ABSTRACT
The respiratory complex I of mitochondria consists of some 40 different subunits which form an L-shaped structure. Perpendicular to a hydrophobic arm embedded in the inner mitochondrial membrane a peripheral arm protrudes into the matrix. Assembly of the complex as studied in the fungus Neurospora crassa involves the formation of discrete intermediates. The matrix arm and the membrane arm are formed independently of each other and are joined in the course of assembly. The membrane arm itself is formed by association of two assembly intermediates, a smaller of 200 kDa and a larger of 350 kDa. The latter is associated with two extra proteins of 84 and 30 kDa which are not constituent parts of mature complex I. Their primary structures show no similarity to known proteins. Mutants generated by disrupting the genes of either of the two proteins accumulate the matrix arm of complex I and the small membrane arm assembly intermediate, but are incapable of forming the large intermediate. In the wild-type, the extra proteins exclusively associate with the large membrane arm assembly intermediate. Pulse-chase labelling experiments showed that the two proteins are repeatedly involved in many assembly cycles of the intermediate. These results indicate that the two proteins are novel chaperones specific for complex I membrane arm assembly.
Subject(s)
Mitochondria/enzymology , Molecular Chaperones/metabolism , NADH, NADPH Oxidoreductases/metabolism , Amino Acid Sequence , Cloning, Molecular , Electron Transport Complex I , Escherichia coli , Molecular Chaperones/genetics , Molecular Chaperones/isolation & purification , Molecular Sequence Data , Neurospora crassa/enzymology , Neurospora crassa/genetics , Neurospora crassa/metabolism , Polymerase Chain Reaction , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
NADH:ubiquinone oxidoreductase, the respiratory chain complex I of mitochondria, is an assembly of some 25 nuclear-encoded and 7 mitochondrially encoded subunits. The complex has an overall L-shaped structure formed by a peripheral arm and an elongated membrane arm. The peripheral arm containing one FMN and at least three iron-sulphur clusters constitutes the NADH dehydrogenase segment of the electron pathway. The membrane arm with at least one iron-sulphur cluster constitutes the ubiquinone reducing segment. We are studying the assembly of the complex in Neurospora crassa. By disrupting the gene of a nuclear-encoded subunit of the membrane arm a mutant was generated that cannot form complex I. The mutant rather pre-assembles the peripheral arm with all redox groups and the ability to catalyse NADH oxidation by artificial electron acceptors. The final assembly of the membrane arm is blocked in the mutant leading to accumulation of complementary assembly intermediates. One intermediate is associated with a protein that is not present in the fully assembled complex I. The results demonstrate that the two arms of complex I are assembled independently on separate pathways, and gave a first insight into the assembly pathway of the membrane arm. It is also shown for the first time that the obligate aerobic fungus N. crassa can grow and respire without an intact complex I. Gene replacement in this fungus is therefore a tool for investigation of this complex.
Subject(s)
Mitochondria/enzymology , NAD(P)H Dehydrogenase (Quinone)/metabolism , Neurospora crassa/metabolism , Blotting, Southern , Blotting, Western , Centrifugation, Density Gradient , Cytochromes/metabolism , Gene Deletion , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/metabolism , Mutagenesis , NAD(P)H Dehydrogenase (Quinone)/genetics , Neurospora crassa/enzymology , Neurospora crassa/genetics , Recombination, Genetic , Transformation, GeneticABSTRACT
This paper draws attention to the different definitions of 'birth centre', and the lack of consensus regarding the physical and organisational characteristics that distinguish a birth centre from conventional labour suites. Two case studies of decision-making about the location of a new birth centre are presented; they illustrate the need to take account of the views of each client and professional group concerned. The need for integrating organisational development with physical planning procedures is emphasised.
