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 IMSlocated 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.Mitochondria consist of two aqueous compartments, the matrix and the intermembrane space (IMS).2 The mitochondrial genome codes only for roughly a dozen different proteins, and the vast majority of mitochondrial proteins are nuclear encoded. Even simple organisms such as bakers' yeast contain several hundred matrix proteins. Matrix proteins are synthesized in the cytosol as precursors with N-terminal presequences (also referred to as matrix-targeting signals), which are processed following translocation. The import of matrix-destined preproteins is mediated by translocases in the outer membrane (TOM complex) and inner membrane (TIM23 complex) in an ATP-and membrane potential-dependent process (for review, see Refs. 1-3). Proteomic studies suggest that positively charged presequences are consistently found on all matrix proteins, although in some cases they are not proteolytically removed (4, 5).So far, ϳ50 different proteins were identified in the IMS of yeast mitochondria, and the list of IMS proteins is rapidly growing (6). The functions of these proteins are diverse. In addition to components involved in mitochondrial respiration, the IMS contains many proteins that transport proteins, metabolites, lipids, or metal ions between both mitochondrial membranes. In addition, several pro-apoptotic components are stored in the IMS and released when the cell death program is triggered (7).Some IMS proteins are synthesized in the cytosol as preproteins carrying bipartite presequences that consist of a matrixtargeting signal followed by a hydrophobic sorting region. The latter serves as a stop-transfer sequence that is inserted into the inner membrane during protein import and removed by IMSlocated proteases (8). Proteins with bipartite presequences embark on the general matrix-directed protein-targeting pathway, from which they are redirected into the IMS. However, many IMS proteins lack N-terminal targeting signals and are sorted into the IMS on a unique import route that differs in many respects from the matrix-targeting pathway. Import of many of these proteins relies on the mitochondrial disulfide relay, which is introduced below. Mitochondrial Disulfide Relay: Mia40 and Erv1The IMS proteins Mia40 (mitochondrial IMS import and assembly pathway 40 kDa) and Erv1 (essential for respiratory growth and viability 1) represent the central com...

Retro-translocation of mitochondrial intermembrane space proteins

“…Mia40 is a receptor, folding catalyst, and disulfide carrier, and the Erv1 protein serves as a sulfhydryl oxidase. The oxidative folding is believed to provide a trapping mechanism that prevents the escape of proteins from the IMS back to the cytosol (10,13,18). Our initial result raised a possibility that the reverse process can also occur, as we observed the relocation of in vitro imported Tim8 from mitochondria to the incubation buffer (13).…”

Mia40 targets cysteines in a hydrophobic environment to direct oxidative protein folding in the mitochondria

“…To examine whether this modification influenced binding, we performed experiments in which the energy donor Trp was placed in Mia40 and the acceptor AEDANS in Cox17*. Four single-cysteine variants of Cox17* were produced and labelled with AEDANS (variants [8][9][10][11]. Their interaction with the wild-type form of Mia40 was then measured in equilibrium titrations, and its kinetics was followed in the stopped-flow instrument by the FRET between the single Trp (W294) of Mia40 and the AEDANS groups of the four Cox17* variants ( Supplementary Fig.…”

“…In fact, the sliding-and-docking model 22 cannot readily account for the formation of the second disulphide in CX 3 C and CX 9 C substrates of Mia40, because the hydrophobic residues of the MISS/ITS motif become buried at the helix interface during folding and are thus no longer available for Mia40 to introduce the second disulphide. To remedy this situation, it was suggested that the second disulphide forms spontaneously independent of Mia40 by reaction with GSSG or with oxygen 11,23 .…”

“…T he intermembrane space (IMS) of the mitochondria was long believed to provide a reducing environment, similar to the cytosol, but recently proteins with disulphide bonds and subsequently a thiol oxidase (Mia40, mitochondrial import and assembly) were discovered in the IMS [1][2][3][4][5][6][7][8][9][10][11][12] . Unlike the other known thiol oxidases, Mia40 does not belong to the thioredoxin family, and its catalytic disulphide is arranged in a unique Cys-Pro-Cys motif.…”

“…Most, but not all, substrates of Mia40, belong to the families of CX 9 C and CX 3 C proteins 3,11,16 . Similar to Mia40 itself, these proteins are composed of two short antiparallel helices that are covalently linked by two disulphide bonds.…”