Import, maturation, and function of SOD1 and its copper chaperone CCS in the mitochondrial intermembrane space

Abstract

Cu, Zn, superoxide dismutase (SOD1) is a ubiquitous enzyme localized in multiple cellular compartments, including mitochondria, where it concentrates in the intermembrane space (IMS). Similar to other small IMS proteins, the import and retention of SOD1 in the IMS is linked to its folding and maturation, involving the formation of critical intra- and intermolecular disulfide bonds. Therefore, the cysteine residues of SOD1 play a fundamental role in its IMS localization. IMS import of SOD1 involves its copper chaperone, CCS, whose mitochondrial distribution is regulated by the Mia40/Erv1 disulfide relay system in a redox-dependent manner: CCS promotes SOD1 maturation and retention in the IMS. The function of SOD1 in the IMS is still unknown, but it is plausible that it serves to remove superoxide released from the mitochondrial respiratory chain. Mutations in SOD1 cause familial amyotrophic lateral sclerosis (ALS), whose pathologic features include mitochondrial bioenergetic dysfunction. Mutant SOD1 localization in the IMS is not dictated by oxygen concentration and the Mia40/Erv1 system, but is primarily dependent on aberrant protein folding and aggregation. Mutant SOD1 localization and aggregation in the IMS might cause the mitochondrial abnormalities observed in familial ALS and could play a significant role in disease pathogenesis.

FIG. 1.

This figure and corresponding figure legend were reprinted by permission from the Macmillan Publishers Ltd; Nature Structural Biology (33), copyright (2001). (A) The yeast SOD1 homodimer (PDB accession code 1SDY) viewed looking down the dimer twofold axis. The copper ion is shown as a blue sphere, and the zinc ion, as a gray sphere. (B) The heterodimer shown with the SOD1 monomer in the same orientation as in (A). The SOD1 monomer is shown in green, and the three domains of the yCCS monomer are shown in magenta (I), blue (II), and yellow (III). (C) The yCCS homodimer (PDB accession code 1QUP) shown in the same orientation as (A and B). Only domains I and II are present in this structure. Domain I is shown in pink, and domain II, in light blue. (D) Superposition of SOD1 in the heterodimer (dark green) with SOD1 in the homodimer (light green). The zinc ion is shown as a gray sphere, and the copper ion (present only in the light green monomer), as a blue sphere. Cys residues involved in disulfide formation are shown as ball-and-stick representations. (E) Superposition of yCCS in the heterodimer (domain I in magenta and domain II in dark blue) with yCCS in the homodimer (domain I in pink and domain II in light blue). The figure was generated by superposing the two domain IIs. The Cys residues from the domain I MHCXXC motif and the domain III CXC motif are shown as ball-and-stick representations. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 2.

Import of SOD1 and CCS into mitochondrial IMS. SOD1 and CCS are imported through the translocator of the outer membrane (TOM) into the mitochondrial IMS in their unfolded apoforms. In the IMS, apoCCS forms intermolecular disulfide bonds with the import receptor, Mia40, which results in the formation of CCS intramolecular disulfide bond that traps the protein in the IMS. Reduced Mia40 is reoxidized by the sulfhydryl oxidase, Erv1, which then donates its electron to cytochrome c to return to the oxidized state. SOD1 interacts with oxidized CCS through a transient intermolecular disulfide bond, which promotes the formation of the intramolecular disulfide, trapping SOD1 in the IMS. The resulting reduced CCS can then be reoxidized by the Mia40/Erv1 system.

FIG. 3.

Proposed pathway of copper distribution to the mitochondrial cupro-enzymes, COX and SOD1 (27). This scheme was kindly provided by Drs. Horn and Barrientos (University of Miami School of Medicine). Copper is translocated across the plasma membrane by specific transporters. Intracellular chaperones are presumably involved in delivering copper to mitochondria; however, the mechanisms of copper translocation across two layers of membranes to the matrix are still unknown. From the matrix pool, copper is translocated to the IMS, where it is distributed between Cmc1p for delivery to the COX copper chaperone system (Cox23, Cox19, Cox17, Sco1 and Sco2), which then loads it into COX subunits 1 and 2, and CCS for delivery to SOD1. Several players and steps in this complex mitochondrial copper-partitioning system, indicated by question marks in the scheme, remain to be elucidated. Nevertheless, this proposed pathway suggests the possibility that CCS-SOD1 and COX17-COX may compete for the same pool of copper in mitochondria.

FIG. 4.

Oxygen regulation of SOD1 and CCS import into the mitochondrial IMS. Both SOD1 and CCS cross the outer membrane through the general import pore (GIP) in their apo forms. In the IMS, mitochondrial CCS promotes folding and retention of SOD1. Under high-oxygen conditions (A), CCS folding is facilitated in the cytosol, where it enhances SOD1 folding and maturation. This allows cytosolic SOD1 to be effective in removal of superoxide produced outside of mitochondria. Under lower oxygen conditions (B), CCS and SOD1 maturation are delayed, and more apo-CCS and apo-SOD1 can enter mitochondria to remove superoxide released in this cell compartment.

FIG. 5.

Aggregation of mutant SOD1 in mitochondria and its potential pathogenic consequences. Mutant SOD1 enters the IMS through the general import pore (GIP) and forms disulfide-linked oligomeric aggregates. Such aggregates have been proposed to cause mitochondrial damage in different ways, including free radical production, NO generation, and copper depletion. These events lead to mitochondrial electron-transfer chain (ETC) dysfunction and loss of membrane potential (ΔΨ), which results in decreased ATP synthesis and impaired calcium uptake through the calcium uniporter.