Darryl Horn

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 CuA site, the primary acceptor of electrons from ferrocytochrome c. The third copper, CuB, is associated with the high‐spin heme a3 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 CuA and CuB 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 CX9C 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 CX9C characteristic of Cox17p and its homologues. The different pathways are reviewed and discussed in the context of both mitochondrial COX assembly and copper homeostasis.

Suppression mechanisms of COX assembly defects in yeast and human: Insights into the COX assembly process

Eukaryotic cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial respiratory chain. COX is a multimeric enzyme formed by subunits of dual genetic origin whose assembly is intricate and highly regulated. In addition to the structural subunits, a large number of accessory factors are required to build the holoenzyme. The function of these factors is required in all stages of the assembly process. They are relevant to human health because devastating human disorders have been associated with mutations in nuclear genes encoding conserved COX assembly factors. The study of yeast strains and human cell lines from patients carrying mutations in structural subunits and COX assembly factors has been invaluable to attain the current state of knowledge, even if still fragmentary, of the COX assembly process. After the identification of the genes involved, the isolation and characterization of genetic and metabolic suppressors of COX assembly defects, reviewed here, have become a profitable strategy to gain insight into their functions and the pathways in which they operate. Additionally, they have the potential to provide useful information for devising therapeutic approaches to combat human disorders associated with COX deficiency.

Mss51 and Ssc1 Facilitate Translational Regulation of Cytochrome c Oxidase Biogenesis

ABSTRACT The intricate biogenesis of multimeric organellar enzymes of dual genetic origin entails several levels of regulation. In Saccharomyces cerevisiae, mitochondrial cytochrome c oxidase (COX) assembly is regulated translationally. Synthesis of subunit 1 (Cox1) is contingent on the availability of its assembly partners, thereby acting as a negative feedback loop that coordinates COX1 mRNA translation with Cox1 utilization during COX assembly. The COX1 mRNA-specific translational activator Mss51 plays a fundamental role in this process. Here, we report that Mss51 successively interacts with the COX1 mRNA translational apparatus, newly synthesized Cox1, and other COX assembly factors during Cox1 maturation/assembly. Notably, the mitochondrial Hsp70 chaperone Ssc1 is shown to be an Mss51 partner throughout its metabolic cycle. We conclude that Ssc1, by interacting with Mss51 and Mss51-containing complexes, plays a critical role in Cox1 biogenesis, COX assembly, and the translational regulation of these processes.

Cmc1p Is a Conserved Mitochondrial Twin CX9C Protein Involved in Cytochrome c Oxidase Biogenesis

ABSTRACT Copper is an essential cofactor of two mitochondrial enzymes: cytochrome c oxidase (COX) and Cu-Zn superoxide dismutase (Sod1p). Copper incorporation into these enzymes is facilitated by metallochaperone proteins which probably use copper from a mitochondrial matrix-localized pool. Here we describe a novel conserved mitochondrial metallochaperone-like protein, Cmc1p, whose function affects both COX and Sod1p. In Saccharomyces cerevisiae, Cmc1p localizes to the mitochondrial inner membrane facing the intermembrane space. Cmc1p is essential for full expression of COX and respiration, contains a twin CX9C domain conserved in other COX assembly copper chaperones, and has the ability to bind copper(I). Additionally, mutant cmc1 cells display increased mitochondrial Sod1p activity, while CMC1 overexpression results in decreased Sod1p activity. Our results suggest that Cmc1p could play a direct or indirect role in copper trafficking and distribution to COX and Sod1p.

Rcl1 Protein, a Novel Nuclease for 18 S Ribosomal RNA Production

Background: rRNAs are produced as precursors and require nucleases for maturation. Results: Recombinant Rcl1 cleaves pre-rRNA at the in vivo A2 site, and mutations abolish cleavage in vivo and in vitro. Conclusion: Rcl1 is the nuclease for co-transcriptional separation of rRNAs for the large and small subunit. Significance: Identification of the nucleases for rRNA production will allow accumulation and study of novel intermediates. In all forms of life, rRNAs for the small and large ribosomal subunit are co-transcribed as a single transcript. Although this ensures the equimolar production of rRNAs, it requires the endonucleolytic separation of pre-rRNAs to initiate rRNA production. In yeast, processing of the primary transcript encoding 18 S, 5.8 S, and 25 S rRNAs has been studied extensively. Nevertheless, most nucleases remain to be identified. Here, we show that Rcl1, conserved in all eukaryotes, cleaves pre-rRNA at so-called site A2, a co-transcriptional cleavage step that separates rRNAs destined for the small and large subunit. Recombinant Rcl1 cleaves pre-rRNA mimics at site A2 in a reaction that is sensitive to nearby RNA mutations that inhibit cleavage in vivo. Furthermore, mutations in Rcl1 disrupt rRNA processing at site A2 in vivo and in vitro. Together, these results demonstrate that the role of Rcl1 in eukaryotic pre-rRNA processing is identical to that of RNase III in bacteria: to co-transcriptionally separate the pre-rRNAs destined for the small and large subunit. Furthermore, because Rcl1 has no homology to other known endonucleases, these data also establish a novel class of nucleases.

Synthesis of cytochrome c oxidase subunit 1 is translationally downregulated in the absence of functional F1F0-ATP synthase

The mitochondrial F(1)F(0)-ATP synthase or ATPase is a key enzyme for aerobic energy production in eukaryotic cells. Mutations in ATPase structural and assembly genes are the primary cause of severe human encephalomyopathies, frequently associated with a pleiotropic decrease in cytochrome c oxidase (COX) activity. We have studied the structural and functional constraints underlying the COX defect using Saccharomyces cerevisiae genetic and pharmacological models of ATPase deficiency. In both yeast Deltaatp10 and oligomycin-treated wild type cells, COX assembly is selectively impaired in the absence of functional ATPase. The COX biogenesis defect does not involve a primary alteration in the expression of the COX subunits as previously suggested but in their maturation and/or assembly. Expression of COX subunit 1, however, is translationally regulated as in most bona fide COX assembly mutants. Additionally, the COX defect in oligomycin-inhibited ATPase-deficient yeast cells, but not in atp10 cells could be partially prevented by partially dissipating the mitochondrial membrane potential using the uncoupler CCCP. Similar results were obtained with oligomycin-treated and ATP12-deficient human fibroblasts respectively. Our findings imply that fully assembled ATPase and its proton pumping function are both required for COX biogenesis in yeast and mammalian cells through a mechanism independent of Cox1p synthesis.

The Conserved Mitochondrial Twin Cx9C Protein Cmc2 Is a Cmc1 Homologue Essential for Cytochrome c Oxidase Biogenesis

Mitochondrial copper metabolism and delivery to cytochrome c oxidase and mitochondrially localized CuZn-superoxide dismutase (Sod1) requires a growing number of intermembrane space proteins containing a twin Cx9C motif. Among them, Cmc1 was recently identified by our group. Here we describe another conserved mitochondrial metallochaperone-like protein, Cmc2, a close homologue of Cmc1, whose function affects both cytochrome c oxidase and Sod1. In the yeast Saccharomyces cerevisiae, Cmc2 localizes to the mitochondrial inner membrane facing the intermembrane space. In the absence of Cmc2, cytochrome c oxidase activity measured spectrophotometrically and cellular respiration measured polarographically are undetectable. Additionally, mutant cmc2 cells display 2-fold increased mitochondrial Sod1 activity, whereas CMC2 overexpression results in Sod1 activity decreased to 60% of wild-type levels. CMC1 overexpression does not rescue the respiratory defect of cmc2 mutants or vice versa. However, Cmc2 physically interacts with Cmc1 and the absence of Cmc2 induces a 5-fold increase in Cmc1 accumulation in the mitochondrial membranes. Cmc2 function is conserved from yeast to humans. Human CMC2 localizes to the mitochondria and CMC2 expression knockdown produces cytochrome c oxidase deficiency in Caenorhabditis elegans. We conclude that Cmc1 and Cmc2 have cooperative but nonoverlapping functions in cytochrome c oxidase biogenesis.

Mia40 Protein Serves as an Electron Sink in the Mia40-Erv1 Import Pathway

Background: Oxidized substrates such as Tim13 acquire two disulfide bonds simultaneously, but Mia40 has one active redox center that accepts two electrons. Results: Mia40 can acquire up to six electrons when oxidizing substrates. Conclusion: Mia40 has the flexibility to accept several electrons. Significance: Mechanistic properties of the MIA pathway are unique compared with redox pathways in the endoplasmic reticulum and bacterial periplasm. A redox-regulated import pathway consisting of Mia40 and Erv1 mediates the import of cysteine-rich proteins into the mitochondrial intermembrane space. Mia40 is the oxidoreductase that inserts two disulfide bonds into the substrate simultaneously. However, Mia40 has one redox-active cysteine pair, resulting in ambiguity about how Mia40 accepts numerous electrons during substrate oxidation. In this study, we have addressed the oxidation of Tim13 in vitro and in organello. Reductants such as glutathione and ascorbate inhibited both the oxidation of the substrate Tim13 in vitro and the import of Tim13 and Cmc1 into isolated mitochondria. In addition, a ternary complex consisting of Erv1, Mia40, and substrate, linked by disulfide bonds, was not detected in vitro. Instead, Mia40 accepted six electrons from substrates, and this fully reduced Mia40 was sensitive to protease, indicative of conformational changes in the structure. Mia40 in mitochondria from the erv1–101 mutant was also trapped in a completely reduced state, demonstrating that Mia40 can accept up to six electrons as substrates are imported. Therefore, these studies support that Mia40 functions as an electron sink to facilitate the insertion of two disulfide bonds into substrates.

Exploring Protein-Protein Interactions Involving Newly Synthesized Mitochondrial DNA-Encoded Proteins

Biogenesis of the mitochondrial respiratory chain enzymes involves the coordinated action of the mitochondrial and nuclear genomes. As a matter of fact, the structural sub-units forming these multimeric enzymes are encoded in both genomes. In addition, the assistance of nuclear encoded factors, termed assembly factors, is necessary to allow for the expression of the mitochondrial DNA-encoded subunits and to facilitate their maturation, membrane insertion, and further assembly into the corresponding enzymatic complex. These processes involve transient interactions among the newly synthesized mitochondrial products and specific assembly factors. The identification and characterization of these interactions can be achieved by the method described here, consisting of pulling down tagged versions of the assembly factors immediately after radiolabeling the mitochondrial translation products in isolated mitochondria, and analyzing the radiolabeled pulled-down material.