Import of Copper chaperone for SOD1 (Ccs1) to inter-membrane space of mitochondria

The activity of Superoxides dismutase 1 (Sod1) is regulated by the copper chaperone for Sod1 (Ccs1), which introduces copper ions and disulfide bonds to Sod1. The majority of both proteins localizes to the cytosol with a minor portion targeted to the inter-membrane space (IMS) of mitochondria. The localization of Sod1 to the IMS depends on Ccs1 which in turn depends for import on Mia40, a component of redox-regulated import machinery. However, Ccs1 is larger than classical Mia40 substrates and does not contain the typical cysteine motif.
Using yeast, we investigated the effect of cysteine mutations in Ccs1 on its cellular distribution and found that mutants with substitutions at the cysteines 27 and 64 do not localize to the IMS. In the IMS, C27 and C64 formed a disulfide bond via interaction to Mia40. In contrast, only a part of the cytosolic Ccs1 molecules had this disulfide bond. In addition, we found that hydrophobic residues in the vicinity of C27 and C64 are required for the import to the IMS, which is well characterized for typical Mia40 substrates. We thus propose that Mia40-mediated disulfide bond formation between C27 and C64 controls the cellular distribution of Ccs1. We speculate that the inefficient interaction with Mia40, stemming from the absence of some typical features of Mia40 substrates, enables Ccs1 to dual-localize to the IMS and cytosol. Although Mia40 dependence of CCS1 localization in mammalian cells has been reported, cysteines corresponding to C27 and C64 in yeast Ccs1 are not conserved in mammalian proteins. This may indicate a weaker interaction between Ccs1 and Mia40 in mammalian cells than in yeast cells. We are currently characterizing this interaction.