Genetic screening in Saccharomyces cerevisiae for large numbers of mitochondrial point mutations which affect structure and function of catalytic subunits of cytochrome‐c oxidase

Abstract

A new search for mitochondrial respiratory deficient mutants (Mit-) has been undertaken in order to accumulate a large number of point mutations in the coding portions of cytochrome-coxidase catalytic subunits and cytochrome b. Therefore, a mitochondrial DNA which retains the exons and lacks all the introns of the cytochrome oxidase subunit I and of the cytochrome-b split genes has been introduced into a strain carrying a nuclear recessive mutation affecting the adeninenucleotide translocator, the opl mutation, which is known to prevent the accumulation of large deletion petite mutants and this was used as the parental strain. After a moderate MnCI, mutagenesis in order to limit multiple mutations, 105 Mit-mutants were isolated from 15000 mutagenised clones in Saccharomyces cerevisiae. Mutations were mapped to the three catalytic subunits encoding genes (COX1, COX2 and COX3) of the cytochrome-c oxidase (70 mutations) and to the cytochrome-b gene (15 mutations). More than 50% of the mutants tested still exhibited mitochondrial translation products (subunits I, I1 and 111), suggesting that they carry a missense mutation, rather than a nonsense mutation which would normally have led to a truncated protein. Mutations in the COXl gene were allocated to four different subregions corresponding to exons 4 and 8 or to groups of exons, exons 1 , 2 , 3 or exons 5,6,7. Seven missense monosubstitution mutations and two frameshift mutations were also identified. The amino acid changes of the missense mutations were located in the vicinity of the CUB-heme a3 binuclear centre ligands.Cytochrome oxidase, the terminal acceptor of the mitochondrial respiratory chain, catalyses the reduction of oxygen into water. Even if composed of up to 13 subunits, only three subunits I, 11, and ID are mitochondrially coded and appear to be constituents of the minimal catalytic subunits. It is an integral protein complex of the mitochondrial inner membrane which contains four redox-active metal centres: Cu,, hemes a-a3 and CU, (for review, see [l]).The ligands of Cu,, likely to be bound to subunit 11 [2], are not known. Six conserved histidines of subunit I are thought to be the ligands for heme a, heme a3 and Cu,. In bacterial cytochrome oxidase [3] and the corresponding cytochrome o of Escherichia coli [4, 51, the ligands for heme a and the CUB-heme a3 binuclear centre bound to subunit I have been proposed on the basis of site-directed-mutagenesis data. An alternative model of bovine oxidase has also been presented which is still consistent with the site-directed-mutagenesis data and in which the third proposed Cu, ligand and that of the heme a3 are exchanged [6].In order to obtain further insight into the core of the cytochrome c oxidase catalytic centre, a large number of mutagenized clones has been screened in the model organism,

The Carboxyl-terminal End of Cox1 Is Required for Feedback Assembly Regulation of Cox1 Synthesis in Saccharomyces cerevisiae Mitochondria

“…Plasmids carrying the cox1 mutations were transformed into rho0 strain NAB69 by high-velocity microprojectile bombardment (25). Transformants were selected by their ability to rescue respiratory growth when mated with a strain carrying a Cox1 D369N mutation, L45 (26). Transformants were mated with XPM10b (containing the cox1⌬::ARG8 m construct) or XPM13a (containing the cox2-62 and the cox1⌬::ARG8 m construct) (13).…”

Dual Functions of Mss51 Couple Synthesis of Cox1 to Assembly of CytochromecOxidase inSaccharomyces cerevisiaeMitochondria

“…Sequences encoding 3xHA or 3xMyc epitope tags were added to the 3Ј ends of nuclear gene coding sequences, without altering mRNA flanking sequences, by pop-in pop-out transformation as described previously (Schneider et al, 1995). Mitochondrial transformation, and integration of altered genes into rho ϩ mtDNA, was as described previously (Perez-Martinez et al, 2003;Bonnefoy et al, 2007) (Bonnefoy and Fox, 2000) or by respiratory growth when mated with a rho ϩ strain carrying a cox1-D369N mutation, L45 (Meunier et al, 1993). Altered mitochondrial genes in the transformants were integrated by homologous recombination into rho ϩ mtDNA by isolating cytoductants issued from crosses of the transformants to either NB40-36a followed by selection for Argϩ growth, or to XPM10b followed by selection for respiratory growth, as appropriate.…”

Insight into the interactions between subunits I and II of the cytochrome c oxidase of the yeast Saccharomyces cerevisiae by means of extragenic complementation

“…The mitochondrial heredity of the reversion was determined before sequencing as described in [9,10]. Mutations have been sequenced directly on DNA segments amplified by polymerase chain reaction [6]. The results are presented in Table I.…”

“…The sequencing of the COX 2 gene of the mutant has been performed as described in [6]. Two mutations were observed, A189V and V22OV.…”

“…The mutation leads to an inability to grow on glycerol medium even although the mutant contains optically-detectable oxidase [6,7].…”