Purification and Preliminary Characterization of Mitochondrial Complex I (NADH:Ubiquinone Reductase) from Broad Bean (Vicia faba L.)

Leterme, S.; Boutry, Marc

doi:10.1104/pp.102.2.435

Cited by 69 publications

(42 citation statements)

References 39 publications

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“…After each chromatography step, about half of the applied NADH:FeCN activity was lost, with even more (about 80%) lost after the sizeexclusion column, possibly due to the delipidation of the enzyme (22). To verify that activity is not fractionated away, all eluted fractions, including the 1 M NaCl wash, were assayed (Figs.…”

Section: Resultsmentioning

confidence: 99%

The plastid ndh genes code for an NADH-specific dehydrogenase: Isolation of a complex I analogue from pea thylakoid membranes

Sazanov

Burrows

Nixon

1998

Proc. Natl. Acad. Sci. U.S.A.

193

125

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The plastid genomes of several plants contain ndh genes-homologues of genes encoding subunits of the proton-pumping NADH:ubiquinone oxidoreductase, or complex I, involved in respiration in mitochondria and eubacteria. From sequence similarities with these genes, the ndh gene products have been suggested to form a large protein complex (Ndh complex); however, the structure and function of this complex remains to be established. Herein we report the isolation of the Ndh complex from the chloroplasts of the higher plant Pisum sativum. The purification procedure involved selective solubilization of the thylakoid membrane with dodecyl maltoside, followed by two anion-exchange chromatography steps and one size-exclusion chromatography step. The isolated Ndh complex has an apparent total molecular mass of approximately 550 kDa and according to SDS͞PAGE consists of at least 16 subunits including NdhA, NdhI, NdhJ, NdhK, and NdhH, which were identified by N-terminal sequencing and immunoblotting. The Ndh complex showed an NADH-and deamino-NADH-specific dehydrogenase activity, characteristic of complex I, when either ferricyanide or the quinones menadione and duroquinone were used as electron acceptors. This study describes the isolation of the chloroplast analogue of the respiratory complex I and provides direct evidence for the function of the plastid Ndh complex as an NADH:plastoquinone oxidoreductase. Our results are compatible with a dual role for the Ndh complex in the chlororespiratory and cyclic photophosphorylation pathways.

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Section: Resultsmentioning

confidence: 99%

The plastid ndh genes code for an NADH-specific dehydrogenase: Isolation of a complex I analogue from pea thylakoid membranes

Sazanov

Burrows

Nixon

1998

Proc. Natl. Acad. Sci. U.S.A.

193

125

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“…4 a and b), allowing collection of sufficient amount for microsequencing. It showed significant homology with the 39-to 40-kDa complex I subunit, nucleus-encoded in all species thus far studied (25)(26)(27)(28), especially with the related species S. tuberosum (Fig. 5).…”

Section: Cms Mitochondria Are Deficient In Nad7 Nad9 and A Nucleus-mentioning

confidence: 91%

Lack of mitochondrial and nuclear-encoded subunits of complex I and alteration of the respiratory chain in Nicotiana sylvestris mitochondrial deletion mutants

Gutierres

Sabar

Lelandais

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

201

172

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We previously have shown that Nicotiana sylvestris cytoplasmic male sterile (CMS) mutants I and II present large mtDNA deletions and that the NAD7 subunit of complex I (the main dehydrogenase of the mitochondrial respiratory chain) is absent in CMS I. Here, we show that, despite a large difference in size in the mtDNA deletion, CMS I and II display similar alterations. Both have an impaired development from germination to f lowering, with partial male sterility that becomes complete under low light. Besides NAD7, two other complex I subunits are missing (NAD9 and the nucleus-encoded, 38-kDa subunit), identified on twodimensional patterns of mitochondrial proteins. Mitochondria isolated from CMS leaves showed altered respiration. Although their succinate oxidation through complex II was close to that of the wild type, oxidation of glycine, a priority substrate of plant mitochondria, was significantly reduced. The remaining activity was much less sensitive to rotenone, indicating the breakdown of Complex I activity. Oxidation of exogenous NADH (coupled to proton gradient generation and partly sensitive to rotenone) was strongly increased. These results suggest respiratory compensation mechanisms involving additional NADH dehydrogenases to complex I. Finally, the capacity of the cyanide-resistant alternative oxidase pathway was enhanced in CMS, and higher amounts of enzyme were evidenced by immunodetection.

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“…The data presented in Tables 3 and 4 indicate that the absence of nd4 and the 3Ј end of nd5 in all dum24 mitochondrial DNA copies is correlated with the loss of complex I activity. In Neurospora crassa , mammals (Walker, 1992), and various higher plants ( Leterme and Boutry, 1993;Herz et al, 1994;Rasmusson et al, 1994), the mitochondrial complex I enzyme is by far the largest complex among the proton translocating enzymes of mitochondria. It comprises at least 30 different subunits, most of which are encoded by nuclear genes and imported from the cytoplasm.…”

Section: Discussionmentioning

confidence: 99%

“…Because a wall-less dum24 strain was not available for this study, NADH:ubiquinone oxidoreductase activity was assayed on membrane-enriched fractions by using duroquinone as an electron acceptor (Leterme and Boutry, 1993). The cells were cultured under light in 300 mL of TAP medium and collected by centrifugation (700g for 10 min) at late-exponential phase.…”

Section: Measurement Of Nadh:ubiquinone Oxidoreductase Activitymentioning

confidence: 99%

Alteration of Dark Respiration and Reduction of Phototrophic Growth in a Mitochondrial DNA Deletion Mutant of Chlamydomonas Lacking cob, nd4, and the 3′ End of nd5

Duby

Matagne

1999

Plant Cell

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We describe here a new type of mitochondrial mutation ( dum24 ; for dark uniparental minus inheritance) of the unicellular photosynthetic alga Chlamydomonas reinhardtii . The mutant fails to grow under heterotrophic conditions and displays reduced growth under both photoautotrophic and mixotrophic conditions. In reciprocal crosses between mutant and wild-type cells, the meiotic progeny only inherit the phenotype of the mating-type minus parent, indicating that the dum24 mutation exclusively affects the mitochondrial genome. Digestion with various restriction enzymes followed by DNA gel blot hybridizations with specific probes demonstrated that dum24 cells contain four types of altered mitochondrial genomes: deleted monomers lacking cob , nd4 , and the 3 Ј end of the nd5 gene; deleted monomers deprived of cob , nd4 , nd5 , and the 5 Ј end of the cox1 coding sequence; and two types of dimers produced by end-to-end fusions between monomers similarly or differently deleted. Due to these mitochondrial DNA alterations, complex I activity, the cytochrome pathway of respiration, and presumably, the three phosphorylation sites associated with these enzyme activities are lacking in the mutant. The low respiratory rate of the dum24 cells results from the activities of rotenoneresistant NADH dehydrogenase, complex II, and alternative oxidase, with none of these enzymes being coupled to ATP production. To our knowledge, this type of mitochondrial mutation has never been described for photosynthetic organisms or more generally for obligate aerobes.

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Purification and Preliminary Characterization of Mitochondrial Complex I (NADH:Ubiquinone Reductase) from Broad Bean (Vicia faba L.)

Cited by 69 publications

References 39 publications

The plastid ndh genes code for an NADH-specific dehydrogenase: Isolation of a complex I analogue from pea thylakoid membranes

The plastid ndh genes code for an NADH-specific dehydrogenase: Isolation of a complex I analogue from pea thylakoid membranes

Lack of mitochondrial and nuclear-encoded subunits of complex I and alteration of the respiratory chain in Nicotiana sylvestris mitochondrial deletion mutants

Alteration of Dark Respiration and Reduction of Phototrophic Growth in a Mitochondrial DNA Deletion Mutant of Chlamydomonas Lacking cob, nd4, and the 3′ End of nd5

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