The promoters of Arabidopsis thaliana genes AtCOX17-1 and -2, encoding a copper chaperone involved in cytochrome c oxidase biogenesis, are preferentially active in roots and anthers and induced by biotic and abiotic stress

AtCOX17 genes encode Arabidopsis thaliana homologs of the yeast metallochaperone Cox17p, involved in the delivery of copper for cytochrome c oxidase (COX) assembly. Two different AtCOX17 genes, located in chromosomes 1 and 3, are present in the Arabidopsis genome. Sequences available in data banks indicate that the presence of two genes is a common feature in monocots, but not in dicots, suggesting that Arabidopsis genes may be the result of a recent duplication. Sequences upstream from the translation start sites of AtCOX17 genes, which include an intron located in the 5' leader region, were introduced into plants in front of the gus gene. For both genes, expression was localized preferentially in young roots and anthers, but almost 10-fold higher β-glucuronidase activity levels were observed in plants transformed with AtCOX17-1 upstream regions. Both promoters were induced to different extents by wounding, treatment of leaves with the bacterial pathogen Pseudomonas syringae and incubation with agents that produce oxidative stress and metals. AtCOX17-2 showed similar responses to these factors, while AtCOX17-1 was more strongly induced by relatively low (10–100 μ M ) copper. The results indicate that both AtCOX17 genes have similar, though not identical, expression characteristics and suggest the existence in their promoters of elements involved in tissue-specific expression and in responses to factors that may produce mitochondrial or cell damage. It can be speculated that Arabidopsis COX17 accumulates under stress conditions to actively replace damaged or inactive cytochrome c oxidase to sustain cyanide-sensitive respiration in plant cells.     

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.     

Mutational analysis of the mitochondrial copper metallochaperone Cox17

The copper metallochaperone Cox17 is proposed to shuttle Cu(I) ions to the mitochondrion for the assembly of cytochrome c oxidase. The Cu(I) ions are liganded by cysteinyl thiolates. Mutational analysis on the yeast Cox17 reveals three of the seven cysteinyl residues to be critical for Cox17 function, and these three residues are present in a Cys-Cys-Xaa-Cys sequence motif. Single substitution of any of these three cysteines with serines results in a nonfunctional cytochrome oxidase complex. Cells harboring such a mutation fail to grow on nonfermentable carbon sources and have no cytochrome c oxidase activity in isolated mitochondria. Wild-type Cox17 purified as untagged protein binds three Cu(I) ions/molecule. Mutant proteins lacking only one of these critical Cys residues retain the ability to bind three Cu(I) ions and are imported within the mitochondria. In contrast, Cox17 molecules with a double Cys --> Ser mutation exhibit no Cu(I) binding but are still localized to the mitochondria. Thus, mitochondrial uptake of Cox17 is not restricted to the Cu(I) conformer of Cox17. COX17 was originally cloned by virtue of complementation of a mutant containing a nonfunctional Cys --> Tyr substitution at codon 57. The mutant C57Y Cox17 fails to accumulate within the mitochondria but retains the ability to bind three Cu(I) ions. A C57S Cox17 variant is functional, and a quadruple Cox17 mutant with C16S/C36S/C47S/C57S substitutions binds three Cu(I) ions. Thus, only three cysteinyl residues are important for the ligation of three Cu(I) ions. A novel mode of Cu(I) binding is predicted.     

Isolation of mutants with aberrant mitochondrial morphology from Arabidopsis thaliana

To identify genes related to plant mitochondrial morphology and dynamics, novel mutants with respect to mitochondrial morphology were isolated from an ethyl methane sulphonate (EMS)-mutated population of Arabidopsis thaliana. Mitochondria were visualized by transforming Arabidopsis with a gene for a fusion protein consisting of GFP and a mitochondria-targeting pre-sequence. From 19,000 M2 populations, 17 mutants were isolated by fluorescent microscopic observations. All mitochondria in these mutants were longer and/or larger than wild-type mitochondria. The approximate chromosomal loci of the mutations of seven mutants that grew well were determined. The mitochondrial phenotypes of six of the mutants were recessive but the mitochondrial phenotype of the seventh mutant was dominant. Chromosomal rough mapping of the seven mutants showed that the mutations occurred at four different loci. At least one of these loci was novel, i.e., it was different from loci of other known mitochondrial morphology mutants of Arabidopsis and different from loci of Arabidopsis homologues of yeast genes related to mitochondrial morphology.     

Isolation of a cDNA encoding the human homolog of COX17, a yeast gene essential for mitochondrial copper recruitment

The COX17 gene of Saccharomyces cerevisiae codes for a cytoplasmic protein essential for the expression of functional cytochrome oxidase. This protein has been implicated in targeting copper to mitochondria. To determine if Cox17p is present in mammalian cells, a yeast strain carrying a null mutation in COX17 was transformed with a human cDNA expression library. All the respiratory competent clones obtained from the transformations carried a common cDNA sequence with a reading frame predicting a product homologous to yeast Cox17p. The cloning of a mammalian COX17 homolog suggests that the encoded product is likely to function in copper recruitment in eucaryotic cells in general. Its presence in humans provides a possible target for genetically inherited deficiencies in cytochrome oxidase. Received: 22 August 1996 / Revised: 31 October 1996     

Identification,expression, and import of components 17 and 23 of the inner mitochondrial membrane translocase from Arabidopsis

Characterization of components 17 and 23 of the inner mitochondrial membrane translocase (TIM17:23) from Arabidopsis indicated that there were three genes present for TIM17 and TIM23 and two for TIM44. AtTIM17 differed from the yeast (Saccharomyces cerevisiae) and mammalian homologs in that two genes encoded proteins that were longer and one gene encoded a shorter protein. All Arabidopsis TIM23 predicted proteins appeared to lack the first 34 amino acids compared with yeast TIM23. All AtTIM17 and AtTIM23 genes were expressed but displayed different tissue and developmental profiles. Complementation of deletion mutants in yeast indicated that for AtTIM17, the extension at the C terminus not present in yeast had to be removed to achieve complementation, whereas for TIM23, a preprotein and amino acid transporter domain had to be present for complementation. Import assays with AtTIM17 and AtTIM23 indicated that they both contained internal signals for integration into the inner mitochondrial membrane in a membrane potential-dependent manner. The C terminus of imported AtTIM17-2 was susceptible to degradation by externally added protease with intact mitochondria. Removal of the 85 C-terminal amino acids resulted in import and full protection of the truncated protein. This suggests that the novel extension at the C terminus of AtTIM17-2 links the outer and inner membrane in a manner analogous to yeast TIM23.     

An<Emphasis Type="Italic"> Arabidopsis</Emphasis> homologue of bacterial RecA that complements an<Emphasis Type="Italic"> E. coli recA</Emphasis> deletion is targeted to plant mitochondria

Homologous recombination results in the exchange and rearrangement of DNA, and thus generates genetic variation in living organisms. RecA is known to function in all bacteria as the central enzyme catalyzing strand transfer and has functional homologues in eukaryotes. Most of our knowledge of homologous recombination in eukaryotes is limited to processes in the nucleus. The mitochondrial genomes of higher plants contain repeated sequences that are known to undergo frequent rearrangements and recombination events. However, very little is known about the proteins involved or the biochemical mechanisms of DNA recombination in plant mitochondria. We provide here the first report of an Arabidopsis thaliana homologue of Escherichia coli RecA that is targeted to mitochondria. The mt recA gene has a putative mitochondrial presequence identified from the A. thaliana genome database. This nuclear gene encodes a predicted product that shows highest sequence homology to chloroplast RecA and RecA proteins from proteobacteria. When fused to the GFP coding sequence, the predicted presequence was able to target the fusion protein to isolated mitochondria but not to chloroplasts. The mitochondrion-specific localization of the mt recA gene product was confirmed by Western analysis using polyclonal antibodies raised against a synthetic peptide from a unique region of the mature mtRecA. The Arabidopsis mt recA gene partially complemented a recA deletion in E. coli, enhancing survival after exposure to DNA-damaging agents. These results suggest a possible role for mt recA in homologous recombination and/or repair in Arabidopsis mitochondria.     

AtCCS is a functional homolog of the yeast copper chaperone Ccs1/Lys7

In plant chloroplasts two superoxide dismutase (SOD) activities occur, FeSOD and Cu/ZnSOD, with reciprocal regulation in response to copper availability. This system presents a unique model to study the regulation of metal-cofactor delivery to an organelle. The Arabidopsis thaliana gene AtCCS encodes a functional homolog to yeast Ccs1p/Lys7p, a copper chaperone for SOD. The AtCCS protein was localized to chloroplasts where it may supply copper to the stromal Cu/ZnSOD. AtCCS mRNA expression levels are upregulated in response to Cu-feeding and senescence. We propose that AtCCS expression is regulated to allow the most optimal use of Cu for photosynthesis.     

The plant biotin synthase reaction. Identification and characterization of essential mitochondrial accessory protein components

In plants, the last step of the biotin biosynthetic pathway is localized in mitochondria. This chemically complex reaction is catalyzed by the biotin synthase protein, encoded by the bio2 gene in Arabidopsis thaliana. Unidentified mitochondrial proteins in addition to the bio2 gene product are obligatory for the reaction to occur. In order to identify these additional proteins, potato mitochondrial matrix was fractionated onto different successive chromatographic columns. Combination experiments using purified Bio2 protein and the resulting mitochondrial matrix subfractions together with a genomic based research allowed us to identify mitochondrial adrenodoxin, adrenodoxin reductase, and cysteine desulfurase (Nfs1) proteins as essential components for the plant biotin synthase reaction. Arabidopsis cDNAs encoding these proteins were cloned, and the corresponding proteins were expressed in Escherichia coli cells and purified. Purified recombinant adrenodoxin and adrenodoxin reductase proteins formed in vitro an efficient low potential electron transfer chain that interacted with the bio2 gene product to reconstitute a functional plant biotin synthase complex. Bio2 from Arabidopsis is the first identified protein partner for this specific plant mitochondrial redox chain.     

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.     

A survey of the Arabidopsis thaliana mitochondrial phosphoproteome

Plant mitochondria play central roles in cellular energy production, metabolism and stress responses. Recent phosphoproteomic studies in mammalian and yeast mitochondria have presented evidence indicating that protein phosphorylation is a likely regulatory mechanism across a broad range of important mitochondrial processes. This study investigated protein phosphorylation in purified mitochondria from cell suspensions of the model plant Arabidopsis thaliana using affinity enrichment and proteomic tools. Eighteen putative phosphoproteins consisting of mitochondrial metabolic enzymes, HSPs, a protease and several proteins of unknown function were detected on 2‐DE separations of Arabidopsis mitochondrial proteins and affinity‐enriched phosphoproteins using the Pro‐Q Diamond phospho‐specific in‐gel dye. Comparisons with mitochondrial phosphoproteomes of yeast and mouse indicate that these three species share few validated phosphoproteins. Phosphorylation sites for seven of the eighteen mitochondrial proteins were characterized by titanium dioxide enrichment and MS/MS. In the process, 71 phosphopeptides from Arabidopsis proteins which are not present in mitochondria but found as contaminants in various types of mitochondrial preparations were also identified, indicating the low level of phosphorylation of mitochondrial components compared with other cellular components in Arabidopsis. Information gained from this study provides a better understanding of protein phosphorylation at both the subcellular and the cellular level in Arabidopsis.     

Mitochondrial localization of AtOXA1, an arabidopsis homologue of yeast Oxa1p involved in the insertion and assembly of protein complexes in mitochondrial inner membrane

Components of some protein complexes present in the inner membrane of mitochondria are encoded in both nuclear and mitochondrial genomes, and correct sorting and assembly of these proteins is necessary for proper respiratory function. Recent studies in yeast suggest that Oxa1p, a protein conserved between prokaryotes and eukaryotes, is an essential factor for protein sorting and assembly into membranes. We previously identified AtOXA1, an Arabidopsis homologue of OXA1 by functional complementation of a yeast oxa1- mutant. In this study, we investigated the genomic organization of AtOXA1 and localization of the AtOXA1 protein. Characterization of the AtOXA1 genomic region indicated that the gene consists of 10 exons and is located on chromosome V. A database search also revealed another gene coding for a putative protein homologous to AtOXA1 on chromosome II. Transient expression of a green fluorescent protein (GFP) fusion in suspension-cultured tobacco cells showed that AtOXA1 is targeted into mitochondria by its N-terminal presequence. Antibodies raised against AtOXA1 recognized a 38-kDa intrinsic protein of the inner mitochondrial membrane. Thus, localization of AtOXA1 in the mitochondrial inner membrane, together with our previous complementation experiment in yeast, suggested that it is a functional homologue of Oxa1p.     

A novel Arabidopsis gene causes Bax-like lethality in Saccharomyces cerevisiae

Overexpression of the mammalian proapoptotic protein Bax induces cell death in plant and yeast cells. The Bax inihibitor-1 (BI-1) gene rescues yeast and plant from Bax-mediated lethality. Using the Arabidopsis BI-1 (AtBI-1) gene controlled by the GAL1 promoter as a cell death suppressor in yeast, Cdf1 (cell growth defect factor-1) was isolated from Arabidopsis cDNA library. Overexpression of Cdf1 caused cell death in yeast, whereas such an effect was suppressed by co-expression of AtBI-1. The Cdf1 protein fused with a green fluorescent protein was localized in the mitochondria and resulted in the loss of mitochondrial membrane potential in yeast. The Bax-resistant mutant BRM1 demonstrated tolerance against Cdf1-mediated lethality, whereas the Deltaatp4 strain was sensitive to Cdf1. Our results suggest that Cdf1 and Bax cause mitochondria-mediated yeast lethality through partially overlapped pathways.