CHAPTER 1 Introduction
1.3 Intracellular copper trafficking
Once copper enters the cell, soluble Cu+-binding metallochaperones bind the ion and deliver it to target proteins through protein-protein interactions (Rosenzweig, 2002). Although it is not clear how these chaperones are loaded with Cu+, it is thought to perhaps be through a direct interaction with the Ctr1 transporter (Xiao et al., 2004). Well characterised pathways of copper delivery include those for CCS (Copper Chaperone for Superoxide dismutase), which delivers
1. 2. 3. 4.
copper to Cu,Zn superoxide dismutase (SOD), as well as for Atox1, which transfers copper to the membrane-bound, copper transporting P-type ATPases in the secretory pathway (Figure 1.4) (Kim et al., 2008; Leary et al., 2009; Lutsenko, 2010). Copper is also required in the mitochondrion for the maturation and activity of cytochrome-c oxidase and Cu,Zn superoxide dismutase. In this regard, the copper metallochaperones Cox11, Cox17, Cox19, Sco1 and Sco2 are essential for the formation of a functional cytochrome-c oxidase complex (Cobine et al., 2006a; Leary et al., 2009). The mechanism by which copper is delivered to the mitochondrion does, however, require further elucidation (Kim et al., 2008; Lutsenko, 2010;
Rees and Thiele, 2004).
1.3.1 Copper trafficking to the mitochondrion
Based on the localisation of Cox17 to the cytosol and mitochondrial intermembrane space, it was originally believed that this small, cysteine-rich protein was responsible for the delivery of copper to the mitochondrion (Beers et al., 1997; Glerum et al., 1996). The simple prediction was that Cox17 would shuttle Cu+ ions into the intermembrane space for use in the assembly of cytochrome-c oxidase. This idea was supported by the finding that purified Cox17 could bind copper (Heaton et al., 2001; Palumaa et al., 2004; Srivastava et al., 1997). However, by tethering Cox17 to the mitochondrial inner membrane only it was established that the cox17Δ cells displayed normal cytochrome-c oxidase activity, thereby reversing the expected respiratory defect. This therefore suggested that perhaps the function of Cox17 was spatially restricted to the intermembrane space (Maxfield et al., 2004). Consequently, it was proposed that Cox17 was still required for the delivery of copper to cytochrome-c oxidase, but it may not be metallated until reaching the intermembrane space. This was supported by the finding that a deletion of COX17 did not decrease the total mitochondria-associated copper levels (Carr and Winge, 2003). It was found that the bulk of mitochondrial copper was localised within the matrix in a soluble, low molecular weight complex (Figure 1.8). This complex contains Cu+, is resistant to proteinase K or trypsin digestion, is anionic and is eluted in a monodisperse fraction on reverse phase HPLC (Cobine et al., 2004). Furthermore, it was found that the complex could be depleted of Cu+ by two heterologously expressed copper binding proteins, human SOD1 or yeast Crs5 metallothionein, suggesting it could be involved in a copper delivery mechanism (Cobine et al., 2006b).
Figure 1.8 Metallochaperone-mediated loading of copper into mitochondrial cytochrome-c oxidase
Copper is shuttled from the cytoplasm to the mitochondrion by an as yet incompletely characterised mechanism. It is thought that a small ligand (denoted L) binds cytosolic copper and delivers it to the mitochondrial intermembrane space (IMS). The Cu+ ions are then bound by Cox17 for transfer to Sco1, which transfers copper to the Cox2 subunit, or to Cox11, which delivers copper to the Cox1 subunit of cytochrome-c oxidase (CCO). The novel copper ligand is thought to perhaps be involved in mitochondrial copper storage as well. Adapted from Kim et al. (2008).
Although the precise mechanism of copper delivery to the mitochondrion remains to be fully elucidated, the importance of copper to cytochrome-c oxidase assembly and function remains well documented (Cobine et al., 2006a; Kim et al., 2008; Leary et al., 2009). Copper is one of the catalytic cofactors in cytochrome-c oxidase, therefore copper insertion into this enzyme is essential for its function (Tsukihara et al., 1996). A number of accessory factors are important for the copper metallation of cytochrome-c oxidase. These include Cox11, Cox17, Sco1 and Sco2 (Figure 1.8), although Sco2 is not required in yeast cells (Cobine et al., 2006a). Three copper ions are inserted into two sites of cytochrome-c oxidase; a single ion into the CuB site and two ions into the CuA site. These sites are located in the Cox1 and Cox2 subunits respectively, which form part of the core cytochrome-c oxidase enzyme. The insertion of copper into the CuA and CuB sites is likely to occur in the intermembrane space with Sco1 and Cox11 implicated as the respective donor molecules (Carr et al., 2002; Hiser et al., 2000; Lode et al., 2000). These two co-metallochaperones obtain copper ions from Cox17 through transient interactions mediated by distinct structural interfaces (Horng et al., 2004). Copper transfer from Cox17 to Sco1 was also found to be coupled with a transfer of electrons, which was thought to perhaps assist with selective copper delivery (Banci et al., 2008a). The essential requirement for Cox17-mediated copper delivery is perhaps best highlighted by embryonic lethality in Cox17 null mice (Takahashi et al., 2002). Additionally yeast cells lacking Cox17 are respiratory deficient due to a lack of cytochrome-c oxidase activity, however, this mutant phenotype can be
suppressed by the addition of high copper salt concentrations to the growth medium (Glerum et al., 1996).
1.3.2 Structural features of Cox17
The Cox17 metallochaperone can readily adopt multiple oligomeric states and is thus capable of forming a number of distinct Cu+ conformers (Arnesano et al., 2005; Heaton et al., 2001;
Voronova et al., 2007a). However, in the intermembrane space the bioactive conformer of Cox17 appears to consist of a single Cu+ ion coordinated to a monomeric protein stabilised by two disulfide bonds (Abajian et al., 2004; Banci et al., 2008b; Voronova et al., 2007b). This was supported by the finding that, in vitro, the partially oxidised state of Cox17 (Cox172S-S), containing a single copper ion, and not the fully oxidised form, containing four copper ions, transfers Cu+ to apoWT-hSco1 (Banci et al., 2007). In this active state, human Cox17 is structurally organised in a coiled-coil-helix-coiled-coil-helix (CHCH) structural motif with a flexible amino terminal tail. The single copper ion bound to this conformer is coordinated by two consecutive cysteine residues at positions C22 and C23 (Banci et al., 2008b). The additional Cox17 cysteine residues are organised in a twin CX9C motif, located at the amino and carboxy- terminal ends of each helix of the CHCH motif, which form two interhelical disulfide bonds (Banci et al., 2008b). It has, however, been suggested for yeast Cox17 that only three of the six conserved cysteine residues are essential for in vivo function (Heaton et al., 2000; Punter and Glerum, 2003). These three essential cysteine residues are present in a C23CxC26 motif and are involved in copper coordination (Abajian et al., 2004; Heaton et al., 2000).
The import of Cox17, into the mitochondrial intermembrane space, is dependent on an oxidative folding mechanism. This is achieved by the TOM complex through a Mia40/Erv1 disulfide-relay process (Mesecke et al., 2005). Mia40 and Erv1 are located within the intermembrane space and mediate the import of small, cysteine-rich Tim proteins in addition to Cox17 (Rissler et al., 2005). Erv1 catalyses the formation of disulfides in Mia40 allowing it to transiently capture imported proteins, containing reduced thiolates, by forming interchain disulfide bonds. The bound polypeptides are then released by disulfide exchange reactions resulting in disulfide bond formation within the imported protein (Mesecke et al., 2005). Due to the highly reducing conditions of the cell cytosol, it is presumed that Cox17 exists in this environment in its fully reduced molten globule state, since this is necessary for its import into the intermembrane space (Mesecke et al., 2005). The two CX9C motifs present in Cox17 are also present in Mia40 (Chacinska et al., 2004), thus it is presumed that the similar folds found in these proteins could be involved in the protein-protein recognition process that occurs during
Cox17 entrapment in the intermembrane space (Banci et al., 2008b). Once in the intermembrane space Cox17 is predominantly matured into the Cox172S-S. This state is presumed to predominate as a result of the more oxidative redox environment of the intermembrane space compared to the cytosol (Voronova et al., 2007b). This redox environment would allow for the reduction of the easily reducible disulfide bond, between the cysteine residues involved in copper binding, whilst the two disulfides in the CHCH motif are highly stable toward reduction and therefore unaffected (Banci et al., 2008b). Cox172S-S would subsequently be loaded with copper in the intermembrane space for transfer to the two co- metallochaperones Sco1 and Cox11. These proteins then load copper into the CuA and CuB
sites of cytochrome-c oxidase.