Targeting of cytochrome b2 into the mitochondrial intermembrane space

Cytochrome b2 contains 2-fold targeting information: an amino-terminal signal for targeting to the mitochondrial matrix, followed by a second cleavable sorting signal that functions in directing the precursor into the mitochondrial intermembrane space. The role of the second sorting sequence was analyzed by replacing one, two or all of the three positively charged amino acid residues which are present at the amino-terminal side of the hydrophobic core by uncharged residues or an acidic residue. With a number of these mutant precursor proteins, processing to the mature form was reduced or completely abolished and at the same time targeting to the matrix space occurred. The accumulation in the matrix depended on a high level of intramitochondrial ATP. At low levels of matrix ATP, the mutant proteins were sorted into the intermembrane space like the wild-type precursors. The results: (i) suggest the existence of one or more matrix components that specifically recognize the second sorting signal and thereby trigger the translocation into the intermembrane space; (ii) indicate that the mutant signals have reduced ability to interact with the recognition component(s) and then embark on the default pathway into the matrix by interacting with mitochondrial hsp70 in conjunction with matrix ATP; (iii) strongly argue against a mechanism by which the hydrophobic segment of the sorting sequence stops translocation in the hydrophobic phase of the inner membrane

Recent results in chiral nuclear dynamics

Some recent developments in the description of nuclear forces and few-nucleon dynamics derived from chiral effective field theory are reviewed.Comment: invited talk at the International Workshop SCGT 02 "Strong Coupling Gauge Theories and Effective Field Theories", 10-13 December 2002, Nagoya, Japa

Cover Crop Productivity in Contrasting Growing Conditions and Influence on the Subsequent Crop

In arable systems, cover crops provide multiple ecosystem services involved in soil protection and fertility. They can efficiently compete against weeds, control erosion or recycle nutrients. The supply of these services is however largely dependent on cover crop growth as most of the services are related to biomass production. The choice of a cover crop adapted to the growing conditions is thus essential. Growing mixtures instead of sole crops is expected to be a way to overcome variable growing conditions and to insure high biomass production.Different field trials have been conducted from 2013 to 2016 in Nyon, Switzerland to assess cover crop biomass production and stability, and their influence on the following crop. Indian mustard, field pea, black oat and phacelia were sown as sole crops and in mixtures with different diversity levels (2, 3 and 4 species). A mixture of 11 species (50% of legumes and 50% of other species) was also tested. Biomass production was assessed about three months after seeding, just before the seeding of the next main crop, in two 0.5 m × 0.5 m quadrats. For each cover crop, a risk of failure, e.g. the probability of producing less than 3 t/ha (minimal biomass allowing to provide the services expected from cover crops), was estimated. Weed pressure was appraised by weed biomass in quadrats. The yield of the following main crop, here winter wheat, was determined after harvesting with a combine harvester, at wheat maturity, in summer. Sole crops showed very contrasting performance according to the growing conditions. Pea was the most productive in low yielding conditions with 2 t/ha while other sole crops produced only 0.5 t/ha. Pea was barely more productive in high yielding conditions, reaching only 3 t/ha when cover crop average was 6 t/ha. By contrast, mustard and oat showed high production potential in these conditions, exceeding 7 t/ha. Pea should thus be favoured when the growing conditions are clearly identified as limiting (low N availability), while oat or mustard should be chosen in favourable conditions. However, in general, conditions are hardly predictable. Our results showed that mixtures should be preferred as they were adapted to a wider range of environments than sole crops, performing well regardless of the conditions and resulting in a lower risk of failure than sole crops. The 11-specie mixture revealed that generally 4 to 5 species are sufficient to insure a good performance regardless of the conditions. Regarding cover crop effect on the following crop, we evidenced the importance of high biomass production for efficient weed control and positive influence on crop yield in no till treatments.Our results showed that cover crop mixtures rather than sole crops should be chosen as they insure high biomass production and thus a good supply of ecosystem services. In addition, cover crop cultivation, even for a short period, proved to be paramount to the maintenance of grain yield and soil fertility on the long term, especially in reduced tillage systems

Characterization of the mitochondrial processing peptidase of Neurospora crassa

The mitochondrial processing peptidase (MPP) of Neurospora crassa is constituted by an alpha- and a beta-subunit. We have purified alpha-MPP after expression in Escherichia coli while beta-MPP was purified from mitochondria. A fusion protein between precytochrome b2 and mouse dihydrofolate reductase was expressed in E. coli, and the purified protein was used as substrate for MPP. Both subunits of MPP are required for processing. MPP removes the matrix targeting signal of cytochrome b2 by a single cut, and the resulting presequence peptide is 31 amino acid residues in length. It acts as a competitive inhibitor of processing but has a approximately 30-fold lower affinity for MPP than the preprotein. Competition assays show that MPP recognizes the COOH- terminal portion of the presequence of cytochrome b2 rather than the NH2-terminal part which has the potential to form an amphiphilic helix. Substitution of arginine in position -2 of the matrix targeting sequence of cytochrome b2 prevents processing but not import of a chimeric precursor. Substitution of the tyrosyl residue in position +1 also prevents processing, indicating that MPP interacts with sequences COOH-terminal to the cleavage site. Non-cleavable preprotein is still recognized by MPP. Our data suggest that processing peptidase and import machinery recognize distinct structural elements in preproteins which, however, can be overlapping

Polypeptides traverse the mitochondrial envelope in an extended state

Most mitochondrial proteins are synthesized as precursors in the cytosol and imported through the contact sites between outer and inner mitochondrial membranes. The molecular mechanism of membrane translocation of precursor proteins is largely unclear. For this report, various hybrid proteins between portions of the precursor of cytochrome b2 and the entire dihydrofolate reductase (DHFR) were accumulated in mitochondrial contact sites. We unexpectedly found that about 30 amino acid residues of the polypeptide chain in transit were sufficient to span both membranes. This suggests linear translocation of the polypeptide chain and presents evidence for a high degree of unfolding of polypeptides traversing the mitochondrial membranes