Mitochondrial disulfide relay: redox-regulated protein import into the intermembrane space

Abstract

99% of all mitochondrial proteins are synthesized in the cytosol, from where they are imported into mitochondria. In contrast to matrix proteins, many proteins of the intermembrane space (IMS) lack presequences and are imported in an oxidation-driven reaction by the mitochondrial disulfide relay. Incoming polypeptides are recognized and oxidized by the IMS-located receptor Mia40. Reoxidation of Mia40 is facilitated by the sulfhydryl oxidase Erv1 and the respiratory chain. Although structurally unrelated, the mitochondrial disulfide relay functionally resembles the Dsb (disufide bond) system of the bacterial periplasm, the compartment from which the IMS was derived 2 billion years ago.

FIGURE 1.

Components of mitochondrial disulfide relay. A, scheme of Mia40. This oxidoreductase contains a helix-loop-helix domain that is stabilized by two structural disulfide bonds. The domain is preceded by a short helical region that contains the redox-active CPC motif. To oxidize its substrates, the CPC motif has to be in an oxidized state. Moreover, the substrate should contain a MISS motif that is capable of binding to the hydrophobic groove formed by the helix-loop-helix domain of Mia40. B, scheme of Erv1. Erv1 is a homodimeric flavoprotein. Each subunit is composed of two domains: a four-helix bundle FAD domain and a flexible shuttle domain. Both domains contain redox-active CXXC motifs. During the reoxidation of Mia40, the CXXC motif in the shuttle domain of Erv1 forms an intermolecular disulfide with Mia40. Subsequently, electrons are passed onto the CXXC motif in the FAD domain of the other subunit of Erv1. From there, electrons are shuttled via the FAD cofactor to cytochrome c. In addition to its redox-active cysteine pairs, Erv1 also contains a structural disulfide bond that stabilizes the four-helix bundle of the FAD domain. The box shows a schematic presentation of the structure of one FAD domain of Erv1.

FIGURE 2.

Import of substrates into IMS of mitochondria. Substrates of the mitochondrial disulfide relay are synthesized on cytosolic ribosomes and are imported post-translationally. Notably, there are indications for a localized synthesis of several precursor proteins close to mitochondria, ensuring an efficient import into the organelle. The binding of mRNAs to the surface receptor Puf3 might restrict synthesis of some proteins to the mitochondrial surface (Class I), whereas others are synthesized on free ribosomes (Class III) (41). Mia40 substrates can traverse the TOM complex only in a reduced and unfolded state; thus, the proteins are likely stabilized by chaperones and zinc ions in the cytosol. As soon as the substrates enter the IMS, they are recognized by the oxidoreductase and import receptor Mia40 via their MISS motifs. The substrates and Mia40 form mixed disulfide bonds, and the substrates are correctly oriented on Mia40. Subsequently, oxidized substrates are released, and the now reduced Mia40 is reoxidized by Erv1, allowing the start of a new import cycle.

FIGURE 3.

Functions of proteins with disulfide bonds in IMS of mitochondria. Proteins with disulfide bonds are indicated in boldface. Names of substrate proteins that depend on the function or presence of disulfide-containing proteins are indicated in blue. See text for details. MCF, mitochondrial carrier family; ROS, reactive oxygen species.