ABSTRACT
Cox11 is an intrinsic mitochondrial membrane protein essential for the assembly of an active cytochrome c oxidase complex. Cox11 is tethered to the mitochondrial inner membrane by a single transmembrane helix. Domain mapping was carried out to determine the functional segments of the Cox11 protein. The C-terminal 189 residue Cu(I)-binding domain is shown to be exposed within the mitochondrial intermembrane space. This orientation was demonstrated by the proteolytic susceptibility of a C-terminal Myc epitope tag in mitoplasts but not intact mitochondria. Fusion of the N terminus of Cox11 to the matrix ribosomal protein Rsm22 results in a functional protein capable of suppressing the respiratory defect of both Deltacox11 cells and Deltarsm22 cells. The functionality of the fusion protein suggests that the Cox11 N terminus projects into the matrix. The fusion of the C-terminal segment of Cox11 to Rsm22 resembles a naturally occurring fusion of Cox11 in Schizosaccharomyces pombe to a sequence homologous to the Saccharomyces cerevisiae Rsm22. Studies on a series of SCO1/COX11 chimeras reveal that the matrix domain of Cox11 lacks a specific function, whereas the Cu(I) binding/donating function requires the yeast Cox11 sequence. The Cu(I)-binding domain from human Cox11 cannot functionally replace the yeast sequence. The copper domain of Cox11 may be an important docking motif for Cox1 or a Cox1-associated protein.
Subject(s)
Membrane Proteins/chemistry , Membrane Proteins/physiology , Ribosomal Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/enzymology , Schizosaccharomyces/enzymology , Amino Acid Motifs , Amino Acid Sequence , Epitopes/chemistry , Genetic Complementation Test , Immunoprecipitation , Mitochondria/metabolism , Mitochondrial Proteins , Molecular Sequence Data , Plasmids/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Proto-Oncogene Proteins c-myc/chemistry , Recombinant Fusion Proteins/chemistry , Ribosomes/metabolism , Saccharomyces cerevisiae/metabolism , Trypsin/pharmacologyABSTRACT
Cox17 is an essential protein in the assembly of cytochrome c oxidase within the mitochondrion. Cox17 is implicated in providing copper ions for formation of CuA and CuB sites in the oxidase complex. To address whether Cox17 is functional in shuttling copper ions to the mitochondrion, Cox17 was tethered to the mitochondrial inner membrane by a fusion to the transmembrane domain of the inner membrane protein, Sco2. The copper-binding domain of Sco2 that projects into the inter-mitochondrial membrane space was replaced with Cox17. The Sco2/Cox17 fusion protein containing the mitochondrial import sequence and transmembrane segment of Sco2 is exclusively localized within the mitochondrion. The Sco2/Cox17 protein restores respiratory growth and normal cytochrome oxidase activity in cox17Delta cells. These studies suggest that the function of Cox17 is confined to the mitochondrial intermembrane space. Domain mapping of yeast Cox17 reveals that the carboxyl-terminal segment of the protein has a function within the intermembrane space that is independent of copper ion binding. The essential C-terminal function of Cox17 maps to a candidate amphipathic helix that is important for mitochondrial uptake and retention of the Cox17 protein. This motif can be spatially separated from the N-terminal copper-binding functional motif. Possible roles of the C-terminal motif are discussed.
Subject(s)
Cation Transport Proteins/chemistry , Cation Transport Proteins/metabolism , Intracellular Membranes/metabolism , Mitochondria/metabolism , Saccharomyces cerevisiae Proteins , Alleles , Amino Acid Motifs , Amino Acid Sequence , Blotting, Western , Chromatography, Gel , Copper Transport Proteins , Cytoplasm/metabolism , Cytosol/metabolism , DNA Mutational Analysis , Electron Transport Complex IV/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Fungal Proteins/chemistry , Genetic Complementation Test , Genetic Vectors , Molecular Chaperones , Molecular Sequence Data , Mutation , Plasmids/metabolism , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Subcellular Fractions/metabolismABSTRACT
The assembly of the copper sites in cytochrome c oxidase involves a series of accessory proteins, including Cox11, Cox17, and Sco1. The two mitochondrial inner membrane proteins Cox11 and Sco1 are thought to be copper donors to the Cu(B) and Cu(A) sites of cytochrome oxidase, respectively, whereas Cox17 is believed to be the copper donor to Sco1 within the intermembrane space. In this report we show Cox17 is a specific copper donor to both Sco1 and Cox11. Using in vitro studies with purified proteins, we demonstrate direct copper transfer from CuCox17 to Sco1 or Cox11. The transfer is specific because no transfer occurs to heterologous proteins, including bovine serum albumin and carbonic anhydrase. In addition, a C57Y mutant of Cox17 fails to transfer copper to Sco1 but is competent for copper transfer to Cox11. The in vitro transfer studies were corroborated by a yeast cytoplasm expression system. Soluble domains of Sco1 and Cox11, lacking the mitochondrial targeting sequence and transmembrane domains, were expressed in the yeast cytoplasm. Metallation of these domains was strictly dependent on the co-expression of Cox17. Thus, Cox17 represents a novel copper chaperone that delivers copper to two proteins.