Yeast Contain a Non-proteinaceous Pool of Copper in the Mitochondrial Matrix

Abstract

The yeast mitochondrion is shown to contain a pool of copper that is distinct from that associated with the two known mitochondrial cuproenzymes, superoxide dismutase (Sod1) and cytochrome c oxidase (CcO) and the copper-binding CcO assembly proteins Cox11, Cox17, and Sco1. Only a small fraction of mitochondrial copper is associated with these cuproproteins. The bulk of the remainder is localized within the matrix as a soluble, anionic, low molecular weight complex. The identity of the matrix copper ligand is unknown, but the bulk of the matrix copper fraction is not protein-bound. The mitochondrial copper pool is dynamic, responding to changes in the cytosolic copper level. The addition of copper salts to the growth medium leads to an increase in mitochondrial copper, yet the expansion of this matrix pool does not induce any respiration defects. The matrix copper pool is accessible to a heterologous cuproenzyme. Co-localization of human Sod1 and the metallochaperone CCS within the mitochondrial matrix results in suppression of growth defects of sod2⌬ cells. However, in the absence of CCS within the matrix, the activation of human Sod1 can be achieved by the addition of copper salts to the growth medium.Copper is an essential cell nutrient acting as a cofactor in nearly 20 enzymes (1). However, excess accumulation of copper ions results in toxicity. Evidence for the effectiveness of copper ions as a toxin comes from its historic use as a fungicide, molluscide, and algicide. Homeostatic mechanisms exist in cells to regulate the cellular concentration of copper ions, thus maintaining copper balance and minimizing deleterious effects. Cells appear to maintain a quota for essential metal ions; this quota is primarily the quantity necessary to metallate the various copper proteins (2, 3). Copper ions are required for at least three key enzymes in Saccharomyces cerevisiae. The cuproenzymes include the cytosolic superoxide dismutase Sod1, 1 the plasma membrane ferroxidase Fet3, and the mitochondrial inner membrane enzyme cytochrome c oxidase (CcO). The copper quota of the yeast S. cerevisiae is about 5 ϫ 10 5 atoms per cell (3, 4).It is unclear what fraction of the 5 ϫ 10 5 copper atoms per cell is from copper in Sod1, Fet3, and CcO. Expression of these copper-binding proteins varies with growth conditions, suggesting that the copper may be distributed differently depending on growth conditions. Clearly, a significant fraction of the cellular copper is associated with Sod1; however, not all Sod1 molecules are metallated (4, 5). Fet3 requires four copper ions for activity, but levels of this protein are dependent on iron status of the medium (6). CcO levels vary depending on whether the cells are grown by fermentation or respiration. In addition, a varying quantity of cellular copper exists bound to two metallothioneins, Cup1 and Crs5 (7,8). Expression of CUP1 and CRS5 is regulated by copper levels through the copper-responsive transcription factor Ace1 (9, 10). An increase in the free Cu(I) ion pool activates Ace1 throu...

Yeast Cox17 Solution Structure and Copper(I) Binding

“…Recent data indicate that Cox17 tethered to the inner mitochondrial membrane is fully functional, however (15). In addition, mitochondrial copper levels are independent of the presence of Cox17 (16). These results suggest that trafficking copper ions between the cytosol and the mitochondrial intermembrane space is not a function of Cox17.…”

Metal Ion availability in mitochondria

“…In yeast and mouse liver 85 and ~70% of the total mitochondrial copper is associated with this matrix CuL complex. Although the identity of the ligand remains unresolved, the matrix CuL pool is accessible to a heterologous cuproenzyme (Cobine et al 2004). The mechanism by which Aft1 (and Aft2) sense cellular iron levels involves the mitochondrion (Chen et al 2004;Chen et al 2002).…”

Copper supplementation increases yeast life span under conditions requiring respiratory metabolism

“…However, the increased copper concentration is not likely to be working by providing copper to Cu/Zn SOD, since copper greatly increased the life span of an SOD1 mutant. It has been demonstrated that yeast contain a pool of copper in the mitochondrial matrix that is not associated with any protein (Cobine et al, 2004). This pool may be used to buffer copper concentration in the cell, or to provide copper to cytochrome c oxidase and the fraction of Cu/ZnSOD present in the intermembrane space (Sturtz et al, 2001).…”