Analysis of wheat mitochondrial complex I purified by a one-step immunoaffinity chromatography
Introduction
The proton-pumping NADH:ubiquinone oxidoreductase or complex I plays a key role in the oxidation of NADH at the entry point in the respiratory chain. This enzyme has been described in the mitochondrial membrane of aerobic eukaryotes like mammals [1] or Neurospora crassa [2] and the membrane-associated equivalent NADH:quinone oxidoreductase has been found in procaryotes such as Paracoccus denitrifians and Escherichia coli [3]. However, many differences have been found between polypeptide compositions and molecular masses of these enzymes. In mammals, complex I consists of about 40 subunits for an estimated molecular mass of around 900 kDa, whereas in N. crassa this enzyme is composed of 32 polypeptides leading to a molecular mass of around 700 kDa. The bacterial NADH:quinone oxidoreductase consists of only 14 subunits. Five iron-sulphur (FeS) clusters have been found in the bacterial enzyme whereas the presence of six has been demonstrated in beef.
To date, the mitochondrial complex I has been purified from only two plant species: broad bean (Vicia faba) [4] and potato (Solanum tuberosum) [5], after solubilization by detergent treatment and successive chromatographic steps including sucrose gradient, gel filtration and hydroxyapatite chromatography. In potato, 32 subunits were recorded among which five subunits were characterised by cross-reactions with heterologous antibodies or N-terminal microsequencing.
Intracellular localisation of the genes that encode complex I subunits can be nuclear or mitochondrial. In mammals and in N. crassa, seven genes are encoded by the mitochondrial DNA (nad1, nad2, nad3, nad4, nad4L, nad5, nad6) [6], [7] whereas the remaining subunits are nuclear-encoded and exported into the mitochondrion. Analysis of the gene content of the mitochondrial DNA of higher plants has shown that, in addition to the NAD1-6 polypeptides, it encodes at least two subunits of complex I which are nuclear-encoded in other organisms, NAD7 [8] and NAD9 [9]. The corresponding subunits of animal complex I, respectively the 49 and 31 kDa subunits, are located in the hydrophilic IP (iron-protein) sub-complex containing two FeS centers [10] whereas all the mitochondrial-encoded subunits belong to the hydrophobic sub-complex (complex Iβ). The presence of nad7 and nad9 genes in the mitochondrial DNA of wheat raises the possibility that other unknown subunits of complex I may also be encoded and expressed in the mitochondria of higher plants.
In man, a number of mutations or deletions affecting mitochondrial-encoded genes of complex I are the cause of severe diseases [1]. Similarly, in plants, DNA recombinations which are frequent in the mitochondrial genomes [11] induce the appearance of specific diseases. This is the case for the maize mutant named NCS2 (non- chromosomal stripe) where a nad4-nad7 chimeric mitochondrial gene is associated with reduced complex I function [12]. In tobacco, a large deletion of the mitochondrial genome resulted in the loss of expression of nad1 and in the alteration of the respiratory function [13]. Such defects in mitochondrial nad gene expression result in abnormally small plants which show a male-sterile phenotype.
Thorough analysis of plant complex I composition and biogenesis requires a high-yield preparation of this enzyme. Therefore, we developed a immunoaffinity method to isolate the whole complex I in non-denaturating conditions, taking advantage of the availability of a serum raised against NAD9, a hydrophilic subunit of the IP fraction [9]. We show that wheat mitochondrial complex I, the first complex I to be purified in monocots, exhibits around 30 subunits of which 12 were shown to have homologies with nuclear- and mitochondrial-encoded complex I subunits of other organisms. As shown by in organello synthesis, only nine of the wheat complex I subunits are encoded by the mitochondrial genome.
Section snippets
Preparation of wheat mitochondrial membranes and membrane complexes
Mitochondrial membranes were directly prepared from commercial wheat germ obtained from an industrial mill (Grand Moulins de Strasbourg, France) as described previously [14] with the following modifications: i) extraction buffer was 0.4 M sucrose, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1 mM EGTA, 0.5 mM PMSF; ii) the last centrifugation at 48000 g was for 1 h and the final resuspension buffer was 25 mM Tris-HCl, pH 7.8, 0.2 M NaCl, 1 mM EDTA, 0.1 mM EGTA, 3% Triton X-100 and 0.5 mM
Separation and analysis of wheat mitochondrial membrane complexes by blue native electrophoresis and detection of NAD9 subunit in complex I
Biochemical methods for purification of plant mitochondrial complex I that have been previously reported, relied essentially on successive chromatographic steps to separate the membrane complexes released from intact mitochondria. Therefore, these methods led to low yields of purified complex I and thus did not allow further extensive investigations. We developed a novel approach based on a one-step immunoaffinity procedure to purify the mitochondrial complex I from a crude mitochondrial
Discussion
In this paper, we analyse a mitochondrial membrane complex purified by a one-step immunoaffinity procedure. Antibodies raised against a hydrophilic subunit located in the peripheral arm of the wheat mitochondrial complex I, NAD9 [9] were first tested for their ability to recognise the whole complex in its native form in non-denaturing gels. In our case, mitochondrial membrane complexes were prepared from wheat germ, a material suitable for direct extraction of mitochondrial membranes, thus
Acknowledgements
Dr. Nicole Chaubet-Gigot is greatly acknowledged for critical reading and helpful comments on the manuscript. We thank Drs. R. de Paepe, J.E. Walker, T. Yagi and J.M. Gualberto for generous gifts of antibodies.
References (32)
The bacterial energy transducing NADH quinone oxidoreductases
Biochim. Biophys. Acta
(1993)- et al.
Purification of the NADH:ubiquinone oxidoreductase (complex-I) of the respiratory chain from the inner mitochondrial membrane of Solanum tuberosum
J. Biol. Chem.
(1994) - et al.
Molecular genetic studies of Complex-I in Neurospora crassa, Aspergillus niger and Escherichia coli
Biochim. Biophys. Acta
(1992) - et al.
The maize mitochondrial genome: dynamic, yet functional
Trends Genet.
(1995) - et al.
Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form
Anal. Biochem.
(1991) - et al.
Gene organization deduced from the complete sequence of liverwort Marchantia polymorpha mitochondrial DNA. A primitive form of plant mitochondrial genome
J. Mol. Biol.
(1992) The NADH-ubiquinone oxidoreductase (Complex-I) of respiratory chains
Q. Rev. Biophys.
(1992)- et al.
The respiratory-chain NADH dehydrogenase (complex I) of mitochondria
Eur. J. Biochem.
(1991) - et al.
Purification and preliminary characterization of mitochondrial Complex-I (NADH-ubiquinone reductase) from broad bean (Vicia faba)
Plant Physiol.
(1993) - et al.
Sequence and organization of the human mitochondrial genome
Nature
(1981)
The NADH dehydrogenase subunit 7 gene is interrupted by four group II introns in the wheat mitochondrial genome
Mol. Gen. Genet.
Higher plant mitochondria encode an homologue of the nuclear-coded 30 kDa subunit of bovine mitochondrial complex I
Eur. J. Biochem.
Isolation of iron-sulfur-containing peptides of NADH:ubiquinone oxydoreductase
Methods Enzymol.
The maize NCS2 abnormal growth mutant has a chimeric nad4-nad7 mitochondrial gene and is associated with reduced complex I function
Genetics
Lack of mitochondrial and nuclear-encoded subunits of complex I and alteration of the respiratory chain in Nicotiana sylvestris mitochondrial deletion mutants
Proc. Natl. Acad. Sci. USA
Structurally unique plant cytochrome c oxidase isolated from wheat germ, a rich source of plant mitochondrial enzymes
Biochemistry
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2011, PhytochemistryCitation Excerpt :For instance, complex III of the respiratory chain includes both subunits of the mitochondrial processing peptidase (MPP) in plants (Braun et al., 1992). Complex I has been biochemically characterized for several different plant species (Combettes and Grienenberger, 1999; Herz et al., 1994; Jänsch et al., 1996; Leterme and Boutry, 1993; Rasmusson et al., 1994; Trost et al., 1995). Electron microscopic (EM) studies recently revealed a very special shape of Arabidopsis complex I because it includes a second matrix-exposed domain which is attached to the membrane arm at a central position (Dudkina et al., 2005).