Translocation of mitochondrially synthesized Cox2 domains from the matrix to the intermembrane space

The N-terminal and C-terminal domains of mitochondrially synthesized cytochrome c oxidase subunit II, Cox2, are translocated through the inner membrane to the intermembrane space (IMS). We investigated the distinct mechanisms of N-tail and C-tail export by analysis of epitope-tagged Cox2 variants encoded in Saccharomyces cerevisiae mitochondrial DNA. Both the N and C termini of a truncated protein lacking the Cox2 C-terminal domain were translocated to the IMS via a pathway dependent upon the conserved translocase Oxa1. The topology of this Cox2 variant, accumulated at steady state, was largely but not completely unaffected in mutants lacking proteins required for export of the C-tail domain, Cox18 and Mss2. C-tail export was blocked by truncation of the last 40 residues from the C-tail domain, indicating that sequence and/or structural features of this domain are required for its translocation. Mss2, a peripheral protein bound to the inner surface of the inner membrane, coimmunoprecipitated with full-length newly synthesized Cox2, whose leader peptide had already been cleaved in the IMS. Our data suggest that the C-tail domain is recognized posttranslationally by a specialized translocation apparatus after the N-tail has been translocated by Oxa1.     

Multiple roles of the Cox20 chaperone in assembly of Saccharomyces cerevisiae cytochrome c oxidase

The Cox2 subunit of Saccharomyces cerevisiae cytochrome c oxidase is synthesized in the mitochondrial matrix as a precursor whose leader peptide is rapidly processed by the inner membrane protease following translocation to the intermembrane space. Processing is chaperoned by Cox20, an integral inner membrane protein whose hydrophilic domains are located in the intermembrane space, and Cox20 remains associated with mature, unassembled Cox2. The Cox2 C-tail domain is exported post-translationally by the highly conserved translocase Cox18 and associated proteins. We have found that Cox20 is required for efficient export of the Cox2 C-tail. Furthermore, Cox20 interacts by co-immune precipitation with Cox18, and this interaction requires the presence of Cox2. We therefore propose that Cox20 binding to Cox2 on the trans side of the inner membrane accelerates dissociation of newly exported Cox2 from the Cox18 translocase, promoting efficient cycling of the translocase. The requirement for Cox20 in cytochrome c oxidase assembly and respiratory growth is partially bypassed by yme1, mgr1 or mgr3 mutations, each of which reduce i-AAA protease activity in the intermembrane space. Thus, Cox20 also appears to stabilize unassembled Cox2 against degradation by the i-AAA protease. Pre-Cox2 leader peptide processing by Imp1 occurs in the absence of Cox20 and i-AAA protease activity, but is greatly reduced in efficiency. Under these conditions some mature Cox2 is assembled into cytochrome c oxidase allowing weak respiratory growth. Thus, the Cox20 chaperone has important roles in leader peptide processing, C-tail export, and stabilization of Cox2.     

Peripheral mitochondrial inner membrane protein, Mss2p, required for export of the mitochondrially coded Cox2p C tail in Saccharomyces cerevisiae

Cytochrome oxidase subunit 2 (Cox2p) is synthesized on the matrix side of the mitochondrial inner membrane, and its N- and C-terminal domains are exported across the inner membrane by distinct mechanisms. The Saccharomyces cerevisiae nuclear gene MSS2 was previously shown to be necessary for Cox2p accumulation. We have used pulse-labeling studies and the expression of the ARG8(m) reporter at the COX2 locus in an mss2 mutant to demonstrate that Mss2p is not required for Cox2p synthesis but rather for its accumulation. Mutational inactivation of the proteolytic function of the matrix-localized Yta10p (Afg3p) AAA-protease partially stabilizes Cox2p in an mss2 mutant but does not restore assembly of cytochrome oxidase. In the absence of Mss2p, the Cox2p N terminus is exported, but Cox2p C-terminal export and assembly of Cox2p into cytochrome oxidase is blocked. Epitope-tagged Mss2p is tightly, but peripherally, associated with the inner membrane and protected by it from externally added proteases. Taken together, these data indicate that Mss2p plays a role in recognizing the Cox2p C tail in the matrix and promoting its export.     

Identification of Cox20p, a novel protein involved in the maturation and assembly of cytochrome oxidase subunit 2

We have identified Cox20p, a 23.8-kDa protein of the mitochondrial inner membrane that is involved in the biogenesis of the yeast cytochrome oxidase complex. Cytochrome oxidase subunit 2 (Cox2p) accumulates as a precursor in cox20 mutants, suggesting a defect in biogenesis of this mitochondrially encoded protein. The inability of cox20 mutants to process the subunit 2 precursor (pCox2p) is not due to impaired export of the protein across the inner membrane or to an inactive Imp1p/Imp2p peptidase. Rather, Cox20p specifically binds the newly synthesized pCox2p, a step required to present the exported pCox2p as a substrate to the Imp1p peptidase. All of the endogenous pCox2p accumulated in an Deltaimp1 mutant, and a small fraction of Cox2p in wild type yeast, is detected in a complex with Cox20p. Following maturation Cox2p remained associated with Cox20p, prior to assembling into the cytochrome oxidase complex. We propose that Cox20p acts as a membrane-bound chaperone necessary for cleavage of pCox2p and for interaction of the mature protein with other subunits of cytochrome oxidase in a later step of the assembly process.     

Membrane translocation of mitochondrially coded Cox2p: distinct requirements for export of N and C termini and dependence on the conserved protein Oxa1p.

To study in vivo the export of mitochondrially synthesized protein from the matrix to the intermembrane space, we have fused a synthetic mitochondrial gene, ARG8m, to the Saccharomyces cerevisiae COX2 gene in mitochondrial DNA. The Arg8mp moiety was translocated through the inner membrane when fused to the Cox2p C terminus by a mechanism dependent on topogenic information at least partially contained within the exported Cox2p C-terminal tail. The pre-Cox2p leader peptide did not signal translocation. Export of the Cox2p C-terminal tail, but not the N-terminal tail, was dependent on the inner membrane potential. The mitochondrial export system does not closely resemble the bacterial Sec translocase. However, normal translocation of both exported domains of Cox2p was defective in cells lacking the widely conserved inner membrane protein Oxa1p.     

Mutations Affecting a Yeast Mitochondrial Inner Membrane Protein, Pnt1p, Block Export of a Mitochondrially Synthesized Fusion Protein from the Matrix

The machinery that inserts mitochondrially encoded proteins into the inner membrane and translocates their hydrophilic domains through the membrane is poorly understood. We have developed a genetic screen for Saccharomyces cerevisiae mutants defective in this export process. The screen is based on the fact that the hydrophilic polypeptide Arg8(m)p is exported from the matrix if it is synthesized within mitochondria as a bifunctional Cox2p-Arg8(m)p fusion protein. Since export of Arg8(m)p causes an Arg(-) phenotype, defective mutants can be selected as Arg(+). Here we show that mutations in the nuclear gene PNT1 block the translocation of mitochondrially encoded fusion proteins across the inner membrane. Pnt1p is a mitochondrial integral inner membrane protein that appears to have two hydrophilic domains in the matrix, flanking a central hydrophobic hairpin-like anchor. While an S. cerevisiae pnt1 deletion mutant was more sensitive to H(2)O(2) than the wild type was, it was respiration competent and able to export wild-type Cox2p. However, deletion of the PNT1 orthologue from Kluyveromyces lactis, KlPNT1, caused a clear nonrespiratory phenotype, absence of cytochrome oxidase activity, and a defect in the assembly of KlCox2p that appears to be due to a block of C-tail export. Since PNT1 was previously described as a gene affecting resistance to the antibiotic pentamidine, our data support a mitochondrial target for this drug.     

Translocation and Assembly of Mitochondrially Coded Saccharomyces cerevisiae Cytochrome c Oxidase Subunit Cox2 by Oxa1 and Yme1 in the Absence of Cox18

Members of the Oxa1/YidC/Alb3 family of protein translocases are essential for assembly of energy-transducing membrane complexes. In Saccharomyces cerevisiae, Oxa1 and its paralog, Cox18, are required for assembly of Cox2, a mitochondrially encoded subunit of cytochrome c oxidase. Oxa1 is known to be required for cotranslational export of the Cox2 N-terminal domain across the inner mitochondrial membrane, while Cox18 is known to be required for post-translational export of the Cox2 C-tail domain. We find that overexpression of Oxa1 does not compensate for the absence of Cox18 at the level of respiratory growth. However, it does promote some translocation of the Cox2 C-tail domain across the inner membrane and causes increased accumulation of Cox2, which remains unassembled. This result suggests that Cox18 not only translocates the C-tail, but also must deliver it in a distinct state competent for cytochrome oxidase assembly. We identified respiring mutants from a cox18Δ strain overexpressing OXA1, whose respiratory growth requires overexpression of OXA1. The recessive nuclear mutations allow some assembly of Cox2 into cytochrome c oxidase. After failing to identify these mutations by methods based on transformation, we successfully located them to MGR1 and MGR3 by comparative hybridization to whole-genome tiling arrays and microarray-assisted bulk segregant analysis followed by linkage mapping. While Mgr1 and Mgr3 are known to associate with the Yme1 mitochondrial inner membrane i-AAA protease and to participate in membrane protein degradation, their absence does not appear to stabilize Cox2 under these conditions. Instead, Yme1 probably chaperones the folding and/or assembly of Oxa1-exported Cox2 in the absence of Mrg1 or Mgr3, since respiratory growth and cytochrome c oxidase assembly in a cox18 mgr3 double-mutant strain overexpressing OXA1 is YME1 dependent.CYTOCHROME c oxidase is the last enzyme in the pathway of aerobic respiration. Its catalytic core consists of the three largest subunits, Cox1, Cox2, and Cox3, which are encoded in mitochondrial DNA (mtDNA) in fungi and animals, and surrounded by nuclear gene products. The synthesis of these subunits and the assembly of active cytochrome oxidase is a highly complex process that requires the action of at least 30 nuclear genes in Saccharomyces cerevisiae (reviewed in Barrientos et al. 2002; Herrmann and Funes 2005; Cobine et al. 2006; Fontanesi et al. 2008). For example, functional expression of the mitochondrial COX2 gene specifically requires, at least, Pet111 to activate mRNA translation; Oxa1 for translocation of the N-terminal domain through the inner membrane; Cox20 to chaperone the processing of the Cox2 leader peptide by the inner membrane protease (Imp1, Imp2, and Som1); Cox18, Mss2, and Pnt1 to translocate the Cox2 C-terminal domain; and Sco1 and Cox17 to insert copper into the CuA site in the C-terminal domain. These functions generate a mature protein with two transmembrane helices in the inner membrane and N- and C-tail domains in the intermembrane space (IMS) that is assembled into the complex in steps involving additional factors.Oxa1 is the founding member of the Oxa1/YidC/Alb3 family of integral membrane proteins that facilitate the insertion of respiratory and energy-transducing complexes into bacterial, mitochondrial, and thylakoid membranes through protein translocase and membrane insertase activities (reviewed in Bonnefoy et al. 2009). Mitochondria of fungi, animals, and plants contain both Oxa1 proteins and paralogously related Cox18 (also known as Oxa2) proteins (Funes et al. 2004; Gaisne and Bonnefoy 2006). These proteins, and bacterial YidC proteins, share similar core topologies with five transmembrane domains. Oxa1 has a large C-terminal domain facing the matrix that interacts with mitochondrial ribosomes (Jia et al. 2003; Szyrach et al. 2003). Bacterial YidC and mitochondrial Cox18 proteins lack this domain.In S. cerevisiae, Oxa1 and Cox18 have distinct functions in the biogenesis of Cox2. Oxa1 is required for translocation of the Cox2 N-tail domain (He and Fox 1997) via a cotranslational mechanism. It is also required for translocation of the C-tail domain, although it is unclear whether this requirement involves direct participation of Oxa1 or reflects a requirement of prior N-tail topogenesis for C-tail export (Bonnefoy et al. 2009). Yeast Oxa1 also participates in the assembly of the ATP synthase (Altamura et al. 1996; Hell et al. 2001; Jia et al. 2007). Yeast Cox18 is not required for N-tail export, but in conjunction with the peripheral inner membrane protein Mss2 and the integral membrane protein Pnt1, Cox18 is required for the export of the Cox2 C-tail post-translationally and has no other known substrate (He and Fox 1999; Broadley et al. 2001; Saracco and Fox 2002; Fiumera et al. 2007). These observations show that Oxa1 alone is not capable of translocating the Cox2 C-tail, whether or not it directly participates in that reaction.S. cerevisiae OXA1 fails to complement cox18 mutations when overexpressed in otherwise wild-type cells (Saracco and Fox 2002). Similarly, COX18 fails to complement oxa1 mutations (L. E. Elliott, H. L. Fiumera and T. D. Fox, unpublished results), confirming that these proteins have distinct functions. While the precise roles of human Oxa1 and Cox18 in human cells have not been established (Stiburek et al. 2007), expression in yeast of cDNAs encoding these human proteins does partially complement the corresponding yeast mutations (Bonnefoy et al. 1994b; Gaisne and Bonnefoy 2006). Furthermore, expression of mitochondrially targeted Escherichia coli YidC in yeast partially complements cox18 mutations but not oxa1 mutations (Preuss et al. 2005). Addition of the yeast Oxa1 C-terminal ribosome-binding domain to YidC allows it to partially complement oxa1 mutations but not cox18 mutations.If Cox2 is correctly inserted into the inner membrane but prevented from assembling into cytochrome oxidase, it is degraded by a pathway largely dependent on Yme1 (Nakai et al. 1995; Pearce and Sherman 1995; Weber et al. 1996). Yme1 is a member of a conserved family of ATP-dependent AAA proteases (reviewed in Koppen and Langer 2007), whose human ortholog functions in yeast (Shah et al. 2000). Yme1 comprises the i-AAA protease, an integral inner membrane protein whose AAA and proteolytic domains are exposed in the IMS (Leonhard et al. 1996) where they interact with exported domains of Cox2 (Graef et al. 2007). When export of the Cox2 C-tail domain is prevented by an mss2 deletion, Cox2 is instead largely degraded by the m-AAA protease (Broadley et al. 2001), an enzyme homologous to Yme1 with catalytic domains in the matrix (Leonhard et al. 1996). The AAA domain of Yme1 exhibits the chaperone-like property of binding to unfolded substrates in isolated mitochondria and in vitro, an interaction that precedes degradation (Leonhard et al. 1999; Graef et al. 2007). However, Yme1 has not previously been shown to participate as a chaperone in the productive folding of mitochondrial proteins in vivo.In this study we have examined the phenotype of a nonrespiring cox18Δ deletion strain overproducing Oxa1 from multiple plasmid-borne copies of the wild-type OXA1 gene. Surprisingly, we found that overproduced Oxa1 does support limited export of the Cox2 C-tail domain, but cytochrome oxidase is not assembled. Thus, in wild-type cells Cox18 appears not only to translocate the Cox2 C-tail, but also to do so in a fashion that promotes its proper folding and/or assembly. Respiring mutants selected from this strain inactivate either of two recently discovered adaptor subunits of the i-AAA protease. Respiratory growth of these strains remains dependent upon Yme1 activity, suggesting that under these conditions Yme1 can function as a chaperone in the assembly of Cox2 into cytochrome oxidase.     

Accumulation of mitochondrially synthesized Saccharomyces cerevisiae Cox2p and Cox3p depends on targeting information in untranslated portions of their mRNAs.

The essential products of the yeast mitochondrial translation system are seven hydrophobic membrane proteins and Var1p, a hydrophilic protein in the small ribosomal subunit. Translation of the membrane proteins depends on nuclearly encoded, mRNA-specific translational activators that recognize the 5'-untranslated leaders of their target mRNAs. These translational activators are themselves membrane associated and could therefore tether translation to the inner membrane. In this study, we tested whether chimeric mRNAs with the untranslated sequences normally present on the mRNA encoding soluble Var1p, can direct functional expression of coding sequences specifying the integral membrane proteins Cox2p and Cox3p. DNA sequences specifying these chimeric mRNAs were inserted into mtDNA at the VAR1 locus and expressed in strains containing a nuclearly localized plasmid that supplies a functional form of Var1p, imported from the cytoplasm. Although cells expressing these chimeric mRNAs actively synthesized both membrane proteins, they were severely deficient in cytochrome c oxidase activity and in the accumulation of Cox2p and Cox3p, respectively. These data strongly support the physiological importance of interactions between membrane-bound mRNA-specific translational activators and the native 5'-untranslated leaders of the COX2 and COX3 mRNAs for localizing productive synthesis of Cox2p and Cox3p to the inner membrane.     

Mutational analysis of the Saccharomyces cerevisiae cytochrome c oxidase assembly protein Cox11p

Cox11p is an integral protein of the inner mitochondrial membrane that is essential for cytochrome c oxidase assembly. The bulk of the protein is located in the intermembrane space and displays high levels of evolutionary conservation. We have analyzed a collection of site-directed and random cox11 mutants in an effort to further define essential portions of the molecule. Of the alleles studied, more than half had no apparent effect on Cox11p function. Among the respiration deficiency-encoding alleles, we identified three distinct phenotypes, which included a set of mutants with a misassembled or partially assembled cytochrome oxidase, as indicated by a blue-shifted cytochrome aa(3) peak. In addition to the shifted spectral signal, these mutants also display a specific reduction in the levels of subunit 1 (Cox1p). Two of these mutations are likely to occlude a surface pocket behind the copper-binding domain in Cox11p, based on analogy with the Sinorhizobium meliloti Cox11 solution structure, thereby suggesting that this pocket is crucial for Cox11p function. Sequential deletions of the matrix portion of Cox11p suggest that this domain is not functional beyond the residues involved in mitochondrial targeting and membrane insertion. In addition, our studies indicate that Deltacox11, like Deltasco1, displays a specific hypersensitivity to hydrogen peroxide. Our studies provide the first evidence at the level of the cytochrome oxidase holoenzyme that Cox1p is the in vivo target for Cox11p and suggest that Cox11p may also have a role in the response to hydrogen peroxide exposure.     

Saccharomyces cerevisiae Cox18 complements the essential Sec-independent function of Escherichia coli YidC

Members of the YidC/Oxa1/Alb3 protein family function in the biogenesis of membrane proteins in bacteria, mitochondria and chloroplasts. In Escherichia coli, YidC plays a key role in the integration and assembly of many inner membrane proteins. Interestingly, YidC functions both in concert with the Sec-translocon and as a separate insertase independent of the translocon. Mitochondria of higher eukaryotes contain two distant homologues of YidC: Oxa1 and Cox18/Oxa2. Oxa1 is required for the insertion of membrane proteins into the mitochondrial inner membrane. Cox18/Oxa2 plays a poorly defined role in the biogenesis of the cytochrome c oxidase complex. Employing a genetic complementation approach by expressing the conserved region of yeast Cox18 in E. coli, we show here that Cox18 is able to complement the essential Sec-independent function of YidC. This identifies Cox18 as a bona fide member of the YidC/Oxa1/Alb3 family.     

Absence of the mitochondrial AAA protease Yme1p restores F0-ATPase subunit accumulation in an oxa1 deletion mutant of Saccharomyces cerevisiae

The nuclear gene OXA1 encodes a protein located within the mitochondrial inner membrane that is required for the biogenesis of both cytochrome c oxidase (Cox) and ATPase. In the absence of Oxa1p, the translocation of the mitochondrially encoded subunit Cox2p to the intermembrane space (also referred to as export) is prevented, and it has been proposed that Oxa1p could be a component of a general mitochondrial export machinery. We have examined the role of Oxa1p in light of its relationships with two mitochondrial proteases, the matrix protease Afg3p-Rca1p and the intermembrane space protease Yme1p, by analyzing the assembly and activity of the Cox and ATPase complexes in Deltaoxa1, Deltaoxa1Deltaafg3, and Deltaoxa1Deltayme1 mutants. We show that membrane subunits of both complexes are specifically degraded in the absence of Oxa1p. Neither Afg3p nor Yme1p is responsible for the degradation of Cox subunits. However, the F(0) subunits Atp4p, Atp6p, and Atp17p are stabilized in the Deltaoxa1Deltayme1 double mutant, and oligomycin-sensitive ATPase activity is restored, showing that the increased stability of the ATPase subunits allows significant translocation and assembly to occur even in the absence of Oxa1p. These results suggest that Oxa1p is not essential for the export of ATPase subunits. In addition, although respiratory function is dispensable in Saccharomyces cerevisiae, we show that the simultaneous inactivation of AFG3 and YME1 is lethal and that the essential function does not reside in their protease activity.     

Protein export across the inner membrane of mitochondria: the nature of translocated domains determines the dependence on the Oxa1 translocase

The biogenesis of mitochondria requires the insertion of both nuclear and mitochondrially encoded proteins into the inner membrane. The inner membrane protein Oxa1 plays an important role in this process. Translocation of the terminal intermembrane space domains of subunit 2 of the cytochrome oxidase complex, Cox2, strictly depends on Oxa1. In contrast, other Oxa1 substrates can be inserted independently of Oxa1 function, although at reduced efficiency. A Saccharomyces cerevisiae mutant containing a large deletion in its mitochondrial genome allowed us to analyze the insertion process of a fusion protein of cytochrome b and Cox2. In this mutant, the N-terminal domain of Cox2 is synthesized as a hairpin loop that is flanked by hydrophobic transmembrane segments on both sides. Both genetic and biochemical evidences indicate that translocation of this region across the inner membrane still requires Oxa1 function. Thus, the position of intermembrane space domains within protein sequences does not appear to determine their dependence on the Oxa1 translocase. Our observations rather suggest that the dependence on Oxa1 correlates with the net charge of the domain that has to be translocated across the lipid bilayer.     

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.     

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.     

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.     

Mutagenesis reveals a specific role for Cox17p in copper transport to cytochrome oxidase

The provision of copper to cytochrome oxidase is one of the requisite steps in the assembly of the holoenzyme. Several proteins are involved in this process including Cox17p, Sco1p, and Cox11p. Cox17p, an 8-kDa protein, is the only molecule thought to be involved in shuttling copper from the cytoplasm into mitochondria. Given the small size of Cox17p, we have taken a random and site-directed mutagenesis approach to studying structure-function relationships in Cox17p. Mutations have been generated in 70% of the Cox17p amino acid residues, with only a small subset leading to a detectable respiration-deficient phenotype. We have characterized the respiration-deficient cox17 mutants and found in addition to the expected cytochrome oxidase deficiency, a specific lack of Cox2p and the presence of a misassembled cytochrome oxidase in a subset of mutants. These results suggest that Cox17p is involved upstream of Sco1p in delivering copper specifically to subunit 2 of cytochrome oxidase and predict the existence of a subunit 1-specific copper chaperone.     

hCOX18 and hCOX19: two human genes involved in cytochrome c oxidase assembly

We identified the human homologues of yCOX18 and yCOX19, two Saccharomyces cerevisiae genes involved in the biogenesis of mitochondrial respiratory chain complexes. In yeast, these two genes are required for the expression of cytochrome c oxidase: Cox18p catalyses the insertion of Cox2p COOH-tail into the mitochondrial inner membrane, and Cox19p is probably involved in metal transport to the intermembrane space. Both hCox18p and hCox19p present significant amino acid identity with the corresponding yeast polypeptides and reveal highly conserved functional domains. In addition, their subcellular localization is analogous to that of the yeast proteins. These data strongly suggest that the human gene products share similar functions with their yeast homologues. These two COX-assembly genes represent new candidates for mutational analysis in patients with isolated COX deficiency of unknown etiology.     

Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae

Mutations in SURF1, the human homologue of yeast SHY1, are responsible for Leigh's syndrome, a neuropathy associated with cytochrome oxidase (COX) deficiency. Previous studies of the yeast model of this disease showed that mutant forms of Mss51p, a translational activator of COX1 mRNA, partially rescue the COX deficiency of shy1 mutants by restoring normal synthesis of the mitochondrially encoded Cox1p subunit of COX. Here we present evidence showing that Cox1p synthesis is reduced in most COX mutants but is restored to that of wild type by the same mss51 mutation that suppresses shy1 mutants. An important exception is a null mutation in COX14, which by itself or in combination with other COX mutations does not affect Cox1p synthesis. Cox14p and Mss51p are shown to interact with newly synthesized Cox1p and with each other. We propose that the interaction of Mss51p and Cox14p with Cox1p to form a transient Cox14p-Cox1p-Mss51p complex functions to downregulate Cox1p synthesis. The release of Mss51p from the complex occurs at a downstream step in the assembly pathway, probably catalyzed by Shy1p.