Subunit II of yeast QH₂:cytochrome‐c oxidoreductase

Abstract

The nucleotide sequence of a 2.5 x 103-base segment of yeast nuclear DNA, containing the structural gene for the 40-kDa subunit I1 of the ubiquinol: cytochrome-c oxidoreductase, has been determined. The region contains only one single reading frame of length sufficient to encode a protein of the size of subunit 11. The mature protein is predicted to have a length of 352 amino acids, with a molecular mass of 38714 Da. It is predominantly hydrophilic, with an overall polarity of 45%. Comparison of the sequence of the reading frame with that derived from direct sequence analysis of the N terminus of the mature 40-kDa protein shows that subunit I1 is synthesized as a longer precursor and shows that the extension is N-terminal. The presequence is 16 amino acids long and it contains a number of positively charged residues and lacks acidic ones. It is also rich in neutral, polar amino acids. S1 nuclease protection analysis of DNA . RNA hybrids identifies two major and one minor transcript of the gene, whose 5' termini map approximately 55, 65 and 75 nucleotides upstream of the initiation codon. Sequences 5' of these termini lack obvious homology to the regulatory sequences of other imported mitochondrial proteins, whose synthesis is controlled by oxygen and by catabolite repression. A mutant lacking a functional subunit I1 gene has been constructed by a one-step gene-disruption procedure. This mutant grows only slowly on glycerol and still displays a low level of QH,: cytochrome-c oxidoreductase activity (approx. 5% of that of wild type). The implications of this finding for the possible role of subunit I1 in the complex are discussed.QH, : cytochrome-c oxidoreductase is a proton-translocating enzyme of the respiratory chain in mitochondria. In yeast it consists of eight to ten subunits [l -31, of which only one, apocytochrome b, is mitochondrially encoded [4, 51. The remaining subunits are encoded in the nucleus. The dual genetic origin of the enzyme poses questions concering the coordinate synthesis, import and assembly of individual subunits. To address these, molecular information about the genes and the subunits is required. This is so far available for subunits I11 (cytochrome cl), IV (cytochrome b) and VI-VIII [6-91 (Maarse, unpublished results). We describe here an analysis of the nuclear gene for subunit 11. Comparison of the nucleotide sequence with a partial amino acid sequence of the N terminus of the mature protein reveals an amino-terminal extension which displays several features common to the presequences of other imported mitochondrial proteins. The remaining protein sequence is predominantly hydrophilic, consistent with previous suggestions that the bulk of the subunit protrudes from the mitochondrial inner membrane into the matrix space MATERIALS AND METHODS Strains and mediaSaccharomyces cerevisiae strains DL1 (a, his3, leu2, ura3) and HR2 (a, his3,leu2, trpl) were used for most experiments. Transformation was carried out as described by Klebe et al. [Ill and transformants were selected on minima...

Core I protein of bovine ubiquinol–cytochrome‐c reductase; an additional member of the mitochondrial‐protein‐processing family

“…The human and rat core I1 cDNA sequences have been published recently [29,301. Bovine core I1 protein shows, as expected, high identity to human (89%) and rat (85%) core I1 proteins and only a moderate identity of 19% to S. cerevisiae core I1 protein [31]. Genomic Southern blots of restricted bovine DNA probed with the 1.6-kb full-length cDNA insert from pBCII 15 show hybridization patterns which indicate one gene for the core I1 protein (data not shown).…”

“…S. cerevisiae mutants of the cor I gene encoding core Iy [36] do not insert heme into apocytochrome b and lack a functional cytochrome-bcl complex. Yeast mutants deficient in core I1 protein [31] show reduced cytochrome-b levels and reduced-bel complex activity. In addition, reconstitution experiments with the N .…”

Inactivation of the gene encoding the 11‐kDa subunit VIII of the ubiquinol–cytochrome‐c oxidoreductase in Saccharomyces cerevisiae

“…Thus the effects of the absence of the 1 1-kDa subunit VIII are mediated post-transcriptionally, at the level of import or, more probably, assembly, since studies on import of individual cytoplasmically synthesized subunits [23,241 give no indication of coupling at that level. With respect to the amount of apocytochrome b and holocytochrome b, the mutant lacking the 11-kDa-subunit gene behaves differently from mutants lacking either the 40-kDaprotein gene [25] or the 44-kDa-protein gene [26]. The latter contains apocytochrome b, but not holocytochrome b whereas the mutant lacking the 40-kDa-protein gene contains trace amounts of both apo-and holocytochrome 6.…”

The Mitochondrial Protein Import Apparatus

“…In Neurospora crassa, but not in yeast, PEP and subunit I are even identical proteins (120). Although the function of subunits I and II has not been exactly determined, they are assumed to participate in assembly and/or stabilization of the bc~-complex (121,122). PEP may bind precursor proteins at the matrix side of the inner membrane and present them to MPP.…”