Biogenesis of proteins of the mitochondrial intermembrane space

All intermembrane space (IMS) proteins are synthesized in the cytosol and have to be imported into mitochondria. Many proteins of the IMS lack typical N-terminal targeting signals and are characterized by a small molecular mass and highly conserved cysteine residues present in characteristic patterns. These proteins cross the outer membrane of mitochondria via the TOM complex and need their cysteine residues for the efficient retention in the IMS. The aim of this study was to analyse whether specific factors are required for the import of these proteins into the mitochondrial IMS. The candidate protein, later termed Mia40 (mitochondrial intermembrane space import and assembly), was structurally and functionally characterized. The experiments presented here confirmed the mitochondrial location of Mia40 and determined its topology. Mia40 contains a classical N-terminal mitochondrial targeting signal followed by a hydrophobic segment. It is anchored in the inner membrane by a hydrophobic stretch and exposes a large C-terminal domain to the IMS. This domain harbours six highly conserved cysteine residues forming a CXC-CX9C-CX9C- motif (X represents non-cysteine amino acid residues). Since Mia40 is essential for viability of yeast, a strain harbouring the MIA40 gene under control of the glucose-repressible GAL10 promoter was used to study the function of Mia40 in mitochondria. Depletion of Mia40 resulted in strongly reduced levels of Tim13, Cox17 and of other IMS proteins with cysteine motifs, which was due to the impairment of their import into mitochondria. Mia40 is directly involved in the translocation of the small IMS proteins with conserved cysteine motifs: the newly imported IMS proteins form mixed disulfide intermediates with Mia40. In mitochondria, the majority of Mia40 is present in the oxidized state, thus allowing the formation of the mixed disulfide intermediates in an isomerization reaction. Subsequently, Mia40 transfers the disulfide bond from the mixed disulfide to the substrate proteins and thereby triggers the folding and the trapping of these proteins in the IMS. Mia40 is left in a partially reduced state and a reoxidation step is required for the next round of import. Erv1 is an essential FAD-containing sulfhydryl oxidase present in the IMS of fungi, plants and animals. The import of Tim13 was less efficient in mitochondria depleted of Erv1 and Mia40 interacted directly with Erv1 via disulfide bonds. In addition, the depletion of Erv1 affected the redox state of Mia40, which accumulated in a partially reduced state, suggesting that Erv1 is required for the recovery of the oxidized state of Mia40. Thus, Mia40 and Erv1 form a disulfide relay system mediating the import of small cysteine-rich proteins into the IMS. Erv1 passes its electrons further to cytochrome c, linking the import of small IMS proteins to the respiratory chain activity. Notably, Erv1 is not only a component but also a substrate of the disulfide relay system. It represents a novel type of substrate of the Mia40-mediated pathway. Thus, this pathway appears to be quite versatile and not limited to proteins with twin CX3C or twin CX9C motifs. The conserved cysteine residues in Mia40 are crucial for its function. Using single and double cysteine mutants of Mia40, it was possible to assign specific roles to each cysteine residue. In the oxidized state of Mia40 all cysteine residues form intramolecular disulfide bonds. The first two cysteine residues in the CPC motif compose a redox-sensitive disulfide bridge and breaking of this disulfide leads to Mia40 in the partially reduced state. The disulfide bond formed by the first two cysteine residues in Mia40 seems to be involved in the interaction with Erv1 and the substrate proteins, suggesting that it is essential for the catalysis of redox reactions of Mia40. The two other disulfide bonds connect the two CX9C fragments in Mia40 and most likely play a structural role. Taken together, the essential protein Mia40 is the central component of a novel translocation pathway. Mia40 together with Erv1 forms a disulfide relay system required for the import of small cysteine-rich proteins into the IMS of mitochondria

The disulfide relay system of mitochondria is connected to the respiratory chain

All proteins of the intermembrane space of mitochondria are encoded by nuclear genes and synthesized in the cytosol. Many of these proteins lack presequences but are imported into mitochondria in an oxidation-driven process that relies on the activity of Mia40 and Erv1. Both factors form a disulfide relay system in which Mia40 functions as a receptor that transiently interacts with incoming polypeptides via disulfide bonds. Erv1 is a sulfhydryl oxidase that oxidizes and activates Mia40, but it has remained unclear how Erv1 itself is oxidized. Here, we show that Erv1 passes its electrons on to molecular oxygen via interaction with cytochrome c and cytochrome c oxidase. This connection to the respiratory chain increases the efficient oxidation of the relay system in mitochondria and prevents the formation of toxic hydrogen peroxide. Thus, analogous to the system in the bacterial periplasm, the disulfide relay in the intermembrane space is connected to the electron transport chain of the inner membrane

A Disulfide Relay System in the Intermembrane Space of Mitochondria that Mediates Protein Import

SummaryWe describe here a pathway for the import of proteins into the intermembrane space (IMS) of mitochondria. Substrates of this pathway are proteins with conserved cysteine motifs, which are critical for import. After passage through the TOM channel, these proteins are covalently trapped by Mia40 via disulfide bridges. Mia40 contains cysteine residues, which are oxidized by the sulfhydryl oxidase Erv1. Depletion of Erv1 or conditions reducing Mia40 prevent protein import. We propose that Erv1 and Mia40 function as a disulfide relay system that catalyzes the import of proteins into the IMS by an oxidative folding mechanism. The existence of a disulfide exchange system in the IMS is unexpected in view of the free exchange of metabolites between IMS and cytosol via porin channels. We suggest that this process reflects the evolutionary origin of the IMS from the periplasmic space of the prokaryotic ancestors of mitochondria

In vivo imaging enables high resolution preclinical trials on patients' leukemia cells growing in mice.

Xenograft mouse models represent helpful tools for preclinical studies on human tumors. For modeling the complexity of the human disease, primary tumor cells are by far superior to established cell lines. As qualified exemplary model, patients' acute lymphoblastic leukemia cells reliably engraft in mice inducing orthotopic disseminated leukemia closely resembling the disease in men. Unfortunately, disease monitoring of acute lymphoblastic leukemia in mice is hampered by lack of a suitable readout parameter

Mia40, a novel factor for protein import into the intermembrane space of mitochondria is able to bind metal ions

AbstractMany proteins located in the intermembrane space (IMS) of mitochondria are characterized by a low molecular mass, contain highly conserved cysteine residues and coordinate metal ions. Studies on one of these proteins, Tim13, revealed that net translocation across the outer membrane is driven by metal-dependent folding in the IMS [1]. We have identified an essential component, Mia40/Tim40/Ykl195w, with a highly conserved domain in the IMS that is able to bind zinc and copper ions. In cells lacking Mia40, the endogenous levels of Tim13 and other metal-binding IMS proteins are strongly reduced due to the impaired import of these proteins. Furthermore, Mia40 directly interacts with newly imported Tim13 protein. We conclude that Mia40 is the first essential component of a specific translocation pathway of metal-binding IMS proteins

Functional characterization of Mia40p, the central component of the disulfide relay system of the mitochondrial intermembrane space

Mia40p and Erv1p are components of a translocation pathway for the import of cysteine-rich proteins into the intermembrane space of mitochondria. We have characterized the redox behavior of Mia40p and reconstituted the disulfide transfer system of Mia40p by using recombinant functional C-terminal fragment of Mia40p, Mia40C, and Erv1p. Oxidized Mia40p contains three intramolecular disulfide bonds. One disulfide bond connects the first two cysteine residues in the CPC motif. The second and the third bonds belong to the twin CX9C motif and bridge the cysteine residues of two CX9C segments. In contrast to the stabilizing disulfide bonds of the twin CX9C motif, the first disulfide bond was easily accessible to reducing agents. Partially reduced Mia40C generated by opening of this bond as well as fully reduced Mia40C were oxidized by Erv1p in vitro. In the course of this reaction, mixed disulfides of Mia40C and Erv1p were formed. Reoxidation of fully reduced Mia40C required the presence of the first two cysteine residues in Mia40C. However, efficient reoxidation of a Mia40C variant containing only the cysteine residues of the twin CX9C motif was observed when in addition to Erv1p low amounts of wild type Mia40C were present. In the reconstituted system the thiol oxidase Erv1p was sufficient to transfer disulfide bonds to Mia40C, which then could oxidize the variant of Mia40C. In summary, we reconstituted a disulfide relay system consisting of Mia40C and Erv1p

The zinc-binding protein Hot13 promotes oxidation of the mitochondrial import receptor Mia40

A disulphide relay system mediates the import of cysteine-containing proteins into the intermembrane space of mitochondria. This system consists of two essential proteins, Mia40 and Erv1, which bind to newly imported proteins by disulphide transfer. A third component, Hot13, was proposed to be important in the biogenesis of cysteine-rich proteins of the intermembrane space, but the molecular function of Hot13 remained unclear. Here, we show that Hot13, a conserved zinc-binding protein, interacts functionally and physically with the import receptor Mia40. It improves the Erv1-dependent oxidation of Mia40 both in vivo and in vitro. As a consequence, in mutants lacking Hot13, the import of substrates of Mia40 is impaired, particularly in the presence of zinc ions. In mitochondria as well as in vitro, Hot13 can be functionally replaced by zinc-binding chelators. We propose that Hot13 maintains Mia40 in a zinc-free state, thereby facilitating its efficient oxidation by Erv1