“Pulling the plug” on cellular copper: The role of mitochondria in copper export

Mitochondria contain two enzymes, Cu,Zn superoxide dismutase (Sod1) and cytochrome c oxidase (CcO), that require copper as a cofactor for their biological activity. The copper used for their metallation originates from a conserved, bioactive pool contained within the mitochondrial matrix, the size of which changes in response to either genetic or pharmacological manipulation of cellular copper status. Its dynamic nature implies molecular mechanisms exist that functionally couple mitochondrial copper handling with other, extramitochondrial copper trafficking pathways. The recent finding that mitochondrial proteins with established roles in CcO assembly can also effect changes in cellular copper levels by modulating copper efflux from the cell supports a mechanistic link between organellar and cellular copper metabolism. However, the proteins and molecular mechanisms that link trafficking of copper to and from the organelle with other cellular copper trafficking pathways are unknown. This review documents our current understanding of copper trafficking to, and within, the mitochondrion for metallation of CcO and Sod1; the pathways by which the two copper centers in CcO are formed; and, the interconnections between mitochondrial function and the regulation of cellular copper homeostasis.     

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.     

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.     

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.     

Cmc1p is a conserved mitochondrial twin CX9C protein involved in cytochrome c oxidase biogenesis

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.     

Coa1 links the Mss51 post-translational function to Cox1 cofactor insertion in cytochrome c oxidase assembly

The assembly of cytochrome c oxidase (CcO) in yeast mitochondria is shown to be dependent on a new assembly factor designated Coa1 that associates with the mitochondrial inner membrane. Translation of the mitochondrial-encoded subunits of CcO occurs normally in coa1Delta cells, but these subunits fail to accumulate. The respiratory defect in coa1Delta cells is suppressed by high-copy MSS51, MDJ1 and COX10. Mss51 functions in Cox1 translation and elongation, whereas Cox10 participates in the biosynthesis of heme a, a key cofactor of CcO. Respiration in coa1Delta and shy1Delta cells is enhanced when Mss51 and Cox10 are coexpressed. Shy1 has been implicated in formation of the heme a3-Cu(B) site in Cox1. The interaction between Coa1 and Cox1, and the physical and genetic interactions between Coa1 and Mss51, Shy1 and Cox14 suggest that Coa1 coordinates the transition of newly synthesized Cox1 from the Mss51:Cox14 complex to the heme a cofactor insertion involving Shy1. coa1Delta cells also display a mitochondrial copper defect suggesting that Coa1 may have a direct link to copper metallation of CcO.     

Function and redox state of mitochondrial localized cysteine-rich proteins important in the assembly of cytochrome c oxidase

The cytochrome c oxidase (CcO) complex of the mitochondrial respiratory chain exists within the mitochondrial inner membrane (IM). The biogenesis of the complex is a multi-faceted process requiring multiple assembly factors that function on both faces of the IM. Formation of the two copper centers of CcO occurs within the intermembrane space (IMS) and is dependent on assembly factors with critical cysteinyl thiolates. Two classes of assembly factors exist, one group being soluble IMS proteins and the second class being proteins tethered to the IM. A common motif in the soluble assembly factors is a duplicated Cx(9)C sequence motif. Since mitochondrial respiration is a major source of reactive oxygen species, control of the redox state of mitochondrial proteins is an important process. This review documents the role of these cysteinyl CcO assembly factors within the IMS and the necessity of redox control in their function.     

Assembly factors and ATP-dependent proteases in cytochrome c oxidase biogenesis

Eukaryotic cytochrome c oxidase (CcO), the terminal enzyme of the energy-transducing mitochondrial electron transport chain is a hetero-oligomeric, heme–copper oxidase complex composed of both mitochondrially and nuclear-encoded subunits. It is embedded in the inner mitochondrial membrane where it couples the transfer of electrons from reduced cytochrome c to molecular oxygen with vectorial proton translocation across the membrane. The biogenesis of CcO is a complicated sequential process that requires numerous specific accessory proteins, so-called assembly factors, which include translational activators, translocases, molecular chaperones, copper metallochaperones and heme a biosynthetic enzymes. Besides these CcO-specific protein factors, the correct biogenesis of CcO requires an even greater number of proteins with much broader substrate specificities. Indeed, growing evidence indicates that mitochondrial ATP-dependent proteases might play an important role in CcO biogenesis. Out of the four identified energy-dependent mitochondrial proteases, three were shown to be directly involved in proteolysis of CcO subunits. In addition to their well-established protein-quality control function these oligomeric proteolytic complexes with chaperone-like activities may function as molecular chaperones promoting productive folding and assembly of subunit proteins. In this review, we summarize the current knowledge of the functional involvement of eukaryotic CcO-specific assembly factors and highlight the possible significance for CcO biogenesis of mitochondrial ATP-dependent proteases.     

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.     

Sulfur ligation in copper enzymes and models

Biological copper-sulfur entities display versatile and unusual coordination chemistry. The role of the sulfur ligation is briefly reviewed through examples from selected copper enzymes and relevant biomimetic models. Copper thiolate complexes are of particular interest because of their key roles in a number of ubiquitous metalloenzymes such as Type I (blue copper proteins) or in the binuclear Cu(A) electrons transfer site found in both cytochrome c oxidase (CcO) and nitrous oxide reductase (N2OR). The possible roles of the S(Met) ligand in monoxygenases are described in relation to recently proposed pathways. Some prospective regarding the biological relevance of disulfide copper ligation and possible radical copper bonds in catalytic cycle are also discussed.     

SOD2 functions downstream of Sch9 to extend longevity in yeast

Signal transduction pathways inactivated during periods of starvation are implicated in the regulation of longevity in organisms ranging from yeast to mammals, but the mechanisms responsible for life-span extension are poorly understood. Chronological life-span extension in S. cerevisiae cyr1 and sch9 mutants is mediated by the stress-resistance proteins Msn2/Msn4 and Rim15. Here we show that mitochondrial superoxide dismutase (Sod2) is required for survival extension in yeast. Deletion of SOD2 abolishes life-span extension in sch9Delta mutants and decreases survival in cyr1:mTn mutants. The overexpression of Sods--mitochondrial Sod2 and cytosolic CuZnSod (Sod1)--delays the age-dependent reversible inactivation of mitochondrial aconitase, a superoxide-sensitive enzyme, and extends survival by 30%. Deletion of the RAS2 gene, which functions upstream of CYR1, also doubles the mean life span by a mechanism that requires Msn2/4 and Sod2. These findings link mutations that extend chronological life span in S. cerevisiae to superoxide dismutases and suggest that the induction of other stress-resistance genes regulated by Msn2/4 and Rim15 is required for maximum longevity extension.     

Leporipoxvirus Cu,Zn-superoxide dismutase (SOD) homologs are catalytically inert decoy proteins that bind copper chaperone for SOD

Many Chordopoxviruses encode catalytically inactive homologs of cellular Cu-Zn superoxide dismutase (SOD). The biological function of these proteins is unknown, although the proteins encoded by Leporipoxviruses have been shown to promote a slow decline in the level of superoxide dismutase activity in virus-infected cells. To gain more insights into their function, we have further characterized the enzymatic and biochemical properties of a SOD homolog encoded by Shope fibroma virus. Shope fibroma virus SOD has retained the zinc binding properties of its cellular homolog, but cannot bind copper. Site-directed mutagenesis showed that it requires at least four amino acid substitutions to partially restore copper binding activity, but even these changes still did not restore catalytic activity. Reciprocal co-immunoprecipitation experiments showed that recombinant Shope fibroma virus SOD forms very stable complexes with cellular copper chaperones for SOD and these observations were confirmed using glutathione-S-transferase tagged proteins. Similar viral SOD/chaperone complexes were formed in cells infected with a closely related myxoma virus, where we also noted that some of the SOD antigen co-localizes with mitochondrial markers using confocal fluorescence microscopy. About 2% of the viral SOD was subsequently detected in gradient-purified mitochondria extracted from virus-infected cells. These poxviral SOD homologs do not form stable complexes with cellular Cu,Zn-SOD or affect its concentration. We suggest that Leporipoxvirus SOD homologs are catalytically inert decoy proteins that are designed to interfere in the proper metallation and activation of cellular Cu,Zn-SOD. This reaction might be advantageous for tumorigenic poxviruses, since higher levels of superoxide have been proposed to have anti-apoptotic and tumorigenic activity.     

Maintaining copper homeostasis: regulation of copper-trafficking proteins in response to copper deficiency or overload

Copper is an essential micronutrient that plays a vital role as a catalytic co-factor for a variety of metalloenzymes. The redox chemistry of copper also makes it a potentially toxic metal if not properly used. Therefore, elaborate mechanisms have evolved for controlling its cellular uptake, elimination, and distribution. In the last decade, our understanding of the systems involved in maintaining copper homeostasis has improved considerably with the characterization of copper transporters that mediate cellular copper uptake or efflux and with the identification of copper chaperones, a family of proteins required for delivering copper to specific targets in the cell. Despite the distinct roles of these proteins in copper trafficking, all seem able to respond to changes in copper status. Here, we describe recent advances in our knowledge of how copper-trafficking proteins respond to copper deficiency or overload in mammalian cells in order to maintain copper balance.     

Ortholog search of proteins involved in copper delivery to cytochrome C oxidase and functional analysis of paralogs and gene neighbors by genomic context

Cytochrome c oxidase (COX) is a multi-subunit enzyme of the mitochondrial respiratory chain. Delivery of metal cofactors to COX is essential for assembly, which represents a long-standing puzzle. The proteins Cox17, Sco1/2, and Cox11 are necessary for copper insertion into CuA and CuB redox centers of COX in eukaryotes. A genome-wide search in all prokaryotic genomes combined with genomic context reveals that only Sco and Cox11 have orthologs in prokaryotes. However, while Cox11 function is confined to COX assembly, Sco acts as a multifunctional linker connecting a variety of biological processes. Multifunctionality is achieved by gene duplication and paralogs. Neighbor genes of Sco paralogs often encode cuproenzymes and cytochrome c domains and, in some cases, Sco is fused to cytochrome c. This led us to suggest that cytochrome c might be relevant to Sco function and the two proteins might jointly be involved in COX assembly. Sco is also related, in terms of gene neighborhood and phylogenetic occurrence, to a newly detected protein involved in copper trafficking in bacteria and archaea, but with no sequence similarity to the mitochondrial copper chaperone Cox17. By linking the assembly system to the copper uptake system, Sco allows COX to face alternative copper trafficking pathways.     

Intracellular sterol dynamics

We review the cellular mechanisms implicated in cholesterol trafficking and distribution. Recent studies have provided new information about the distribution of sterols within cells, including analysis of its transbilayer distribution. The cholesterol interaction with other lipids and its engagement in various trafficking processes will determine its proper level in a specific membrane; making the cholesterol distribution uneven among the various intracellular organelles. The cholesterol content is important since cholesterol plays an essential role in membranes by controlling their physicochemical properties as well as key cellular events such as signal transduction and protein trafficking. Cholesterol movement between cellular organelles is highly dynamic, and can be achieved by vesicular and non-vesicular processes. Various studies have analyzed the proteins that play a significant role in these processes, giving us new information about the relative importance of these two trafficking pathways in cholesterol transport. Although still poorly characterized in many trafficking routes, several potential sterol transport proteins have been described in detail; as a result, molecular mechanisms for sterol transport among membranes start to be appreciated.     

Selective membrane protein trafficking: vectorial flow and filter.

Membrane proteins trafficking along cellular pathways encounter molecular filters. These filters can introduce them to new pathways and thus direct them towards new destinations. Many proteins carry molecular signals within their cytoplasmic domains that allow them to be selected by the filters. The detailed characterization of these signals is providing new insights into the pathways themselves and indicating the locations of other, as yet unidentified filters.     

Spatial proteomics of vesicular trafficking: coupling mass spectrometry and imaging approaches in membrane biology

In plants, membrane compartmentalization requires vesicle trafficking for communication among distinct organelles. Membrane proteins involved in vesicle trafficking are highly dynamic and can respond rapidly to changes in the environment and to cellular signals. Capturing their localization and dynamics is thus essential for understanding the mechanisms underlying vesicular trafficking pathways. Quantitative mass spectrometry and imaging approaches allow a system-wide dissection of the vesicular proteome, the characterization of ligand-receptor pairs and the determination of secretory, endocytic, recycling and vacuolar trafficking pathways. In this review, we highlight major proteomics and imaging methods employed to determine the location, distribution and abundance of proteins within given trafficking routes. We focus in particular on methodologies for the elucidation of vesicle protein dynamics and interactions and their connections to downstream signalling outputs. Finally, we assess their biological applications in exploring different cellular and subcellular processes.     

Calcium and copper transport ATPases: analogies and diversities in transduction and signaling mechanisms

The calcium transport ATPase and the copper transport ATPase are members of the P-ATPase family and retain an analogous catalytic mechanism for ATP utilization, including intermediate phosphoryl transfer to a conserved aspartyl residue, vectorial displacement of bound cation, and final hydrolytic cleavage of Pi. Both ATPases undergo protein conformational changes concomitant with catalytic events. Yet, the two ATPases are prototypes of different features with regard to transduction and signaling mechanisms. The calcium ATPase resides stably on membranes delimiting cellular compartments, acquires free Ca²⁺ with high affinity on one side of the membrane, and releases the bound Ca²⁺ on the other side of the membrane to yield a high free Ca²⁺ gradient. These features are a basic requirement for cellular Ca²⁺ signaling mechanisms. On the other hand, the copper ATPase acquires copper through exchange with donor proteins, and undergoes intracellular trafficking to deliver copper to acceptor proteins. In addition to the cation transport site and the conserved aspartate undergoing catalytic phosphorylation, the copper ATPase has copper binding regulatory sites on a unique N-terminal protein extension, and has also serine residues undergoing kinase assisted phosphorylation. These additional features are involved in the mechanism of copper ATPase intracellular trafficking which is required to deliver copper to plasma membranes for extrusion, and to the trans-Golgi network for incorporation into metalloproteins. Isoform specific glyocosylation contributes to stabilization of ATP7A copper ATPase in plasma membranes.