Mitochondrial protein import is essential for all eukaryotes and mediated by hetero-oligomeric protein translocases thought to be conserved within all eukaryotes. We have identified and analysed the function and architecture of the non-conventional outer membrane (OM) protein translocase in the early diverging eukaryote Trypanosoma brucei. It consists of six subunits that show no obvious homology to translocase components of other species. Two subunits are import receptors that have a unique topology and unique protein domains and thus evolved independently of the prototype receptors Tom20 and Tom70. Our study suggests that protein import receptors were recruited to the core of the OM translocase after the divergence of the major eukaryotic supergroups. Moreover, it links the evolutionary history of mitochondrial protein import receptors to the origin of the eukaryotic supergroups.

Trypanosoma brucei is a unicellular parasite that causes devastating diseases in humans and animals. It diverged from most other eukaryotes very early in evolution and, as a consequence, has an unusual mitochondrial biology. Moreover, mitochondrial functions and morphology are highly regulated throughout the life cycle of the parasite. The outer mitochondrial membrane defines the boundary of the organelle. Its properties are therefore key for understanding how the cytosol and mitochondria communicate and how the organelle is integrated into the metabolism of the whole cell. We have purified the mitochondrial outer membrane of T. brucei and characterized its proteome using label-free quantitative mass spectrometry for protein abundance profiling in combination with statistical analysis. Our results show that the trypanosomal outer membrane proteome consists of 82 proteins, twothirds of which have never been associated with mitochondria before.

In Bradyrhizobium japonicum, a gene named nnrR was identified which encodes a protein with high similarity to FNR/CRP-type transcriptional regulators. Mutant strains carrying an nnrR null mutation were unable to grow anaerobically in the presence of nitrate or nitrite, and they lacked both nitrate and nitrite reductase activities. Anaerobic activation of an nnrR-lacZ fusion required FixLJ and FixK 2 . In turn, N oxide-mediated induction of nir and nor genes encoding nitrite and nitric oxide reductase, respectively, depended on NnrR. Thus, NnrR expands the FixLJ-FixK 2 regulatory cascade by an additional control level which integrates the N oxide signal required for maximal induction of the denitrification genes.

Mitochondria cannot form de novo but require mechanisms allowing their inheritance to daughter cells. In contrast to most other eukaryotes Trypanosoma brucei has a single mitochondrion whose single-unit genome is physically connected to the flagellum. Here we identify a β-barrel mitochondrial outer membrane protein, termed tripartite attachment complex 40 (TAC40), that localizes to this connection. TAC40 is essential for mitochondrial DNA inheritance and belongs to the mitochondrial porin protein family. However, it is not specifically related to any of the three subclasses of mitochondrial porins represented by the metabolite transporter voltage-dependent anion channel (VDAC), the protein translocator of the outer membrane 40 (TOM40), or the fungi-specific MDM10, a component of the endoplasmic reticulum-mitochondria encounter structure (ERMES). MDM10 and TAC40 mediate cellular architecture and participate in transmembrane complexes that are essential for mitochondrial DNA inheritance. In yeast MDM10, in the context of the ERMES, is postulated to connect the mitochondrial genomes to actin filaments, whereas in trypanosomes TAC40 mediates the linkage of the mitochondrial DNA to the basal body of the flagellum. However, TAC40 does not colocalize with trypanosomal orthologs of ERMES components and, unlike MDM10, it regulates neither mitochondrial morphology nor the assembly of the protein translocase. TAC40 therefore defines a novel subclass of mitochondrial porins that is distinct from VDAC, TOM40, and MDM10. However, whereas the architecture of the TAC40-containing complex in trypanosomes and the MDM10-containing ERMES in yeast is very different, both are organized around a β-barrel protein of the mitochondrial porin family that mediates a DNA-cytoskeleton linkage that is essential for mitochondrial DNA inheritance.organelle | parasitic protozoa

Two oxygen-responsive regulatory systems controlling numerous symbiotic genes in Bradyrhizobium japonicum were assayed in free-living cultures for their capacity to activate target genes under different oxygen conditions. NifA-and FixLJ-controlled target genes showed disparate relative expression patterns. Induction of NifA-dependent genes was observed only at oxygen concentrations below 2% in the gas phase, whereas that of FixLJ-controlled targets progressively increased when the oxygen concentration was lowered from 21 to 5, 2, or 0.5%. We propose that this reflects a response to a gradient of increasing oxygen deprivation as bacteria invade their host during root nodule development.