The primary structure of the iron-sulfur subunit of ubiquinol-cytochrome c reductase from Neurospora, determined by cDNA and gene sequencing

Abstract

The primary structure of the iron-sulfur subunit of ubiquinol -cytochrome c reductase from Neurosporu mitochondria was determined by cDNA and genomic DNA sequencing. A first cDNA was identified from a cDNA bank cloned in Escherichia coli by hybridization selection of mRNA, cell-free protein synthesis and immunoadsorption. Further cDNA and geonomic DNA were identified by colony filter hybridization. The N-terminal sequence of the mature protein was determined by automated Edman degradation. From the sequence a molecular mass of 24749 Da results for the precursor protein and of 21 556 Da for the mature protein. The presequence consists of 32 amino acids with four arginines as the only charged residues. The mature protein consists of 199 amino acids. It is characterized by a small N-terminal hydrophilic part of 29 residues, a hydrophobic stretch of 25 residues and a large C-terminal hydrophilic domain of 145 residues. The only four cysteines of the protein, which are assumed to bind the 2Fe-2S cluster, are located in a moderate hydrophobic region of this large domain. Cysteines 3 and 4 are unusually arranged in that they are separated by only one proline. From sequence data the arrangement of the subunit in the membrane is deduced.Ubiquinol -cytochrome c reductase (cytochrome reductase) is a proton-translocating enzyme complex of the oxidative phosphorylation system in mitochondria. The enzyme isolated from Neurosporu consists of the cytochromes b and cl, an iron-sulfur subunit (Rieske 2Fe-2S protein, [l]) and six subunits without redox centers [2, 31.The three-dimensional structure of cytochrome reductase, the arrangement of the structure in the mitochondrial inner membrane and the topography of most of the subunits within the structure have been studied by electron microscopy of twodimensional crystals, neutron diffraction of enzyme/detergent preparations and biochemical characterization of isolated subunits [3 -71. With regard to the iron-sulfur subunit the studies showed that the subunit extends from the membrane with a large domain which carries the 2Fe-2S cluster into the intermembrane space of mitochondria and is anchored to the bilayer only by a small protein part.The import of the cytoplasmically synthesized subunits of the Neurospora cytochrome reductase (all except cytochrome b, which is a mitochondrial coded protein) into mitochondria has been studied recently [8]. The subunits, except the 14000-Da subunit, are synthesized as larger precursors and proteolytically processed during or after their import into mitochondria. Similar results were obtained for the yeast enzyme [9 -121.In this article we report on the primary structure of the iron-sulfur subunit of Neurospora cytochrome reductase in its

Synthesis and Assembly of Mitochondrial Proteins

“…The segment of the prepiece identified by von Heijne (1986a) as having the highest hydrophobic moment (18 residue window, Eisenberg et al, 1984) when plotted in a helical wheel projection (Schiffer and Edmundson, 1967) is indicated by a box. Proteins: (1) 70 kDa protein (yeast), Hase et al (1983); (2) cytochrome c peroxidase (yeast), Kaput et al (1982), Reid et al (1982); (3) cytochrome b 2 (yeast), Guiard (1985), Gasser et al (1982b); (4) Rieske Fe/S of bc, complex (N. crassa), Harnisch et al (1985), Hartl et al (1986); (5) cytochrome d (yeast), Sadler et al (1984), Gasser et al (1982b); (6) cytochrome oxidase V (yeast), Koerner et aJ. (1985); (7) cytochrome oxidase V (N. crassa), Sachs et al (1986); (8) cytochrome oxidase IV (bovine), Lomax et al (1984); (9) cytochrome P-450 (SCC) (bovine), Morohashi et al (1984); (10) cytochrome oxidase VI (yeast), Wright et al (1984) ; (11) ATPase IX (N. crassa), Viebrock et al (1982), Schmidt et al (1984); (12) ATPase IX-Pl (bovine), Gay and Walker (1985); (13) ATPase IX-P2 (bovine), Gay and Walker (1985); (14) cytochrome oxidase IV (yeast), Maarse et al (1984); (15) citrate synthase (yeast), Suissa et al (1984); (16) Ornithine aminotransferase (rat), Mueckler and Pitot (1985), Simmaco et al (1986); (17) aspartate aminotransferase (chicken), Jaussi et al (1985) ; (18) aspartate aminotransferase (porcine), Joh et al (1985); (19) Mn-superoxide dismutase (yeast), Marres et al (1985); (20) ATPase F, 5 subunit (N. crassa), Kruse and Sebald (1984); (21) 5-aminolevulinate synthase (yeast), Keng et al (1986), Urban-Grimal et al (1986); (22) alcohol dehydrogenase iso-III (...…”

“…Indeed, the equivalent Fe/S protein from the photosynthetic bacteria Rhodopseudomonas sphaeroides is synthesized in the bacterial cytoplasm (comparable to the mitochondrial matrix) and transferred across the photosynthetic membrane to the side opposite the cytoplasm where, as in mitochondria, it is topologically opposed to the Fj part of ATPase. This transfer is accompanied by a reduction in molecular weight (Gabellini et al, 1985) which may be equivalent to processing of the mitochon- Harnisch et al (1985) (N. crassa Fe/S protein of bcx complex), Gabellini and Sebald (1986) (R. sphaeroides Fe/S protein of bcx complex and cytochrome c,), Sadler et al (1984) (yeast cytochrome c^, Lederer and Simon (1974) (N. crassa cytochrome c), Daldal et al (1986) …”

Flower-enhanced expression of a nuclear-encoded mitochondrial respiratory protein is associated with changes in mitochondrion number.

“…The nuclear genes encoding the mitochondrial RlSP have been cloned from Neurospora (Harnisch et al, 1985), Saccharomyces cerevisiae (Beckmann et al, 1987), rat (Nishikimi et al, 1989), human (Nishikimi et al, 1990), and cow…”

The primary structure of cytochrome c1 from Neurospora crassa

“…2). The presumed start codon d(ATG) is preceded by a short sequence d(CACC) (not shown), similar to the start codons of other Neurospora mRNAs [ll, 13,[19][20][21][22][23]. The presequence consists of 70 amino acids corresponding to a molecular mass of 7220 Da.…”