Cox17 is functional when tethered to the mitochondrial inner membrane

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.     

Specific copper transfer from the Cox17 metallochaperone to both Sco1 and Cox11 in the assembly of yeast cytochrome C oxidase

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.     

Yeast Sco1, a protein essential for cytochrome c oxidase function is a Cu(I)-binding protein

Sco1 is a conserved essential protein, which has been implicated in the delivery of copper to cytochrome c oxidase, the last enzyme of the electron transport chain. In this study, we show for the first time that the purified C-terminal domain of yeast Sco1 binds one Cu(I)/monomer. X-ray absorption spectroscopy suggests that the Cu(I) is ligated via three ligands, and we show that two cysteines, present in a conserved motif CXXXC, and a conserved histidine are involved in Cu(I) ligation. The mutation of any one of the conserved residues in Sco1 expressed in yeast abrogates the function of Sco1 resulting in a non-functional cytochrome c oxidase complex. Thus, the function of Sco1 correlates with Cu(I) binding. Data obtained from size-exclusion chromatography experiments with mitochondrial lysates suggest that full-length Sco1 may be oligomeric in vivo.     

Mitochondrial copper metabolism and delivery to cytochrome c oxidase

Metals are essential elements of all living organisms. Among them, copper is required for a multiplicity of functions including mitochondrial oxidative phosphorylation and protection against oxidative stress. Here we will focus on describing the pathways involved in the delivery of copper to cytochrome c oxidase (COX), a mitochondrial metalloenzyme acting as the terminal enzyme of the mitochondrial respiratory chain. The catalytic core of COX is formed by three mitochondrially-encoded subunits and contains three copper atoms. Two copper atoms bound to subunit 2 constitute the Cu(A) site, the primary acceptor of electrons from ferrocytochrome c. The third copper, Cu(B), is associated with the high-spin heme a(3) group of subunit 1. Recent studies, mostly performed in the yeast Saccharomyces cerevisiae, have provided new clues about 1) the source of the copper used for COX metallation; 2) the roles of Sco1p and Cox11p, the proteins involved in the direct delivery of copper to the Cu(A) and Cu(B) sites, respectively; 3) the action mechanism of Cox17p, a copper chaperone that provides copper to Sco1p and Cox11p; 4) the existence of at least four Cox17p homologues carrying a similar twin CX(9)C domain suggestive of metal binding, Cox19p, Cox23p, Pet191p and Cmc1p, that could be part of the same pathway; and 5) the presence of a disulfide relay system in the intermembrane space of mitochondria that mediates import of proteins with conserved cysteines motifs such as the CX(9)C characteristic of Cox17p and its homologues. The different pathways are reviewed and discussed in the context of both mitochondrial COX assembly and copper homeostasis.     

HCC1, the Arabidopsis homologue of the yeast mitochondrial copper chaperone SCO1, is essential for embryonic development

The Arabidopsis HCC1 gene is a homologue of the copper chaperone SCO1 from the yeast Saccharomyces cerevisiae. SCO1 (synthesis of cytochrome c oxidase 1) encodes a mitochondrial protein that is essential for the correct assembly of complex IV in the respiratory chain. GUS analyses showed HCC1 promoter activity in vascular tissue, guard cells, hydathodes, trichome support cells, and embryos. HCC1 function was studied in two hcc1 T-DNA insertion lines, hcc1-1 and hcc1-2. Gametophyte development was not affected by the disruption of HCC1, but homozygous hcc1-1 and hcc1-2 embryos became arrested at various developmental stages, mostly at the heart stage. Both the wild-type HCC1 gene and the modified gene coding for the C-terminally SNAP-tagged HCC1 were able to complement the embryo-lethal phenotype of the hcc1-1 line. Localization of the SNAP-tagged HCC1 in transgenic lines identified HCC1 as a mitochondrial protein. To determine if HCC1 is a functional homologue to Sco1p, the respiratory-deficient yeast sco1 mutant was transformed with chimeric constructs containing different combinations of HCC1 and SCO1 sequences. One of the resulting chimeric proteins restored respiration in the yeast mutant. This protein had the N-terminal mitochondrial targeting signal and the single transmembrane domain derived from Sco1p and the C-terminal half (including the copper-binding motif) derived from HCC1. Growth of the complemented yeast mutant was enhanced by the addition of copper to the medium. The data demonstrate that HCC1 is essential for embryo development in Arabidopsis, possibly due to its role in cytochrome c oxidase assembly.     

Mitochondrial copper metabolism in yeast: interaction between Sco1p and Cox2p

Yeast mitochondrial Sco1p is required for the formation of a functional cytochrome c oxidase (COX). It was suggested that Sco1p aids copper delivery to the catalytic center of COX. Here we show by affinity chromatography and coimmunoprecipitation that Sco1p interacts with subunit Cox2p. In addition we provide evidence that Sco1p can form homomeric complexes. Both homomer formation and binding of Cox2p are neither dependent on the presence of copper nor affected by mutations of His-239, Cys-148 or Cys-152. These amino acids, which are conserved among the members of the Sco1p family, have been suggested to act in the reduction of the cysteines in the copper binding center of Cox2p and are discussed as ligands for copper.     

Transport of proteins to the mitochondrial intermembrane space: the ''matrix-targeting'' and the ''sorting'' domains in the cytochrome c1 presequence.

We reported earlier that the yeast cytochrome c1 presequence (length: 61 amino acids) directs attached proteins to the mitochondrial intermembrane space and that it appears to contain two functional domains: a 'matrix-targeting' domain, and a 'sorting' domain. We have now used gene manipulation together with two different in vivo import assays to map these two domains within the cytochrome c1 presequence. The 'matrix-targeting' domain is contained within the N-terminal 16 residues (or less); by itself, it directs attached proteins to the matrix. The 'sorting' domain extends into the C-terminal 13 residues of the presequence; while it does not mediate intracellular protein transport by itself, it acts together with the preceding 'matrix-targeting' sequence in sorting attached proteins into the intermembrane space. On replacing the authentic 'matrix-targeting' sequence with artificial sequences of different lengths we found that sorting of proteins between the outer membrane and the intermembrane space is not exclusively determined by the length of the N-terminal 'matrix-targeting' sequence.     

The presequences of two imported mitochondrial proteins contain information for intracellular and intramitochondrial sorting

Gene fusion experiments were used to identify signals that direct imported precursor proteins to specific intramitochondrial locations in yeast. The amino terminus of alcohol dehydrogenase III (ADHIII, a mitochondrial matrix enzyme) transported attached mouse dihydrofolate reductase (DHFR, a cytosolic enzyme) into the mitochondrial matrix. The presequence of cytochrome c1 (a mitochondrial inner membrane protein protruding into the intermembrane space) transported attached DHFR into the intermembrane space. The first half of the cytochrome c1 presequence, which resembles the ADHIII presequence, is a matrix-targeting sequence: it transported attached DHFR into the matrix. The second half of the cytochrome c1 presequence contains a stretch of 19 uncharged amino acids and may thus be a stop-transfer sequence. We conclude that intramitochondrial sorting involves matrix-targeting and stop-transfer sequences within the cleavable presequence.     

A conserved α helix of Bcs1, a mitochondrial AAA chaperone,is required for the Respiratory Complex III maturation

Bcs1 is a transmembrane chaperone in the mitochondrial inner membrane, and is required for the mitochondrial Respiratory Chain Complex III assembly. It has been shown that the highly-conserved C-terminal region of Bcs1 including the AAA ATPase domain in the matrix side is essential for the chaperone function. Here we describe the importance of the N-terminal short segment located in the intermembrane space in the Bcs1 function. Among the N-terminal 44 amino acid residues of yeast Bcs1, the first 37 residues are dispensable whereas a hydrophobic amino acid in the residue 38 is essential for integration of Rieske Iron-sulfur Protein into the premature Complex III from the mitochondrial matrix. Substitution of the residue 38 by a hydrophilic amino acid residue affects conformation of Bcs1 and interactions with other proteins. The evolutionarily-conserved short α helix of Bcs1 in the intermembrane space is an essential element for the chaperone function.     

Yeast contain a non-proteinaceous pool of copper in the mitochondrial matrix

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 sod2Delta 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.     

Solution structure of Sco1: a thioredoxin-like protein Involved in cytochrome c oxidase assembly

Sco1, a protein required for the proper assembly of cytochrome c oxidase, has a soluble domain anchored to the cytoplasmic membrane through a single transmembrane segment. The solution structure of the soluble part of apoSco1 from Bacillus subtilis has been solved by NMR and the internal mobility characterized. Its fold places Sco1 in a distinct subgroup of the functionally unrelated thioredoxin proteins. In vitro Sco1 binds copper(I) through a CXXXCP motif and possibly His 135 and copper(II) in two different species, thus suggesting that copper(II) is adventitious more than physiological. The Sco1 structure represents the first structure of this class of proteins, present in a variety of eukaryotic and bacterial organisms, and elucidates a link between copper trafficking proteins and thioredoxins. The availability of the structure has allowed us to model the homologs Sco1 and Sco2 from S. cerevisiae and to discuss the physiological role of the Sco family.     

The P174L mutation in human Sco1 severely compromises Cox17-dependent metallation but does not impair copper binding

Sco1 is a metallochaperone that is required for copper delivery to the Cu(A) site in the CoxII subunit of cytochrome c oxidase. The only known missense mutation in human Sco1, a P174L substitution in the copper-binding domain, is associated with a fatal neonatal hepatopathy; however, the molecular basis for dysfunction of the protein is unknown. Immortalized fibroblasts from a SCO1 patient show a severe deficiency in cytochrome c oxidase activity that was partially rescued by overexpression of P174L Sco1. The mutant protein retained the ability to bind Cu(I) and Cu(II) normally when expressed in bacteria, but Cox17-mediated copper transfer was severely compromised both in vitro and in a yeast cytoplasmic assay. The corresponding P153L substitution in yeast Sco1 was impaired in suppressing the phenotype of cells harboring the weakly functional C57Y allele of Cox17; however, it was functional in sco1delta yeast when the wild-type COX17 gene was present. Pulse-chase labeling of mitochondrial translation products in SCO1 patient fibroblasts showed no change in the rate of CoxII translation, but there was a specific and rapid turnover of CoxII protein in the chase. These data indicate that the P174L mutation attenuates a transient interaction with Cox17 that is necessary for copper transfer. They further suggest that defective Cox17-mediated copper metallation of Sco1, as well as the subsequent failure of Cu(A) site maturation, is the basis for the inefficient assembly of the cytochrome c oxidase complex in SCO1 patients.     

Erv1p from Saccharomyces cerevisiae is a FAD-linked sulfhydryl oxidase

The yeast ERV1 gene encodes a small polypeptide of 189 amino acids that is essential for mitochondrial function and for the viability of the cell. In this study we report the enzymatic activity of this protein as a flavin-linked sulfhydryl oxidase catalyzing the formation of disulfide bridges. Deletion of the amino-terminal part of Erv1p shows that the enzyme activity is located in the 15 kDa carboxy-terminal domain of the protein. This fragment of Erv1p still binds FAD and catalyzes the formation of disulfide bonds but is no longer able to form dimers like the complete protein. The carboxy-terminal fragment contains a conserved CXXC motif that is present in all homologous proteins from yeast to human. Thus Erv1p represents the first FAD-linked sulfhydryl oxidase from yeast and the first of these enzymes that is involved in mitochondrial biogenesis.     

Novel insights into phenotype and mitochondrial proteome of yeast mutants lacking proteins Sco1p or Sco2p

The yeast Saccharomyces cerevisiae is a facultative anaerobe and its mitochondrial morphology is linked to its metabolic activity. The Sco proteins (Sco1p and Sco2p) were characterized as proteins required for copper delivery to cytochrome c oxidase. Our results indicated a higher fermentative capacity of the sco1-Δ mutant in comparison to the control and the sco2-Δ mutant strains. The mitochondrial proteome analysis showed that the sco1-Δ mutant down-regulated components of the respiratory chain, the TCA cycle and transport of metabolites across the mitochondrial membrane. This evidence suggests that the absence of Sco1p causes irreversible damage to the mitochondria.     

Functional analysis of the domains in Cox11

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.     

Analysis of the sorting signals directing NADH-cytochrome b5 reductase to two locations within yeast mitochondria.

Mitochondrial NADH-cytochrome b5 reductase (Mcr1p) is encoded by a single nuclear gene and imported into two different submitochondrial compartments: the outer membrane and the intermembrane space. We now show that the amino-terminal 47 amino acids suffice to target the Mcr1 protein to both destinations. The first 12 residues of this sequence function as a weak matrix-targeting signal; the remaining residues are mostly hydrophobic and serve as an intramitochondrial sorting signal for the outer membrane and the intermembrane space. A double point mutation within the hydrophobic region of the targeting sequence virtually abolishes the ability of the precursor to be inserted into the outer membrane but increases the efficiency of transport into the intermembrane space. Import of Mcr1p into the intermembrane space requires an electrochemical potential across the inner membrane, as well as ATP in the matrix, and is strongly impaired in mitochondria lacking Tom7p or Tim11p, two components of the translocation machineries in the outer and inner mitochondrial membranes, respectively. These results indicate that intramitochondrial sorting of the Mcr1 protein is mediated by specific interactions between the bipartite targeting sequence and components of both mitochondrial translocation systems.     

Copper trafficking to the mitochondrion and assembly of copper metalloenzymes

Copper is required within the mitochondrion for the function of two metalloenzymes, cytochrome c oxidase (CcO) and superoxide dismutase (Sod1). Copper metallation of these two enzymes occurs within the mitochondrial intermembrane space and is mediated by metallochaperone proteins. Cox17 is a key copper donor to two accessory proteins, Sco1 and Cox11, to form the two copper centers in the mature CcO complex. Ccs1 is the necessary metallochaperone for the copper metallation of Sod1 in the IMS as well as within the cytoplasm where the bulk of Sod1 resides. Copper ions used in the metallation of CcO and Sod1 appear to be provided by a novel copper pool within the mitochondrial matrix. This review documents copper ion shuttling within the mitochondrion and the proteins that mediate assembly of active CcO and Sod1.     

Characterization of YpmQ, an accessory protein required for the expression of cytochrome c oxidase in Bacillus subtilis

A search of the Bacillus subtilis genome identifies a potential homolog, ypmQ, of the inner mitochondrial membrane protein Sco1 from yeast. Sco1 has been found to aid the delivery of copper to cytochrome c oxidase. B. subtilis expresses two members of the cytochrome oxidase family, a cytochrome c oxidase that has two copper centers, Cu(A) and Cu(B), and a menaquinol oxidase that has only Cu(B). Deletion of ypmQ in B. subtilis depresses expression of cytochrome c oxidase but not menaquinol oxidase. Levels of cytochrome c oxidase recover when copper is added to the growth medium of the DeltaypmQ strain or when ypmQ is expressed from a plasmid. Neither treatment affects the amount or activity of menaquinol oxidase. YpmQ in which two conserved cysteines are replaced by serines and a conserved histidine is replaced by alanine do not complement the deletion of ypmQ even though these mutant forms are found in the membrane extract at a level similar to the wild type protein. We propose that the two cysteines and the histidine are critical for the function of YpmQ and suggest they are involved in copper exchange between YpmQ and the Cu(A) site of cytochrome c oxidase.     

Mitochondrial matrix copper complex used in metallation of cytochrome oxidase and superoxide dismutase

A mitochondrial matrix copper ligand (CuL) complex, conserved in mammalian cells, is the likely source of copper for assembly of cytochrome c oxidase (CcO) and superoxide dismutase 1 (Sod1) within the intermembrane space (IMS) in yeast. Targeting the copper-binding proteins human Sod1 and Crs5 to the mitochondrial matrix results in growth impairment on non-fermentable medium caused by decreased levels of CcO. This effect is reversed by copper supplementation. Matrix-targeted Crs5 diminished Sod1 protein within the IMS and impaired activity of an inner membrane tethered human Sod1. Copper binding by the matrix-targeted proteins attenuates levels of the CuL complex without affecting total mitochondrial copper. These data suggest that attenuation of the matrix CuL complex via heterologous competitors limits available copper for metallation of CcO and Sod1 within the IMS. The ligand also exists in the cytoplasm in an apparent metal-free state.     

Identification and characterization of the mitochondrial membrane sorting signals in phosphatidylserine decarboxylase 1 from Saccharomyces cerevisiae

Phosphatidylserine decarboxylase 1 (Psd1p) catalyzes the formation of the majority of phosphatidylethanolamine (PE) in the yeast Saccharomyces cerevisiae. Psd1p is localized to mitochondria, anchored to the inner mitochondrial membrane (IMM) through membrane spanning domains and oriented towards the mitochondrial intermembrane space. We found that Psd1p harbors at least two inner membrane-associated domains, which we named IM1 and IM2. IM1 is important for proper orientation of Psd1p within the IMM (Horvath et al., J. Biol. Chem. 287 (2012) 36744–55), whereas it remained unclear whether IM2 is important for membrane-association of Psd1p. To discover the role of IM2 in Psd1p import, processing and assembly into the mitochondria, we constructed Psd1p variants with deletions in IM2. Removal of the complete IM2 led to an altered topology of the protein with the soluble domain exposed to the matrix and to decreased enzyme activity. Psd1p variants lacking portions of the N-terminal moiety of IM2 were inserted into IMM with an altered topology. Psd1p variants with deletions of C-terminal portions of IM2 accumulated at the outer mitochondrial membrane and lost their enzyme activity. In conclusion we showed that IM2 is essential for full enzymatic activity, maturation and correct integration of yeast Psd1p into the inner mitochondrial membrane.