ABSTRACT
The copper chaperone for superoxide dismutase 1 (Ccs1) provides an important cellular function against oxidative stress. Ccs1 is present in the cytosol and in the intermembrane space (IMS) of mitochondria. Its import into the IMS depends on the Mia40/Erv1 disulfide relay system, although Ccs1 is, in contrast to typical substrates, a multidomain protein and lacks twin Cx(n)C motifs. We report on the molecular mechanism of the mitochondrial import of Saccharomyces cerevisiae Ccs1 as the first member of a novel class of unconventional substrates of the disulfide relay system. We show that the mitochondrial form of Ccs1 contains a stable disulfide bond between cysteine residues C27 and C64. In the absence of these cysteines, the levels of Ccs1 and Sod1 in mitochondria are strongly reduced. Furthermore, C64 of Ccs1 is required for formation of a Ccs1 disulfide intermediate with Mia40. We conclude that the Mia40/Erv1 disulfide relay system introduces a structural disulfide bond in Ccs1 between the cysteine residues C27 and C64, thereby promoting mitochondrial import of this unconventional substrate. Thus the disulfide relay system is able to form, in addition to double disulfide bonds in twin Cx(n)C motifs, single structural disulfide bonds in complex protein domains.
Subject(s)
Cysteine , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/metabolism , Molecular Chaperones , Oxidoreductases Acting on Sulfur Group Donors/metabolism , Protein Transport/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Signal Transduction/genetics , Cysteine/chemistry , Cysteine/metabolism , Cytosol/metabolism , Disulfides/metabolism , Gene Expression Regulation, Fungal , Mitochondria/genetics , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Precursor Protein Import Complex Proteins , Mitochondrial Proteins/genetics , Models, Molecular , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Mutation , Oxidation-Reduction , Oxidoreductases Acting on Sulfur Group Donors/genetics , Plasmids , Protein Folding , Protein Interaction Domains and Motifs , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Superoxide Dismutase-1 , Transduction, GeneticABSTRACT
Cells protect themselves against oxygen stress and reactive oxygen species. An important enzyme in this process is superoxide dismutase, Sod1, which converts superoxide radicals into water and hydrogen peroxide. The biogenesis of functional Sod1 is dependent on its copper chaperone, Ccs1, which introduces a disulfide bond and a copper ion into Sod1. Ccs1 and Sod1 are present in the cytosol but are also found in the mitochondrial intermembrane space (IMS), the compartment between the outer and the inner membrane of mitochondria. Ccs1 mediates mitochondrial localization of Sod1. Here, we report on the biogenesis of the fractions of Ccs1 and Sod1 present in mitochondria of Saccharomyces cerevisiae. The IMS of mitochondria harbors a disulfide relay system consisting of the import receptor Mia40 and the thiol oxidase Erv1, which drives the import of substrates with conserved cysteine residues arranged in typical twin Cx(3)C and twin Cx(9)C motifs. We show that depletion of Mia40 results in decreased levels of Ccs1 and Sod1. On the other hand, overexpression of Mia40 increased the mitochondrial fraction of both proteins. In addition, the import rates of Ccs1 were enhanced by increased levels of Mia40 and reduced upon depletion of Mia40. Mia40 forms mixed disulfides with Ccs1, suggesting a role of Mia40 for the generation of disulfide bonds in Ccs1. We suggest that the disulfide relay system transfers disulfide bonds via Mia40 to Ccs1, which then shuttles disulfide bonds to Sod1. In conclusion, the disulfide relay system is crucial for the import of Ccs1, thereby affecting the transport of Sod1, and it can control the distribution of Ccs1 and Sod1 between the IMS of mitochondria and the cytosol.
