Das Disulfidbrücken-Transfer-System der Mitochondrien

Nahezu alle mitochondrialen Proteine sind im Zellkern kodiert und werden im Zytosol synthetisiert. Die Komplexe, die den Import von Außenmembran-, Innenmembran- und Matrixproteinen katalysieren, sind relativ gut untersucht. Der Import von Proteinen des mitochondrialen Intermembranraums ist dagegen weniger gut verstanden. Im Rahmen dieser Arbeit wurde der Importmechanismus für lösliche Intermembranraumproteine untersucht, die durch konservierte Cysteinmotive charakterisiert sind. Nach Mia40 konnte Erv1 als zweite Komponente dieses Importweges identifiziert werden. Mia40 interagiert mit neu importierten Intermembranraumproteinen mit konservierten Cysteinmotiven über Disulfidbrücken. Die Ausbildung dieser Disulfidbrücken ist essentiell für den Import der Proteine. Erv1 interagiert direkt mit Mia40 und erhält es im oxidierten, aktiven Zustand. Nur Oxidiertes Mia40 wirkt als Importrezeptor, der ein gemischtes Disulfid mit neu importierten Proteinen bildet. Durch Isomerisierung überträgt Mia40 seine Disulfidbrücke schließlich auf das Substratprotein, was zur Reduktion von Mia40 und zur stabilen Faltung des Substratproteins führt. Um importkompetentes Mia40 zu regenerieren, muss die Oxidation von Mia40 durch Erv1 erfolgen. Da in früheren Arbeiten reduziertes Tim13 in vivo nachgewiesen wurde, wurde in dieser Arbeit untersucht, ob sich möglicherweise ein Reduktionsschritt an den Mia40-Erv1-abhängigen Importprozess anschließt. Das Intermembranraumprotein Hot13 wurde zuvor als Assemblierungsfaktor für die kleinen Tim-Proteine beschrieben. Im Rahmen dieser Arbeit konnte aber keine Reduktaseaktivität von Hot13 nachgewiesen werden. Hot13 ist allerdings in der Lage, die Oxidation von Mia40 wahrscheinlich durch die Bindung von Zink zu unterstützen. Die Oxidation des metallfreien Mia40 wird so vereinfacht. Auf der Suche nach einer Reduktase im mitochondrialen Intermembranraum wurden zwei neuartige Glutaredoxine identifiziert, Grx6 und Grx7. Grx6 und Grx7 wurden allerdings im cis-Golgi-Apparat lokalisiert und konnten somit für den Importprozess in Mitochondrien ausgeschlossen werden. Dennoch sind sie aufgrund der Lokalisation im sekretorischen Transportweg von besonderem Interesse und ihre Glutaredoxinaktivität konnte in vitro nachgewiesen werden

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

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

Highlighting New Possibilities: A Comparison of SLOT and CT in the Analysis of 3D Printed Optical Elements

Novel printing techniques for optical elements produce characteristics in these optical elements which can sometimes not be resolved with classic testing or imaging methods. As highly individualized components often have a significant cost associated with them, destructive methods are unwanted. For this work, Scanning Laser Optical Tomography (SLOT) is considered to overcome these challenges and provide knowledge and data which would otherwise not be accessible. SLOT is directly compared to µCT which is often the go-to imaging modality for small, printed optics. Advantages such as the possibility to measure fluorescence with volumetric resolution are presented in this work in the application of a functionalized silicone waveguide. Interactions between the excitation light and the sample are also measured in SLOT in form of absorption. The importance of absorption is highlighted with the example of a 3D printed glass block which has light refractive layers that are invisible in µCT measurements. SLOT should be considered as a quality control tool by scientists and manufactures of printed optical elements as it has many advantages over µCT

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

Role of tryptophan residues of Erv1: Trp95 and Trp183 are important for its folding and oxidase function

Erv1 is an FAD-dependent sulphydryl oxidase of the ERV/ALR sub-family, and an essential component of the mitochondrial import and assembly pathway. Erv1 contains six tryptophan residues, which are all located in the highly conserved C-terminal FAD-binding domain. Though important structural roles were predicted for the invariable Trp95, no experimental study has been reported. In this study, we investigated the structural and functional roles of individual Trp residues of Erv1. Six single Trp-to-Phe yeast mutant strains were generated and their effects on cell viability were tested at various temperatures. Then, the mutants were purified from E. coli. Their effects on folding, FAD-binding, and Erv1 activity were characterised. Our results showed that Erv1W95F has the strongest effect on the stability and function of Erv1, and followed by Erv1W183F. Erv1W95F results in a decrease of the Tm of Erv1 by 23°C, a significant loss of the oxidase activity, and thus causing cell growth defects at both 30°C and 37°C. Erv1W183F induces changes in the oligomerisation state of Erv1, along with a pronounced effect on the stability of Erv1 and its function at 37°C, whilst the other mutants had no clear effect on the function of Erv1 including the highly conserved Trp157 mutant. Finally, computational analysis indicates that Trp95 plays a key role in stabilising the isoalloxazine ring to interact with Cys133. Taken together, this study provided important insights into the molecular mechanism of how sulfhydryl oxidases use FAD in catalyzing disulfide bond formation

An intermolecular disulfide-based light switch for chloroplast psbD gene expression in Chlamydomonas reinhardtii

Expression of the chloroplast psbD gene encoding the D2 protein of the photosystem II reaction center is regulated by light. In the green alga Chlamydomonas reinhardtii, D2 synthesis requires a high-molecular-weight complex containing the RNA stabilization factor Nac2 and the translational activator RBP40. Based on size exclusion chromatography analyses, we provide evidence that light control of D2 synthesis depends on dynamic formation of the Nac2/RBP40 complex. Furthermore, 2D redox SDS-PAGE assays suggest an intermolecular disulfide bridge between Nac2 and Cys11 of RBP40 as the putative molecular basis for attachment of RBP40 to the complex in light-grown cells. This covalent link is reduced in the dark, most likely via NADPH-dependent thioredoxin reductase C, supporting the idea of a direct relationship between chloroplast gene expression and chloroplast carbon metabolism during dark adaption of algal cells. © 2012 Blackwell Publishing Ltd.España Ministerio de Ciencia e Innovación BIO20010-15430Junta de Andalucía BIO-182 and CVI-591

A novel intermembrane space–targeting signal docks cysteines onto Mia40 during mitochondrial oxidative folding

A nine-residue intermembrane-targeting signal brings the active Cys of substrate proteins into contact with Mia40 oxidase for folding and import into mitochondria

Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli

<p>Abstract</p> <p>Background</p> <p>The formation of native disulfide bonds is a complex and essential post-translational modification for many proteins. The large scale production of these proteins can be difficult and depends on targeting the protein to a compartment in which disulfide bond formation naturally occurs, usually the endoplasmic reticulum of eukaryotes or the periplasm of prokaryotes. It is currently thought to be impossible to produce large amounts of disulfide bond containing protein in the cytoplasm of wild-type bacteria such as <it>E. coli </it>due to the presence of multiple pathways for their reduction.</p> <p>Results</p> <p>Here we show that the introduction of Erv1p, a sulfhydryl oxidase and FAD-dependent catalyst of disulfide bond formation found in the inter membrane space of mitochondria, allows the efficient formation of native disulfide bonds in heterologously expressed proteins in the cytoplasm of <it>E. coli </it>even without the disruption of genes involved in disulfide bond reduction, for example <it>trxB </it>and/or <it>gor</it>. Indeed yields of active disulfide bonded proteins were higher in BL21 (DE3) pLysSRARE, an <it>E. coli </it>strain with the reducing pathways intact, than in the commercial Δ<it>gor </it>Δ<it>trxB </it>strain rosetta-gami upon co-expression of Erv1p.</p> <p>Conclusions</p> <p>Our results refute the current paradigm in the field that disruption of at least one of the reducing pathways is essential for the efficient production of disulfide bond containing proteins in the cytoplasm of <it>E. coli </it>and open up new possibilities for the use of <it>E. coli </it>as a microbial cell factory.</p