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: costel.darie@mssm.edu (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 (http://www.matrixscience.com). 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. 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