Analysis of COX2 mutants reveals cytochrome oxidase subassemblies in yeast

Abstract

Cytochrome oxidase catalyses the reduction of oxygen to water. The mitochondrial enzyme contains up to 13 subunits, 11 in yeast, of which three, Cox1p, Cox2p and Cox3p, are mitochondrially encoded. The assembly pathway of this complex is still poorly understood. Its study in yeast has been so far impeded by the rapid turnover of unassembled subunits of the enzyme. In the present study, immunoblot analysis of blue native gels of yeast wild-type and Cox2p mutants revealed five cytochrome oxidase complexes or subcomplexes: a, b, c, d and f; a is likely to be the fully assembled enzyme; b lacks Cox6ap; d contains Cox7p and/or Cox7ap; f represents unassembled Cox1p; and c, observed only in the Cox2p mutants, contains Cox1p, Cox3p, Cox5p and Cox6p and lacks the other subunits. The identification of these novel cytochrome oxidase subcomplexes should encourage the reexamination of other yeast mutants.

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

“…The assembly pathway is not understood in detail. In mammals, analysis of mutant and drug-treated cell lines indicates that Cox1 is a component of the earliest assembly intermediates (Nijtmans et al, 1998;Williams et al, 2004), and similar analysis in yeast is consistent with this idea (Horan et al, 2005).An important function of this assembly process may be to prevent incompletely assembled components of cytochrome c oxidase from generating damaging reactive oxygen species, before they are contained by the holoenzyme. Indeed, mutations in several yeast genes required for cytochrome c oxidase assembly cause hypersensitivity to hydrogen peroxide (Pungartnik et al, 1999;Williams et al, 2005;Banting and Glerum, 2006), and a key component of the reactive prooxidant species is Cox1 .…”

Evidence for a Pro-oxidant Intermediate in the Assembly of Cytochrome Oxidase

“…Residual levels of unassembled Cox1 exist in CcO mutant yeast strains (35), including cox11⌬ cells (27). The pro-oxidant is probably a heme A-Cox1 complex.…”

“…Degradation kinetics of newly synthesized Cox1 (B), Cox2 (C), Cox3 (D), and Cob1 (E) proteins was evaluated by densitometry. Degradation is assessed as a percentage of 35 S radioactivity in each band during the chase to the pulse time.…”

“…The presence of AFG1 did not alter the 35 S incorporation into Cox1, Cox2, or Cox3 during the pulse phase of the reaction (Fig. 9A), but AFG1 did accentuate the diminution in labeled Cox1, Cox2, and Cox3 during the chase (Fig.…”

Mechanisms of mitochondrial translational regulation

“…Early studies on COX biogenesis in rat liver mitochondria (12) and analysis of COX assembly intermediates in wild-type (13) and COX-deficient human cells (14) have delineated a model of the COX assembly pathway characterized by at least three discrete subcomplexes that probably represent rate-limiting steps in the process (7). COX assembly intermediates similar to those described in mammalian cells have been detected in yeast mutants (15,16), suggesting that the process is highly conserved across species. Cox1 constitutes the first assembly intermediate.…”