Assembly of NADH: ubiquinone reductase (complex I) in Neurospora mitochondria. Independent pathways of nuclear-encoded and mitochondrially encoded subunits.
NADH:ubiquinone reductase, the respiratory chain complex I of mitochondria, consists of some 25 nuclear-encoded and seven mitochondrially encoded subunits, and contains as redox groups one FMN, probably one internal ubiquinone and at least four iron-sulphur clusters. We are studying the assembly of the enzyme in Neurospora crassa. The flux of radioactivity in cells that were pulse-labelled with [35S]methionine was followed through immunoprecipitable assembly intermediates into the holoenzyme. Labelled polypeptides were observed to accumulate transiently in a Mr 350,000 intermediate complex. This complex contains all mitochondrially encoded subunits of the enzyme as well as subunits encoded in the nucleus that have no homologous counterparts in a small, merely nuclear-encoded form of the NADH:ubiquinone reductase made by Neurospora crassa cells poisoned with chloramphenicol. With regard to their subunit compositions, the assembly intermediate and small NADH:ubiquinone reductase complement each other almost perfectly to give the subunit composition of the large complex I. These results suggest that two pathways exist in the assembly of complex I that independently lead to the preassembly of two major parts, which subsequently join to form the complex. One preassembled part is related to the small form of NADH:ubiquinone reductase and contributes most of the nuclear-encoded subunits, FMN, three iron-sulphur clusters and the site for the internal ubiquinone. The other part is the assembly intermediate and contributes all mitochondrially encoded subunits, one iron-sulphur cluster and the catalytic site for the substrate ubiquinone. We discuss the results with regard to the evolution of the electron pathway through complex I.
Tuschen
G
G
Universität Düsseldorf, Institut für Biochemie, Federal Republic of Germany.
Sackmann
U
U
Nehls
U
U
Haiker
H
H
Buse
G
G
Weiss
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D002467:Cell Nucleus / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D002851:Chromatography, High Pressure Liquid; D004271:DNA, Fungal / Q000235:genetics; D004272:DNA, Mitochondrial / Q000235:genetics; D005800:Genes, Fungal; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D009243:NAD / Q000378:metabolism*; D016660:NAD(P)H Dehydrogenase (Quinone); D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D011808:Quinone Reductases / Q000235:genetics / Q000378:metabolism*
D004271:DNA, Fungal; D004272:DNA, Mitochondrial; D009097:Multienzyme Complexes; D009243:NAD; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases