Transmembrane organization of mitochondrial NADH dehydrogenase as revealed by radiochemical labelling and cross-linking

Patel, S.; Cleeter, M. W. J.; Ragan, C I

doi:10.1042/bj2560529

Cited by 14 publications

(7 citation statements)

References 17 publications

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“…This is clearly indicated by the presence of several low-Mr products containing 'flavoprotein' or 'iron-protein' subunits whose other constituent must be a subunit of the hydrophobic domain. Further evidence came from the TID experiments and are consistent with our view [1,18] that the larger 'flavoprotein' and 'iron-protein' subunits are partially surrounded by subunits of the hydrophobic domain.…”

Section: Discussionsupporting

confidence: 84%

“…In the following paper [18] we describe the use of cross-linking agents to study the sidedness of some of the Complex I subunits.…”

Section: Discussionmentioning

confidence: 99%

“…In the present paper we describe the preparation of antisera to several other subunits and their use in analysing the products of cross-linking. In the following paper [18], we describe the use of impermeable crosslinking agents to study the topology of Complex I in the mitochondrial membrane.…”

Section: Introductionmentioning

confidence: 99%

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Structural studies on mitochondrial NADH dehydrogenase using chemical cross-linking

Patel

Ragan

1988

Biochemical Journal

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The structure of bovine heart mitochondrial NADH dehydrogenase was investigated by cross-linking constituent subunits with disuccinimidyl tartrate, (ethylene glycol)yl bis(succinimidyl succinate) and dimethyl suberimidate. Cross-linked products were identified by Western blotting with monospecific antisera to nine subunits of the enzyme. Cross-links between subunits within the flavoprotein, iron-protein and hydrophobic domains of the enzyme were identified. Cross-linking between the 75 kDa iron-protein-domain subunit and the 51 kDa flavoprotein-domain subunit was modulated by the substrate NADH. Cross-linking of subunits of the iron-protein and flavoprotein domains to constituents of the hydrophobic domain was also found. This was further substantiated by photolabelling subunits of the latter region, which were in contact with the membrane lipid, with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. One such subunit of Mr 19,000 could be cross-linked to components of the iron-protein domain.

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Section: Discussionsupporting

confidence: 84%

“…In the following paper [18] we describe the use of cross-linking agents to study the sidedness of some of the Complex I subunits.…”

Section: Discussionmentioning

confidence: 99%

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Structural studies on mitochondrial NADH dehydrogenase using chemical cross-linking

Patel

Ragan

1988

Biochemical Journal

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“…5). This observation is consistent with the evidence from radiolabeling and crosslinking experiments (26) and from immunochemical experiments (27) that indicates that this subunit is not transmembranous but is only partially embedded in the membrane, being exposed largely on the matrix side of the membrane.…”

Section: Discussionsupporting

confidence: 80%

cDNA-derived amino acid sequence of the NADH-binding 51-kDa subunit of the bovine respiratory NADH dehydrogenase reveals striking similarities to a bacterial NAD(+)-reducing hydrogenase.

Patel

Aebersold

Attardi

1991

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

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A AgtlO bovine brain and a Agtll bovine heart cDNA library were screened with oligonucleotide probes corresponding to partial protein sequences directly determined from the isolated 51-kDa subunit of the bovine respiratorychain NADH dehydrogenase. Clones were isolated that encode a protein of 464 amino acids containing all the 11 partial tryptic peptide sequences determined from the 51-kDa subunit. The size and amino acid composition ofthis protein agree with those determined for the purified 51-kDa subunit. Furthermore, this protein contains a putative NADH-binding domain, a possible FMN-binding site, and a putative binding site for an iron-sulfur duster. The above evidence indicates that the cloned protein is the 51-kDa subunit or its precursor. A search for sequence similarity with proteins in the Protein Identification Resource data base has revealed that the 51-kDa subunit has 32% amino acid sequence identity with a major portion of the a subunit of the soluble NAD+-reducing hydrogenase from Akaligenes eutrophus. In particular, there are three segments of high sequence similarity (70-88%) between the two proteins which correspond to the three ligand-binding sites.The NADH:ubiquinone oxidoreductase or rotenone-sensitive NADH dehydrogenase (EC 1.6.5.3) is the first enzyme of the mitochondrial respiratory chain (for a review see ref. 1). The molecular genetic analysis of this enzyme complex (complex I) started a few years ago with the discovery that 7 of the -25 subunits of the human enzyme (2, 3) and 6 of the -22 subunits of the Neurospora crassa enzyme (4) are encoded in mitochondrial DNA. Recent efforts have been directed toward the cloning and structural analysis of nuclear-encoded subunits of the flavoprotein and iron-protein fragments of the enzyme. Of the 3 polypeptides that constitute the bovine flavoprotein fragment-i.e., the 51-kDa, 24-kDa, and 9-kDa subunits (1, 5)-the cDNA for the 24-kDa subunit has already been cloned (6, 7). Similarly, of the 6 polypeptides identified in the ironprotein fragment (1), the cDNAs for the 49-kDa (8) and the 75-kDa (9) subunit have been cloned.In the present work, the cDNA for the 51-kDa subunit of the flavoprotein fragment has been cloned by screening cDNA libraries with oligonucleotides corresponding to partial protein sequences directly determined from the isolated subunit.* A search for sequence similarities with proteins in the Protein Identification Resource data base has unexpectedly revealed the existence in the 51-kDa subunit of regions strikingly similar to segments of the soluble NAD+-reducing hydrogenase of Alcaligenes eutrophus (10). MATERIALS AND METHODSIsolation and Sequence Analysis of the 51-kDa Subunit. Complex I was isolated from bovine heart mitochondria according to Hatefi et al. (11) and resolved by perchlorate into the flavoprotein fragment, the iron-protein fragment, and the hydrophobic protein fragment, as described (12). Since preliminary tests indicated that the NH2 terminus of the 51-kDa subunit was blocked, the sequence of internal...

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“…As expected, in the mitochondrial matrix we also identified "soluble" subunits of the electron transport chain that are well known to be exposed on the matrix face of the inner membrane and could have been partially removed from the membrane upon sonication (26,27). This is only one of the possible explanations for the identification of small soluble polypeptides of cytochrome c oxidase (28). Indeed the matrix fraction could reasonably contain inner membrane vesicles as partial crosscontamination is unavoidable in mitochondrial preparation with the presently available isolation procedures.…”

Section: Survey Of Rat Mitochondrial Proteins-mentioning

confidence: 99%

Quantitative Proteomic Comparison of Rat Mitochondria from Muscle, Heart, and Liver

Forner

Foster

Campanaro

et al. 2006

Molecular & Cellular Proteomics

261

232

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Mitochondria, through oxidative phosphorylation, are the primary source of energy production in all tissues under aerobic conditions. Although critical to life, energy production is not the only function of mitochondria, and the composition of this organelle is tailored to meet the specific needs of each cell type. As an organelle, the mitochondrion has been a popular subject for proteomic analysis, but quantitative proteomic methods have yet to be applied to tease apart subtle differences among mitochondria from different tissues or muscle types. Here we used mass spectrometry-based proteomics to analyze mitochondrial proteins extracted from rat skeletal muscle, heart, and liver tissues. Based on 689 proteins identified with high confidence, mitochondria from the different tissues are qualitatively quite similar. However, striking differences emerged from the quantitative comparison of protein abundance between the tissues. Furthermore we applied similar methods to analyze mitochondrial matrix and intermembrane space proteins extracted from the same mitochondrial source, providing evidence for the submitochondrial localization of a number of proteins in skeletal muscle and liver. Several proteins not previously thought to reside in mitochondria were identified, and their presence in this organelle was confirmed by protein correlation profiling. Hierarchical clustering of microarray expression data provided further evidence that some of the novel mitochondrial candidates identified in the proteomic survey might be associated with mitochondria. These data reveal several important distinctions between mitochondrial and submitochondrial proteomes from skeletal muscle, heart, and liver Different mammalian tissues have distinct energy needs, and mitochondria morphology can vary widely, although the structure is not exclusively linked to respiration (1). Morphology and structure of mitochondria in mammalian skeletal muscle, heart, and liver are very different (2). In skeletal muscle mitochondria are distributed between sarcomeres and tightly embedded in the microfilaments (F-actin) and microtubules. Indeed the intimate association of mitochondria with the myofilaments minimizes diffusion distance and facilitates conversion of chemical energy to mechanical work. Mammalian skeletal muscle is a highly specialized tissue composed of fibers with a diverse range of properties. The aerobic type I fibers contain many mitochondria and rely on oxidative phosphorylation for ATP. Type IIa fibers are very rich in small mitochondria and are characterized by a high capacity for oxidative ATP generation. These mitochondria can only oxidize glycolytic products unlike those in type I fibers that can also utilize fatty acids and ketones. Type IIb fibers rely upon anaerobic glycolysis, contain few mitochondria, and have high levels of glycogen (3). Given these functional differences it is reasonable to assume that mitochondrial and mitochondria-related proteins are present in different amounts depending on the specific energy requirement...

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Transmembrane organization of mitochondrial NADH dehydrogenase as revealed by radiochemical labelling and cross-linking

Cited by 14 publications

References 17 publications

Structural studies on mitochondrial NADH dehydrogenase using chemical cross-linking

Structural studies on mitochondrial NADH dehydrogenase using chemical cross-linking

cDNA-derived amino acid sequence of the NADH-binding 51-kDa subunit of the bovine respiratory NADH dehydrogenase reveals striking similarities to a bacterial NAD(+)-reducing hydrogenase.

Quantitative Proteomic Comparison of Rat Mitochondria from Muscle, Heart, and Liver

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