Sara Sjöling

Mitochondrial protein import in plants. Signals, sorting, targeting, processing and regulation.

Mitochondrial biogenesis requires a coordinated expression of both the nuclear and the organellar genomes and specific intracellular protein trafficking, processing and assembly machinery. Most mitochondrial proteins are synthesised as precursor proteins containing an N-terminal extension which functions as a targeting signal, which is proteolytically cleaved off after import into mitochondria. We review our present knowledge on components and mechanisms involved in the mitochondrial protein import process in plants. This encompasses properties of targeting peptides, sorting of precursor proteins between mitochondria and chloroplasts, signal recognition, mechanism of translocation across the mitochondrial membranes and the role of cytosolic and organellar molecular chaperones in this process. The mitochondrial protein processing in plants is catalysed by the mitochondrial processing peptidase (MPP), which in contrast to other sources, is integrated into the bc1 complex of the respiratory chain. This is the most studied component of the plant import machinery characterised to date. What are the biochemical consequences of the integration of the MPP into an oligomeric protein complex and how are several hundred presequences of precursor proteins with no sequence similarities and no consensus for cleavage, specifically cleaved off by MPP? Finally we will address the emerging area of the control of protein import into mitochondria.

The metagenomics of disease-suppressive soils - experiences from the METACONTROL project

Soil teems with microbial genetic information that can be exploited for biotechnological innovation. Because only a fraction of the soil microbiota is cultivable, our ability to unlock this genetic complement has been hampered. Recently developed molecular tools, which make it possible to utilize genomic DNA from soil, can bypass cultivation and provide information on the collective soil metagenome with the aim to explore genes that encode functions of key interest to biotechnology. The metagenome of disease-suppressive soils is of particular interest given the expected prevalence of antibiotic biosynthetic clusters. However, owing to the complexity of soil microbial communities, deciphering this key genetic information is challenging. Here, we examine crucial issues and challenges that so far have hindered the metagenomic exploration of soil by drawing on experience from a trans-European project on disease-suppressive soils denoted METACONTROL.

Mitochondrial targeting peptides in plants

Proteins function at distinct sites in the cell, yet most are primarily synthesized in the cytosol and must therefore be transported intracellularly to their final destination. Mitochondrial targeting peptides are localized at the N-terminus of the precursor proteins and most are cleaved off after import into mitochondria. Cleavage is catalysed by the general mitochondrial processing peptidase, which, in plants, is integrated into the bc1 complex of the respiratory chain. This peptidase recognizes the cleavage sites of nearly 1000 precursor proteins that have no sequence similarity.

The ubiquinol cytochrome c oxidoreductase complex of spinach leaf mitochondria is involved in both respiration and protein processing

Abstract Nuclear encoded mitochondrial precursor proteins are cleaved to mature size products by the general mitochondrial processing peptidase (MPP). In contrast to non-plant tissues where MPP is located in the matrix, the general processing activity of potato tuber (storage tissue) mitochondria has been shown to constitute an integral part of the isolated cytochrome c reductase complex of the respiratory chain. Here we show isolation of MPP from photosynthetic tissue, spinach leaf mitochondria, starting from the total membrane processing extract using extraction with dodecyl-β-maltoside followed by FPLC anion-exchange and gel filtration chromatography. The total spinach leaf MPP is found in the fractions containing the cytochrome c reductase complex and is shown to be an integral part of the complex. No processing activity has been found in any other fractions. The isolated cytochrome c reductase complex is shown to process three precursor proteins of different intramitochondrial localisation, the F 1 β subunit of ATP synthase (extrinsic membrane protein on matrix side), the Rieske FeS protein (integral membrane protein facing intermembrane space) and the malate dehydrogenase (matrix protein). The processing activity is totally inhibited by EDTA and orthophenanthroline. Our results together with the results in potato mitochondria show that integration of MPP into the cytochrome c reductase is a general phenomenon for plants. The complex consists of ten protein bands on SDS-PAGE of 61, 54, 52, 34, 32, 26, 15, 12, 11 and 10 kDa. The 61, 54 and 52 kDa bands correspond to Core proteins, the 32 kDa band to cytochrome b and the 26 kDa band to Rieske FeS protein as estimated by immunological methods. The three Core proteins are shown to be immunologically related to MPP from other sources, the Core 1 protein corresponding to β-MPP and the Core 2 and Core 3 proteins corresponding to α-MPP, which in comparison to MPP in potato mitochondria indicates species-dependent differences in the appearence of the processing components. Furthermore, the processing activity of the isolated and membrane-bound spinach cytochrome c reductase complex is shown to be inhibited by antimycin A and myxothiazol, electron transfer inhibitors of the complex. The inhibition of processing is, however, not correlated to the inhibition of electron transfer through the complex or to the redox state of the complex.

Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen suppressive soil

Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study, we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF(103) of the isolate Streptomyces mutomycini and/or Streptomyces clavifer. There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.

Signals Required for the Import and Processing of the Alternative Oxidase into Mitochondria

The critical residues involved in targeting and processing of the soybean alternative oxidase to plant and animal mitochondria was investigated. Import of various site-directed mutants into soybean mitochondria indicated that positive residues throughout the length of the presequence were important for import, not just those in the predicted region of amphiphilicity. The position of the positive residues in the C-terminal end of the presequence was also important for import. Processing assays of the various constructs with purified spinach mitochondrial processing peptidase showed that all the −2-position mutants had a drastic effect on processing. In contrast to the import assay, the position of the positive residue could be changed for processing. Deletion mutants confirmed the site-directed mutagenesis data in that an amphiphilic α-helix was not the only determinant of mitochondrial import in this homologous plant system. Import of these constructs into rat liver mitochondria indicated that the degree of inhibition differed and that the predicted region of amphiphilic α-helix was more important with rat liver mitochondria. Processing with a rat liver matrix fraction showed little inhibition. These results are discussed with respect to targeting specificity in plant cells and highlight the need to carry out homologous studies and define the targeting requirements to plant mitochondria.

Active bacterial community structure along vertical redox gradients in Baltic Sea sediment

Community structures of active bacterial populations were investigated along a vertical redox profile in coastal Baltic Sea sediments by terminal-restriction fragment length polymorphism (T-RFLP) and clone library analysis. According to correspondence analysis of T-RFLP results and sequencing of cloned 16S rRNA genes, the microbial community structures at three redox depths (179, -64 and -337 mV) differed significantly. The bacterial communities in the community DNA differed from those in bromodeoxyuridine (BrdU)-labelled DNA, indicating that the growing members of the community that incorporated BrdU were not necessarily the most dominant members. The structures of the actively growing bacterial communities were most strongly correlated to organic carbon followed by total nitrogen and redox potentials. Bacterial identification by sequencing of 16S rRNA genes from clones of BrdU-labelled DNA and DNA from reverse transcription polymerase chain reaction showed that bacterial taxa involved in nitrogen and sulfur cycling were metabolically active along the redox profiles. Several sequences had low similarities to previously detected sequences, indicating that novel lineages of bacteria are present in Baltic Sea sediments. Also, a high number of different 16S rRNA gene sequences representing different phyla were detected at all sampling depths.

Mitochondrial protein import in plants

Mitochondrial biogenesis requires a coordinated expression of both the nuclear and the organellar genomes and specific intracellular protein trafficking, processing and assembly machinery. Most mitochondrial proteins are synthesised as precursor proteins containing an N-terminal extension which functions as a targeting signal, which is proteolytically cleaved off after import into mitochondria. We review our present knowledge on components and mechanisms involved in the mitochondrial protein import process in plants. This encompasses properties of targeting peptides, sorting of precursor proteins between mitochondria and chloroplasts, signal recognition, mechanism of translocation across the mitochondrial membranes and the role of cytosolic and organellar molecular chaperones in this process. The mitochondrial protein processing in plants is catalysed by the mitochondrial processing peptidase (MPP), which in contrast to other sources, is integrated into the bc1 complex of the respiratory chain. This is the most studied component of the plant import machinery characterised to date. What are the biochemical consequences of the integration of the MPP into an oligomeric protein complex and how are several hundred presequences of precursor proteins with no sequence similarities and no consensus for cleavage, specifically cleaved off by MPP? Finally we will address the emerging area of the control of protein import into mitochondria.

High 16S rDNA bacterial diversity in glacial meltwater lake sediment, Bratina Island, Antarctica

The microbial diversity in maritime meltwater pond sediments from Bratina Island, Ross Sea, Antarctica was investigated by 16S rDNA-dependent molecular phylogeny. Investigations of the vertical distribution, phylogenetic composition, and spatial variability of Bacteria and Archaea in the sediment were carried out. Results revealed the presence of a highly diverse bacterial population and a significantly depth-related composition. Assessment of 173 partial 16S rDNA clones analyzed by amplified rDNA restriction analysis (ARDRA) using tetrameric restriction enzymes (HinP1I 5′G∇CGC3′and Msp I. 5′C∇CGG3′, BioLabs) revealed 153 different bacterial OTUs (operational taxonomic units). However, only seven archaeal OTUs were detected, indicating low archaeal diversity. Based on ARDRA results, 30 bacterial clones were selected for sequencing and the sequenced clones fell into seven major lineages of the domain Bacteria; the α, γ, and δ subdivisions of Proteobacteria, the Cytophaga–Flavobacterium–Bacteroides, the Spirochaetaceae, and the Actinobacteria. All of the archaeal clones sequenced belonged to the group Crenarchaeota and phylogenetic analysis revealed close relationships with members of the deep-branching Group 1 Marine Archaea.

Bifunctional role of the bc1 complex in plants. Mitochondrial bc1 complex catalyses both electron transport and protein processing.

Nuclear encoded mitochondrial precursor proteins are cleaved to mature size products by the general mitochondrial processing peptidase (MPP). In contrast to non‐plant sources where MPP is a matrix enzyme, the plant mitochondrial MPP is localised in the inner membrane and constitutes an integral part of the bc1 complex of the respiratory chain. Core proteins of the complex are immunologically related and show high sequence similarity to the MPP subunits from non‐plant sources. The bc1 complex in plants is thus bifunctional, being involved both in respiration and in protein processing.