These experiments
suggest that the Ndh complex contains at least 14 sub-
units, four of them (NdhH, -K, -J and -E) identified by
Ndh antibodies (Fig. 4A). The tentative assignment of
the Ndh subunits (based on their theoretical molecular
mass and western blotting results) is shown in Fig. 4B.
Analysis of the Ndh subunits by mass
spectrometry
To confirm the results obtained by Ndh antibodies, the
gel bands that resulted from BN ⁄ PAGE (1D) and Tri-
cine ⁄ PAGE (2D) or from CN ⁄ PAGE (1D), BN ⁄ PAGE
(2D) and Tricine ⁄ PAGE (3D), and which correspon-
ded to Ndh subunits, were further analyzed by mass
spectrometry (MALDI-TOF-MS). The mass spectro-
metry measurements were submitted to the MASCOT
database, as described in the Methods section. To
avoid obtaining false positive data, our search parame-
ters were reduced to only one fixed modification
(carbamidomethyl-cysteine), one variable modification

(methionine-sulphoxide), a maximum of one missed
cleavage and 100 p.p.m. mass tolerance. The mass
spectra with the identified Ndh polypeptides are shown
in Fig. 5.
SS
-H
-K
-J
-E
Ndh
kDa
97-
66-
45-
30-
20-
14-
CBB
AB
NdhB
NdhD
NdhC
NdhA
NdhF
NdhG
NdhI
75 kDa
51 kDa
23 kDa
?

?
?
WB CBB
Fig. 4. Three-dimensional isolation of the Ndh complex from MS chloroplasts. (A) The MS chloroplasts were separated on CN ⁄ PAGE (1D)
and the band corresponding to the Ndh complex was excised and run in a second BN ⁄ PAGE (2D). The gel piece containing the Ndh complex
was further separated on Tricine ⁄ PAGE (3D). The Tricine ⁄ PAGE gel was divided in three pieces and two of them were silver- (SS) and
Coomassie- (CBB) stained. The third gel piece was electroblotted (WB) and immunodecorated with Ndh antibodies (indicated on the right).
On the left, the apparent molecular mass is shown (kDa). (B) Tentative assignment of the Ndh subunits in the CBB stained Tricine ⁄ PAGE
gel piece already shown in Fig. 4A. The theoretical molecular mass of the Ndh subunits is: NdhA (40 kDa) NdhB (56 kDa), NdhC (14 kDa)
NdhD (56 kDa), NdhE (12 kDa), NdhF (83 kDa), NdhG (18 kDa), NdhH (45 kDa), NdhI (21 kDa), NdhJ (18 kDa), NdhK (29 kDa). The 75, 51
and 23 kDa subunits are assigned as the soluble subcomplex of the Ndh complex. The unassigned polypeptides with a molecular mass of
10, 16 and 22 kDa are marked with question marks (?).
C. C. Darie et al. Maize Ndh complex
FEBS Journal 272 (2005) 2705–2716 ª 2005 FEBS 2709
The MASCOT database search of a trypsin-digested
gel band detected by NdhH antibodies identified the
46-kDa maize NdhH polypeptide with seven peptides
matched, two of them with one missed cleavage. The
marked peaks with m ⁄ z of 1352.75 (calculated, 1352.75),
1045.59 (calculated, 1045.54), 1832.88 (calculated,
1832.84), 1315.65 (calculated, 1315.66), 1568.69 (calcula-
ted, 1568.71), 1937.03 (calculated, 1936.97) and 1946.25
(calculated, 1946.19) from the mass spectrum from
Fig. 5A corresponded to peptides SIIQYLPYVTR,
A
B
C
Fig. 5. Analysis of the Ndh subunits by MALDI-TOF-MS. Spectra of NdhH (A), NdhI (B) and NdhJ (C) are shown. The identified peptides in
each spectrum are marked with an asterisk. The monoisotopic peaks represent the mass ⁄ charge (m ⁄ z) ratio.
Maize Ndh complex C. C. Darie et al.

2710 FEBS Journal 272 (2005) 2705–2716 ª 2005 FEBS
ASGIQWDLR, KIDPYESYNQFDWK, IPGGPYEN
LEAR, AKNPEWNDFEYR (one missed cleavage),
GELGIYLVGDDSLFPWR and IRPPGFINLQILP-
QLVK (one missed cleavage). All of these peptides were
part of the maize NdhH subunit.
Database analysis of a MALDI-TOF-MS measure-
ment of a 21-kDa trypsin-digested gel band (indicated
NdhI in Fig. 4B) identified the maize NdhI poly-
peptide with a molecular mass of 21 kDa, with five
peptides matched, one of them with an oxidized methi-
onine. The peaks with m ⁄ z of 1749.83 (calculated,
1749.92), 1373.62 (calculated, 1373.74), 1482.66 (cal-
culated, 1482.77), 1457.66 (calculated, 1457.74) and
1736.82 (calculated, 1736.92) from the mass spectrum
from Fig. 5B corresponded to peptides YIGQSFII
TLSHTNR, LPITIHYPYEK, VCPIDLPLVDWR
(cysteine modified by iodoacetamide: carbamidometh-
yl-cysteine), HELNYNQIALSR and LPISIMGDY
TIQTIR (methionine oxidized to methionine-sulphox-
ide) identified as part of maize NdhI (21 kDa).
Finally, a MASCOT database search of a trypsin-
digested gel band detected by NdhJ antibodies identi-
fied the 18-kDa maize NdhJ polypeptide, with four
matched peptides (one of them with one missed clea-
vage), which covered 28% of the protein. The peaks
with m ⁄ z of 1189.71 (calculated, 1189.64), 2616.25 (cal-
culated, 2616.13), 1782.80 (calculated, 1782.77) and
1725.86 (calculated, 1725.77) from the mass spectrum
from Fig. 5C corresponded to peptides IPSVFWVWR,

SADFQERESYDMVGISYDNHPR (one missed
cleavage), ESYDMVGISYDNHPR, DYITPNFYEIQ
DAH, which were part of the 18-kDa maize NdhJ
protein.
By using these 2D (BN ⁄ PAGE and Tricine ⁄ PAGE)
and 3D (CN ⁄ PAGE, BN ⁄ PAGE and Tricine ⁄ PAGE)
experiments, we were able to determine that the Ndh
complex contains at least 14 subunits, some of them
identified by Ndh antibodies. Moreover, by combining
the gel electrophoresis methods with mass spectrome-
try, we were able to identify five (NdhE-, H, -I, -J, and
-K) out of 14 Ndh subunits.
Discussion
The minimal bacterial complex I homologous to the
chloroplast Ndh complex contains 14 subunits (Nuo
A–N), with a molecular mass of 550 kDa. Seven sub-
units form a membrane subcomplex and four subunits
form a connecting subcomplex. The remaining three
subunits form a soluble subcomplex, which harbors
the binding and oxidation site for NADH [1,26,27].
The maize chloroplast contains 11 ndh genes enco-
ding 11 polypeptides (NdhA–K) [28]. Seven subunits
(NdhA–G) form the membrane subcomplex, while the
remaining four subunits (NdhH–K) form the connect-
ing subcomplex, both of them homologous to the bac-
terial subcomplexes. The theoretical molecular mass of
the Ndh complex, calculated from its 11 subunits, is
almost 400 kDa. NdhA–G (the membrane subcom-
plex) accounts for 290 kDa and NdhH–K (the con-
necting subcomplex) accounts for 110 kDa.

Previous reports regarding the molecular mass of the
Ndh complex are ambiguous. While some groups
reported that the molecular mass of this complex is
550–580 kDa [2–7], other groups have reported detec-
tion of the Ndh complex with a molecular mass
between 800 and 1000 kDa [8,9].
By using BN ⁄ PAGE and Tricine ⁄ PAGE or
CN ⁄ PAGE, BN ⁄ PAGE and Tricine ⁄ PAGE, we found
that the molecular mass of the Ndh complex from
both MS and BS chloroplasts is 550 kDa. In addition,
we found that the Ndh complex (from both MS and
BS chloroplasts) may be in monomeric (550 kDa) as
well as in dimeric form (1000–1100 kDa). Similar
results were obtained with the semipurified Ndh com-
plex isolated by anion exchange followed by gel filtra-
tion, or by sucrose gradient combined with BN ⁄ PAGE
(data not shown). Our results confirm some previously
reported results [2–7], but disagree with other reports
[8,9], and suggest that the 1000-kDa Ndh complex des-
cribed by these groups was actually a dimeric form.
The observation that the molecular mass of the Ndh
complex monomer is similar in both MS and BS
chloroplast types led us to hypothesize that the archi-
tecture of the Ndh complex in other plastid types is
similar.
We also found that the Ndh complex splits into a
300-kDa subcomplex (corresponding to the membrane
subcomplex, detected by NdhE antibodies) and a
250-kDa subcomplex (detected by NdhH, -J and -K
antibodies). The 250-kDa subcomplex contains NdhH,

-I, -J and -K subunits. However, the theoretical mole-
cular mass of these subunits is 110 kDa, suggesting
that the difference up to 250 kDa may be the electron
input module (the soluble subcomplex), which in the
bacterial Ndh complex contains three polypeptides (75,
51 and 23-kDa subunits). Alternatively, the 250-kDa
subcomplex contains two copies of NdhH, -I, -J, and
-K, as suggested [29].
By using BN ⁄ PAGE and Tricine ⁄ PAGE, we deter-
mined that the Ndh complex contains a minimum of
14 subunits (with a molecular mass between 10 and
70 kDa). Some of them were detected by Ndh anti-
bodies and some by mass spectrometry. Similarly,
using CN ⁄ PAGE, BN ⁄ PAGE, and Tricine ⁄ PAGE, we
also determined that the Ndh complex contains at
C. C. Darie et al. Maize Ndh complex
FEBS Journal 272 (2005) 2705–2716 ª 2005 FEBS 2711
least 14 subunits, partly detected by Ndh antibodies
and mass spectrometry. Since there are only 11 plas-
tidal ndh genes, this suggests that the detected extra
proteins are encoded in the nucleus, and may repre-
sent the electron input module, similar with the bac-
terial 75, 55 and 23-kDa homologue. Indeed, Quiles
and his colleagues [7,9], reported that in oat and bar-
ley, the Ndh complex contains one nuclear-encoded
polypeptide homologous to bacterial 55-kDa protein.
Later, the same group [30], compared the plastidal
Ndh complex and mitochondrial Complex I from the
same plant (barley) by western blotting, and reported
that both complexes contained the electron input

module (the soluble subcomplex) containing polypep-
tides homologous to bacterial 24, 51, and 75-kDa pro-
teins. Quiles and colleagues [30] also suggested that
these nuclear gene products could contain a dual
targeting sequence, which allows them to be targeted
to both mitochondria and chloroplasts. However,
these reports were based only on western blotting
experiments and to confirm this statement, identifi-
cation of these polypeptides in further studies will be
necessary.
Based on the number and the molecular mass of
the polypeptides obtained by BN ⁄ PAGE and Tricine ⁄
PAGE or CN ⁄ PAGE, BN ⁄ PAGE and Tricine ⁄ PAGE,
we suggest that the 250-kDa subcomplex does not
contain contains two copies of NdhH, -I, -J, and -K
polypeptides, as previously reported [29], but contains
the soluble subcomplex (the electron input device).
Our results could explain why the theoretical
molecular mass of the Ndh complex is 400 kDa and its
determined mass is 520–550 kDa. These data could
also explain why the experimental number of Ndh
polypeptides [determined by (1) BN ⁄ PAGE and Tri-
cine ⁄ PAGE and (2) CN⁄ PAGE, BN ⁄ PAGE and Tri-
cine ⁄ PAGE in both MS and BS chloroplasts] is at
least 14, despite the theoretical number of the encoding
genes. Moreover, the number of Ndh subunits
observed in plastids from different plants is close to
our number: at least 15 polypeptides in oat [7], 16
polypeptides in pea [4] and 14 polypeptides in maize
[31]. We also provide evidence that the number of the

polypeptides from the Ndh complex in MS and
C3-type chloroplasts is similar to Ndh proteins from
BS chloroplasts.
Recently, Promeenade et al. [29], found that the cy-
anobacterial Ndh complex contains two extra subunits
(slr1623 and sll1262), unrelated to the subunits of the
minimal bacterial complex I, but homologous to two
nuclear-encoded maize Ndh proteins, as detected in an
Ndh complex preparation by Funk et al. [31]. If we
calculate that the Ndh complex contains 11 plastidal-
encoded subunits, three (mitochondrial-related) nuc-
lear-encoded subunits (the soluble subcomplex), and
two (cyanobacterial-related) nuclear-encoded subunits
(slr1623 and sll1262), we should conclude that the
minimal Ndh complex from higher plants contains at
least 16 subunits.
MALDI-TOF-MS [32,33] is a useful tool for the
analysis and identification of proteins [34–36]. Unfor-
tunately, most groups that have tried to assign the
polypeptide pattern of the plastidal Ndh complex
failed because of the low protein yield obtained for
further protein analysis. Although we overcame this
problem, we were still unsuccessful in the assignment
of all Ndh subunits, probably because of the technical
difficulties inherent in assigning the highly hydropho-
bic membrane subunits.
It should be mentioned that the electron input
device of the Ndh complex from the chloroplast could
be different than the corresponding one from cyano-
bacteria, since the last one contains subunits with a

molecular mass between 9 and 50 kDa [29], compared
with the Ndh subunits, which have molecular masses
between 10 and 70 kDa. In addition, cyanobacteria
contain the protein complexes for both the photosyn-
thetic and respiratory functions on the same mem-
brane.
In conclusion, we demonstrated that one ndhE gene
is expressed in three different plastid types: MS and
BS chloroplasts and ET. Furthermore, we were able to
determine the molecular mass of the Ndh complex
monomer (550 kDa) and dimer (100–1100 kDa). Also,
the Ndh complex splits into a 300-kDa membrane sub-
complex (containing NdhE) and a 250-kDa subcom-
plex (containing NdhH, -J and -K). The 250-kDa
subcomplex contains the connecting subcomplex
(NdhH, -I, -J and -K) in a monomeric form and per-
haps at least three nuclear-encoded subunits. We were
also able to determine that the Ndh complex contains
at least 14 different polypeptides (with a molecular
mass between 10 and 70 kDa), five of them (NdhH, -I,
-J, -K and -E) identified by western blotting and mass
spectrometry. The three-dimensional CN ⁄ PAGE,
BN ⁄ PAGE and Tricine ⁄ PAGE method we have des-
cribed should allow the isolation of large amounts of
pure Ndh complex from maize chloroplasts for further
structural and functional studies. This may include
identification of further Ndh subunits, as well as deter-
mination of the substrate specificity and function of
the Ndh complex.
We hope that further analysis of the Ndh subunits

by N-terminal sequence analysis or mass spectrometry
(MALDI-TOF-MS and MS ⁄ MS) will reveal the
complete subunit composition of this incompletely
Maize Ndh complex C. C. Darie et al.
2712 FEBS Journal 272 (2005) 2705–2716 ª 2005 FEBS
characterized plastidal Ndh complex. Providing more
sequence information will also give us more insights
about the function of the Ndh complex, and will be a
focus of future studies.
Experimental procedures
Materials
Maize (Zea mays, L, Perceval, Deutsche Saatveredlung
Lippstadt Bremen GmbH, Lippstadt, Germany). The
pBluescript KS vector was from Stratagene. The pGEX-6P-
2 vector and ECL immunoblotting kit were from Amer-
sham Pharmacia Biotech (Freiburg, Germany). The antisera
were raised in rabbits at Charles River Deutschland GmbH
(Sulzfeld, Germany). The Protean II cell for native PAGE
was from Bio-Rad (Munich, Germany). Poly(vinylidene
difluoride) membranes were from Immobilon-P (Millipore,
Billerica, MA, USA). Trypsin, horseradish peroxidase con-
jugated to secondary antibody (goat anti-rabbit IgG) and
all other chemicals were from Sigma-Aldrich (Munich,
Germany).
Plant material and isolation of maize chloroplasts
Maize seeds were grown in a green house at 24 °C during
an 18 h photoperiod of white light. All experiments were
carried out with 14-day-old plants. The leaves were harves-
ted 3–5 h after the beginning of the photoperiod. Intact
MS and BS chloroplasts were isolated on a Percoll step

gradient as described [37]. All procedures were performed
at 4 °C and all materials were also kept at this temperature.
The leaves (13–18 g, second leaf, upper 5–10 cm) (after
excising the middle vascular system) were cut into small
pieces 3–5 mm and left for 2–4 h at 4 °C. The leaves were
mixed with 100 mL buffer (350 mm sorbitol; 10 mm
EDTA; 1 mm MgCl
2
;20mm Hepes pH 8.0) and cut in a
mixer for 15 s at speed 3 and passed through a 600 lm
nylon mesh. The solution contained MS chloroplasts and
was centrifuged for 5 min at 6000 r.p.m., at 4 °C, using a
GS3 rotor. The resulting pellet was resuspended in the
same buffer, washed again and applied to a 40 ⁄ 80% Percoll
step gradient. The lower band contained the intact MS
chloroplasts. The procedure was similar for isolation of
ET, except that the maize was grown in complete darkness.
The retained material was mixed two more times (the last
time only with 50 mL buffer and mixed 8 s). The superna-
tant from both steps was passed through a 100-lm nylon
mesh and the retained material was immersed in digestion
buffer [0.35 m sorbitol, 1 mm KH
2
PO
4
,10mm Mes ⁄ KOH
pH 6.0, 0.3% (w ⁄ v) macerozym, 2% (w ⁄ v) cellulase] and
shuttled for 45 min at 25 °C and 120 r.p.m. The BS chloro-
plasts were released from the cells by mechanical treatment
and added to a preformed percoll gradient (30 ⁄ 80%),

followed by centrifugation for 10 min at 4500 r.p.m. using
an HB4 rotor, at 4 °C. The intact chloroplasts (lower band)
were washed with 10 mL buffer for 2 min at 2000 r.p.m.
(HB4 rotor), and the pellet was used as a starting mater-
ial for BN ⁄ PAGE, CN ⁄ PAGE, Tricine ⁄ PAGE and
SDS ⁄ PAGE.
Production of Ndh antibodies
Full-length cDNA was amplified by PCR and every ndh
gene was cloned in the SmaI restriction site of pBluescript
KS, sequenced for PCR errors and transformed into
XL1Blue competent cells. After amplification of the vector
containing the genes (Qiagen midi protocol), the ndh genes
were excised with SalI and XhoI restriction enzymes, puri-
fied and subcloned by T4 ligase into pGEX-6P-2 vector,
already linearized with SalI and dephosphorylated with calf
intestinal phosphatase. The GST–Ndh fusion proteins were
then overexpressed as inclusion bodies in Escherichia coli
(BL21). The expression of the fusion proteins in bacteria
was routinely monitored by western blotting using horse-
radish peroxidase coupled to glutathione-S-transferase
(GST) antibodies. The expressed GST–Ndh proteins were
then purified by preparative SDS ⁄ PAGE, electroeluted
from excised gel bands and used to raise antisera in rabbits.
Native PAGE – CN
/
PAGE and BN
/
PAGE
CN ⁄ BN ⁄ PAGE was carried out in the Protean II cell (Bio-
Rad) following an in-house optimized protocol of the pub-

lished method [38–40]. Briefly, the starting material was run
on a separating gel (gradient 5–13% acrylamide) in both
CN ⁄ PAGE and BN ⁄ PAGE. Compared with CN ⁄ PAGE, in
the cathode buffer of BN ⁄ PAGE, Coomassie blue dye was
used. For transition from CN ⁄ PAGE to BN ⁄ PAGE, the
CN ⁄ PAGE band was excised, oriented horizontally and
run in BN ⁄ PAGE. At first, a special cathode buffer was
used (100 mm glycine, 20 mm bis ⁄ Tris pH 8.1, 0.002%
Coomassie blue) until the protein complexes ran out of the
gel piece, followed by a substitution with the regular
BN ⁄ PAGE cathode buffer.
Tricine
/
PAGE for the second
/
third dimension
Gel lanes of the CN ⁄ PAGE or BN ⁄ PAGE with separated
protein complexes were excised and implanted horizontally
on denaturing gels for resolution of proteins in a second
(CN- ⁄ BN ⁄ PAGE) or a third (CN ⁄ and BN ⁄ PAGE)
dimension. For the separation of proteins with low
molecular mass, the Tricine ⁄ SDS ⁄ PAGE was performed
as described [41]. Before electrophoresis, the gel strips
were incubated with denaturation solution [1% (w ⁄ v)
SDS ⁄ 1% (v ⁄ v) 2-mercaptoethanol] for 2 h under moderate
shaking.
C. C. Darie et al. Maize Ndh complex
FEBS Journal 272 (2005) 2705–2716 ª 2005 FEBS 2713
SDS
/

PAGE and immunoblot analysis
The isolated MS and BS chloroplasts were separated by
denaturing SDS ⁄ PAGE [42]. The gels were stained with
Coomassie dye or electroblotted onto poly(vinylidene diflu-
oride) membranes. The immune reaction was performed by
incubation of the membranes with a mixture of primary
antibodies (NdhE, -H, -J and -K) in a dilution of 1 : 1000.
Before a mixture of Ndh antibodies was used, their individ-
ual specificity was tested. The horseradish peroxidase
conjugated to secondary antibody was used for immunode-
tection, performed by ECL immunoblotting kit according
to the manufacturer’s instructions.
Enzymatic digestion of Ndh subunits
Digestion of gel pieces containing individual Ndh polypep-
tides with trypsin was carried as described [43]. Gel pieces
containing Ndh subunits were incubated with 60% (v ⁄ v)
acetonitrile for 20 min, dried completely in a SpeedVac
evaporator, and rehydrated for 10 min with digestion buffer
(25 mm ammonium bicarbonate, pH 8.0). This procedure
was repeated three times. After drying, gel pieces were
again rehydrated in digestion buffer containing 10 mm
dithiothreitol and incubated for 1 h at 56 °C. Following
reduction, cysteine residues were blocked by replacing the
dithiothreitol solution with 100 mm iodoacetamide in
25 mm ammonium bicarbonate pH 8.0, for 45 min at room
temperature with occasional vortexing. Gel pieces were
dehydrated, dried, and rehydrated twice. Dried gel pieces
were then digested overnight at 37 °C in digestion buffer
containing 15 ngÆlL
)1

trypsin and 5 mm calcium chloride.
When the digestion was complete, the peptides were extrac-
ted twice from gel pieces by addition of 300 lL of 60%
acetonitrile ⁄ 5% formic acid (v ⁄ v ⁄ v) in 25 mm ammonium
bicarbonate, pH 8.0, and shaking for 60–90 min at room
temperature. Solutions containing peptides of Ndh subunits
were dried and used for MALDI-TOF-MS in reflective
mode.
MALDI-TOF-MS analysis of Ndh peptides
The measurements were carried out on a Reflex III TOF
system (Bruker Daltonics, Leipzig, Germany) reflectron,
equipped with a nitrogen laser (337 nm). The dry samples
were dissolved with 2% (v ⁄ v) trifluoroacetic acid. A mix-
ture of nitrocellulose and alpha cyano-4-hydroxycinnamic
acid was used as matrix. After drying, samples were ana-
lyzed either undiluted or at suitable dilutions (1 : 10). After
ionization, the ions were measured in the mass ⁄ charge
range of m ⁄ z ¼ 700–3200 and time-to-mass conversion was
achieved by using external or internal calibration. Peaks
detected by MALDI-TOF-MS corresponded to monoiso-
topic mass ⁄ charge (m ⁄ z) peptides. Calibration differences
for these measurements were generally under 100 p.p.m.
Before analysis of the mass spectra, the peaks that resul-
ted from trypsin autolysis (e.g. m ⁄ z 2163, 2185, 2273), as
well as other peaks observed in the blank sample (peptides
that resulted from trypsin digest of a gel piece containing
no protein) were subtracted.
Analysis of the mass spectra was performed using pep-
tidemass fingerprinting search algorithm from the MAS-
COT search database (). The

parameters used in the MASCOT search were: database
(NCBI, MSDB, OWL, Swissprot and EST), organism (all
entries and Viridiplants), fixed modifications (carbamido-
methyl), variable modifications (oxidation of methionine to
methionine sulfoxide), missed cleavages (0 and 1) and mass
tolerance (100 and 150 p.p.m.).
Acknowledgements
We thank Drs Alisa G. Woods and Ike Woods
(Padure Biomedical Consulting, Brooklyn, USA) for
discussion and editing the manuscript and Dr Ch.
Peters and Patric Hoert (University of Freiburg, Ger-
many) for providing the Reflex III for measurements
and for the mass spectrometry measurements. We also
thank to the Deutsche Forschungsgemeinschaft for
financial support, grant SFB388 ⁄ A1. Dr Costel C.
Darie received support from the Graduiertenkolleg
‘Biochemie der Enzyme’, Freiburg, Germany.
References
1 Friedrich T, Steinmu
¨
ller K & Weiss H (1995) The pro-
ton-pumping respiratory complex I of bacteria and
mitochondria and its homologue in chloroplasts. FEBS
Lett 367, 107–111.
2 Burrows PA, Sazanov LA, Svab Z, Maliga P & Nixon PJ
(1998) Identification of a functional respiratory complex
in chloroplasts through analysis of tobacco mutants con-
taining disrupted plastid ndh genes. EMBO J 17, 868–
876.
3 Guera A, De Nova PG & Sabater B (2000) Identifica-

tion of the Ndh (NAD(P)H-plastoquinone-oxidoreduc-
tase) complex in etioplast membranes of Barley: changes
during photomorphogenesis of chloroplasts. Plant Cell
Physiol 41, 49–59.
4 Sazanov LA, Burrows PA & Nixon PJ (1998) The plastid
ndh genes code for an NADH-specific dehydrogenase:
isolation of a complex I analogue from pea thylakoid
membranes. Proc Natl Acad Sci USA 95, 1319–1324.
5 Sazanov LA, Burrows PA & Nixon PJ (1998) The
chloroplast Ndh complex mediates the dark reduction
of the plastoquinone pool in response to heat stress in
tobacco leaves. FEBS Lett 429, 115–118.
6 Guedeney G, Corneille S, Cuine S & Peltier G (1996)
Evidence for an association of ndhB, ndhJ gene products
Maize Ndh complex C. C. Darie et al.
2714 FEBS Journal 272 (2005) 2705–2716 ª 2005 FEBS
and ferredoxin-NADP-reductase as components of a
chloroplastic NAD(P)H dehydrogenase complex. FEBS
Lett 378, 277–280.
7 Quiles MJ, Molina NC & Cuello J (2002) Isolation of
an NADH dehydrogenase complex not associated to
ferredoxin-NADP oxidoreductase from oat stroma thy-
lakoids. J Plant Physiol 159, 457–464.
8 Elortza F, Asturian JA & Arizmendi JM (1999) Chloro-
plast NADH dehydrogenase from Pisum sativum: char-
acterization of its activity and cloning of ndhK gene.
Plant Cell Physiol 40, 149–154.
9 Quiles MJ, Garcia A & Cuello J (2000) Separation by
blue-native PAGE and identification of the whole
NAD(P)H dehydrogenase complex from barley stroma

thylakoids. Plant Physiol Biochem 38, 225–232.
10 Berger S, Ellersiek U, Westhoff P & Steinmuller K
(1993) Studies on the expression of NDH-H, a subunit
of the NAD(P)H-plastoquinone-oxidoreductase of
higher-plant chloroplasts. Planta 190, 25–31.
11 Steinmetz AA, Castroviejo M, Sayre RT & Bogorad L
(1986) Protein PS II-G. An additional component of PS
II identified through its plastid gene in maize. J Biol
Chem 261, 2485–2488.
12 Wu M, Nie ZQ & Yang J (1989) The 18 kDa protein
that binds to the chloroplast DNA replicative origin is
an iron-sulfur protein related to a subunit of NADH
dehydrogenase. Plant Cell 1, 551–557.
13 Martin M, Casano LM & Sabater B (1996) Identifica-
tion of the product of ndhA gene as a thylakoid protein
synthesized in response to photooxidative treatment.
Plant Cell Physiol 37, 293–298.
14 Catala R, Sabater B & Guera A (1997) Expression of
the plastid ndhF gene product in photosynthetic and
non-photosynthetic tissues of developing Barley seed-
lings. Plant Cell Physiol 38, 1382–1388.
15 Woo KC, Anderson JM, Boardmann NK, Downton
WJS, Osmond CB & Thorne SW (1970) Deficient
photosystem II in agranal bundle sheath chloroplasts of
C
4
plants. Proc Natl Acad Sci USA 67, 18–25.
16 Chapman KSR, Berry JA & Hatch MD (1980) Photosyn-
thetic metabolism in bundle sheath cells of the C4 species
Zea mays: sources of ATP and NADPH and the contri-

bution of PS II. Arch Biochem Biophys 202, 330–341.
17 Leegood RC, Crowther D, Walker DA & Hind G
(1981) Photosynthetic electron transport in the bundle
sheath of maize. FEBS Lett 126, 89–92.
18 Kubicki A, Funk E, Westhoff P & Steinmuller K (1996)
Differential expression of plastome-encoded ndh genes
in mesophyll and bundle-sheath chloroplasts of the C4
plant Sorghum bicolor indicates that the complex
I-homologous NAD(P)H-plastoquinone oxidoreductase
is involved in cyclic electron transport. Planta 199, 276–
281.
19 Shikanai T, Endo T, Hashimoto T, Yamada Y, Asada
K & Yokota A (1998) Directed disruption of the
tobacco ndhB gene impairs cyclic electron flow around
photosystem I. Plant Biol 95, 9705–9709.
20 Joet T, Cournac L, Horvath EM, Medgyesy P & Peltier G
(2001) Increased sensitivity of photosynthesis to anti-
mycin A induced by inactivation of the chloroplast
ndhB gene. Evidence for a participation of the NADH-
dehydrogenase complex to cyclic electron flow around
photosystem I. Plant Physiol 125, 1919–1929.
21 Joet T, Cournac L, Peltier G & Havaux M (2002)
Cyclic electron flow around PS I in C (3) plants. In vivo
control by the redox state of chloroplasts and involve-
ment of the NADH-dehydrogenase complex. Plant
Physiol 128, 760–769.
22 Horvath EM, Peter SO, Joet T, Rumeau D, Cournac L,
Horvath GV, Kavanagh TA, Schafer C, Peltier G &
Medgyesy P (2000) Targeted inactivation of the plastid
ndhB gene in tobacco results in an enhanced sensitivity

of photosynthesis to moderate stomatal closure. Plant
Physiol 123, 1337–1350.
23 Kofer W, Koop HU, Wanner G & Steimu
¨
ller K
(1998) Mutagenesis of the genes encoding subunits A,
C, H, I, J and K of the plastid NAD(P)H-plastoqui-
none-oxidoreductase in tobacco by polyethylene
glycol-mediated plastome transformation. Mol Gen
Genet 258, 166–173.
24 Casano LM, Zapata JM, Martin M & Sabater B (2000)
Chlororespiration and poising of cyclic electron trans-
port: plastoquinone as electron transporter between
thylakoid NADH dehydrogenase and peroxidase. J Biol
Chem 275, 942–948.
25 Pieulle L, Guedeney G, Cassier-Chauvat C, Jeanjean R,
Chauvat F & Peltier G (2000) The gene encoding the
NdhH subunit of type 1 NAD(P)H dehydrogenase is
essential to survival of synechocystis PCC6803. FEBS
Lett 487, 272–276.
26 Friedrich T (1998) The NADH: ubiquinone oxidoreduc-
tase (complex I) from Escherichia coli. Biochem Biophys
Acta 1364, 134–146.
27 Friedrich T & Scheide D (2000) The respiratory com-
plex I of bacteria, archaea and eucarya and its module
common with membrane-bound multisubunit hydroge-
nases. FEBS Lett 479, 1–5.
28 Maier RM, Neckermann K, Igloi GL & Ko
¨
ssel H

(1995) Complete sequence of the maize chloroplast
genome: gene content, hotspots of divergence and fine
tuning of genetic information by transcript editing.
J Mol Biol 251, 614–628.
29 Prommeenate P, Lennon AM, Markert C, Hippler M &
Nixon PJ (2004) Subunit composition of NDH-1 com-
plexes of Synechocystis sp. PCC 6803: identification of
two new ndh gene products with nuclear-encoded
homologues in the chloroplast Ndh complex. J Biol
Chem 279, 28165–28173.
30 Quiles MJ, Garcı
´
a A & Cuello J (2003) Comparison of
the thylakoidal NAD(P)H dehydrogenase complex and
C. C. Darie et al. Maize Ndh complex
FEBS Journal 272 (2005) 2705–2716 ª 2005 FEBS 2715
the mitochondrial complex I separated from barley
leaves by blue-native PAGE. Plant Sci 164, 541–547.
31 Funk E, Scha
¨
fer E & Steinmu
¨
ller K (1999) Characteri-
zation of the complex I-homologous NAD(P)H-plasto-
quinone-oxidoreductase (NDH-complex) of maize
chloroplasts. J Plant Physiol 154, 16–23.
32 Kaufmann R (1995) Matrix-assisted laser desorption ⁄
ionization (MALDI) mass spectrometry: a novel
analytical tool in molecular biology and biotechnology.
J Biotechnol 41, 155–175.

33 Hillenkamp F, Karas M, Beavis RC & Chait BT (1991)
Matrix-assisted laser desorption ⁄ ionization mass spectr-
ometry of biopolymers. Anal Chem 63, 2791–2800.
34 Patterson SD (1995) Matrix-assisted laser-desorp-
tion ⁄ ionization mass spectrometric approaches for the
identification of gel-separated proteins in the 5–50 pmol
range. Electrophoresis 16, 1104–1114.
35 Shevchenko A, Wilm M, Vorm O & Mann M (1996)
Mass spectrometric sequencing of proteins from silver-
stained polyacrylamide gels. Anal Chem 68, 850–858.
36 Wilm M, Shevchenko A, Houthave T, Breit S, Schwei-
gerer L, Fotsis T & Mann M (1996) Femtomole sequen-
cing of proteins from polyacrylamide gels by nano
electrospray mass spectrometry. Nature 379, 466–469.
37 Kubicki A, Steinmu
¨
ller K & Westhoff P (1994) Differ-
ential transcription of plastome-encoded genes in the
mesophyll and bundle-sheath chloroplasts of the mono-
cotyledonous NADP-malic enzyme-type C4 plants
maize and Sorghum. Plant Mol Biol 25, 669–679.
38 Scha
¨
gger H & von Jagow G (1991) Blue native electro-
phoresis for isolation of membrane protein complexes in
enzymatic active form. Anal Biochem 199, 223–231.
39 Scha
¨
gger H, Cramer WA & von Jagow G (1994) Analy-
sis of molecular masses and oligomeric states of protein

complexes by blue native electrophoresis and isolation
of membrane protein complexes by two-dimensional
native electrophoresis. Anal Biochem 217, 220–230.
40 Ku
¨
gler M, Ja
¨
nsch L, Kruft V, Schmitz UK & Braun HP
(1997) Analysis of the chloroplast protein complexes
by blue-native polyacrylamide gel electrophoresis
(BN-PAGE). Photosynthesis Res 53, 35–44.
41 Scha
¨
gger H & von Jagow G (1987) Tricine-sodium
dodecyl sulfate-polyacrylamide gel electrophoresis
for the separation of proteins in the range from 1 to
100 kDa. Anal Biochem 166, 368–379.
42 Laemmli UK (1970) Cleavage of structural proteins
during the assembly of the head of bacteriophageT4.
Nature (Lond) 227, 680–685.
43 Darie CC, Biniossek ML, Jovine L, Litscher ES &
Wassarman PM (2004) Structural characterization of fish
egg vitelline envelope proteins by mass spectrometry.
Biochemistry 43, 7459–7478.
Maize Ndh complex C. C. Darie et al.
2716 FEBS Journal 272 (2005) 2705–2716 ª 2005 FEBS