Two novel disaccharides, rutinose and methylrutinose, are involved in carbon metabolism in Datisca glomerata.

Datisca glomerata forms nitrogen-fixing root nodules in symbiosis with soil actinomycetes from the genus Frankia. Analysis of sugars in roots, nodules and leaves of D. glomerata revealed the presence of two novel compounds that were identified as alpha-L-rhamnopyranoside-(1 --> 6)-D-glucose (rutinose) and alpha-L-rhamnopyranoside-(1 --> 6)-1-O-beta-D-methylglucose (methylrutinose). Rutinose has been found previously as a/the glycoside part of several flavonoid glycosides, e.g. rutin, also of datiscin, the main flavonoid of Datisca cannabina, but had not been reported as free sugar. Time course analyses suggest that both rutinose and methylrutinose might play a role in transient carbon storage in sink organs and, to a lesser extent, in source leaves. Their concentrations show that they can accumulate in the vacuole. Rutinose, but not methylrutinose, was accepted as a substrate by the tonoplast disaccharide transporter SUT4 from Arabidopsis. In vivo (14)C-labeling and the study of uptake of exogenous sucrose and rutinose from the leaf apoplast showed that neither rutinose nor methylrutinose appreciably participate in phloem translocation of carbon from source to sink organs, despite rutinose being found in the apoplast at significant levels. A model for sugar metabolism in D. glomerata is presented.

The disulfide relay of the intermembrane space of mitochondria: an oxygen-sensing system?

The intermembrane space of mitochondria contains many proteins that lack classical mitochondrial targeting sequences. Instead, these proteins often show characteristic patterns of cysteine residues that are critical for their accumulation in the organelle. Import of these proteins is catalyzed by two essential components, Mia40 and Erv1. Mia40 is a protein in the intermembrane space that directly binds newly imported proteins via disulfide bonds. By reorganization of these bonds, intramolecular disulfide bonds are formed in the imported proteins, which are thereby released from Mia40 into the intermembrane space. Because folded proteins are unable to traverse the import pore of the outer membrane, this leads to a permanent location of these proteins within the mitochondria. During this reaction, Mia40 becomes reduced and needs to be re-oxidized to regain its activity. Oxidation of Mia40 is carried out by Erv1, a conserved flavine adenine dinucleotide (FAD)-binding sulfhydryl oxidase. Erv1 directly interacts with Mia40 and shuttles electrons from reduced Mia40 to oxidized cytochrome c, from whence they flow through cytochrome oxidase to molecular oxygen. The connection of the disulfide relay with the respiratory chain not only significantly increases the efficiency of the oxidase activity, but also prevents the formation of potentially deleterious hydrogen peroxide. The oxidative activity of Erv1 strongly depends on the oxygen concentration in mitochondria. Erv1, therefore, may function as a molecular switch that adapts mitochondrial activities to the oxygen levels in the cell.

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.

A novel group of glutaredoxins in the cis-Golgi critical for oxidative stress resistance.

Glutaredoxins represent a ubiquitous family of proteins that catalyze the reduction of disulfide bonds in their substrate proteins by use of reduced glutathione. In an attempt to identify the full complement of glutaredoxins in baker's yeast, we found three so-far uncharacterized glutaredoxin-like proteins that we named Grx6, Grx7, and Grx8. Grx6 and Grx7 represent closely related monothiol glutaredoxins that are synthesized with N-terminal signal sequences. Both proteins are located in the cis-Golgi, thereby representing the first glutaredoxins found in a compartment of the secretory pathway. In contrast to formerly described monothiol glutaredoxins, Grx6 and Grx7, showed a high glutaredoxin activity in vitro. Grx6 and Grx7 overlap in their activity and deletion mutants lacking both proteins show growth defects and a strongly increased sensitivity toward oxidizing agents such as hydrogen peroxide or diamide. Our observations suggest that Grx6 and Grx7 do not play a general role in the oxidative folding of proteins in the early secretory pathway but rather counteract the oxidation of specific thiol groups in substrate proteins.

Two novel monothiol glutaredoxins from Saccharomyces cerevisiae provide further insight into iron-sulfur cluster binding, oligomerization, and enzymatic activity of glutaredoxins.

Two novel monothiol glutaredoxins from yeast (ScGrx6 and ScGrx7) were identified and analyzed in vitro. Both proteins are highly suited to study structure-function relationships of glutaredoxin subclasses because they differ from all monothiol glutaredoxins investigated so far and share features with dithiol glutaredoxins. ScGrx6 and ScGrx7 are, for example, the first monothiol glutaredoxins showing an activity in the standard glutaredoxin transhydrogenase assay with glutathione and bis-(2-hydroxyethyl)-disulfide. Steady-state kinetics of ScGrx7 with glutathione and cysteine-glutathione disulfide are similar to dithiol glutaredoxins and are consistent with a ping-pong mechanism. In contrast to most other glutaredoxins, ScGrx7 and ScGrx6 are able to dimerize noncovalently. Furthermore, ScGrx6 is the first monothiol glutaredoxin shown to directly bind an iron-sulfur cluster. The cluster can be stabilized by reduced glutathione, and its loss results in the conversion of tetramers to dimers. ScGrx7 does not bind metal ions but can be covalently modified in Escherichia coli leading to a mass shift of 1090 +/- 14 Da. What might be the structural requirements that cause the different properties? We hypothesize that a G(S/T)x3 insertion between a highly conserved lysine residue and the active site cysteine residue could be responsible for the abrogated transhydrogenase activity of many monothiol glutaredoxins. In addition, we suggest an active site motif without proline residues that could lead to the identification of further metal binding glutaredoxins. Such different properties presumably reflect diverse functions in vivo and might therefore explain why there are at least seven glutaredoxins in yeast.

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.

We 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.

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

Many 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 . 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.