Additional file 4: Table S2. of Evolution of the Tim17 protein family

The overall distribution of Tim17 family protein in eukaryotes. (XLS 135 kb

Additional file 6: Table S4. of Evolution of the Tim17 protein family

Prokaryotic proteins carrying a PRAT motif [(G/A)X2(F/Y)X10RX3DX6(G/A/S)GX3G]. (PDF 133 kb

Additional file 3: Figure S1. of Evolution of the Tim17 protein family

Protein sequence alignment of Tim22 from Homo sapiens, Saccharomyces cerevisiae and Neurospora crassa and HP20 and HP30 from Arabidopsis thaliana as performed by MAFFT [38]. The identical and similar residues were highlighted by turquoise and green color, respectively. The threshold value for shading was set to 50 %. (PDF 41 kb

Additional file 5: Figure S2. of Evolution of the Tim17 protein family

Protein sequence alignment of Romo1/Mgr2, Tim17 and Tim23 from Saccharomyces cerevisiae, Allomyces macrogynus, Homo sapiens, Monodelphis domestica and Trichoplax adherens as performed by MAFFT [38]. The grey cylinders denote the transmembrane regions. The identical and similar residues were highlighted by turquoise and green color, respectively. The threshold value for shading was set to 50 %. (PDF 25 kb

The Mitochondrion-Like Organelle of <em>Trimastix pyriformis</em> Contains the Complete Glycine Cleavage System

<div><p>All eukaryotic organisms contain mitochondria or organelles that evolved from the same endosymbiotic event like classical mitochondria. Organisms inhabiting low oxygen environments often contain mitochondrial derivates known as hydrogenosomes, mitosomes or neutrally as mitochondrion-like organelles. The detailed investigation has shown unexpected evolutionary plasticity in the biochemistry and protein composition of these organelles in various protists. We investigated the mitochondrion-like organelle in <i>Trimastix pyriformis,</i> a free-living member of one of the three lineages of anaerobic group Metamonada. Using 454 sequencing we have obtained 7 037 contigs from its transcriptome and on the basis of sequence homology and presence of N-terminal extensions we have selected contigs coding for proteins that putatively function in the organelle. Together with the results of a previous transcriptome survey, the list now consists of 23 proteins – mostly enzymes involved in amino acid metabolism, transporters and maturases of proteins and transporters of metabolites. We have no evidence of the production of ATP in the mitochondrion-like organelle of <i>Trimastix</i> but we have obtained experimental evidence for the presence of enzymes of the glycine cleavage system (GCS), which is part of amino acid metabolism. Using homologous antibody we have shown that H-protein of GCS localizes into vesicles in the cell of <i>Trimastix</i>. When overexpressed in yeast, H- and P-protein of GCS and cpn60 were transported into mitochondrion. In case of H-protein we have demonstrated that the first 16 amino acids are necessary for this transport. Glycine cleavage system is at the moment the only experimentally localized pathway in the mitochondrial derivate of <i>Trimastix pyriformis</i>.</p> </div

H-protein of GCS localizes into vesicles (putative mitochondrion-like organelles) in <i>Trimastix pyriformis</i>.

<p>A) Immunofluorescence microscopy of the <i>Trimastix pyriformis</i> cell. The green signal from antiH-protein (human) co-localizes with red signal from the antiH-protein (<i>Trimastix</i>). The DNA is stained blue with Hoechst. B) Western blot on the cellular fractions of <i>Trimastix pyriformis.</i> The lines represent pure bacteria <i>Citrobacter</i> sp. from the culture (Bact), high speed pellet of <i>Trimastix</i> (HSP), supernatant of <i>Trimastix</i> (Sup), total lysate of <i>Trimastix</i> (Total).</p

Over-expression of <i>Trimastix</i> proteins in yeast.

<p>The over-expression of GFP tagged proteins of <i>Trimastix</i> in <i>Saccharomyces cerevisiae</i>. The columns represent the signals from GFP tag (green), MitoTracker (red), merged GFP and MitoTracker and DIC. Rows represent individual proteins: cpn60, P1-protein of GCS, H-protein of GCS and H-protein of GCS truncated of the first 16 amino acids.</p

Mitosomal and hydrogenosomal localization of HaloTagged proteins.

<p>Immunofluorescence analyses of <i>G. intestinalis</i> and <i>T. vaginalis</i> transformants expressing GiIscU-Halo and TvFtx-Halo fusion, respectively. Cells were incubated with TMR-Halo ligand (red), washed and fixed for immunofluorescence analysis. A) TMR-Halo labeled <i>G. intestinalis</i> cells were fixed and labeled by anti-Tom40 specific polyclonal antibodies (green). B) TMR-Halo labeled <i>T. vaginalis</i> cells were fixed and decorated by anti-malic enzyme specific polyclonal antibodies (green). Nuclei were stained with DAPI (blue).</p

Live imaging of mitosomes and hydrogenosomes.

<p>Halo-TMR labeled organelles were followed in living cells. A) Labeled <i>G. intestinalis</i> cells were allowed to attach to the bottom of the well and directly observed while B) the labeled <i>T. vaginalis</i> cells were mounted in 2% agarose and then submitted to microscopy. Five different snapshots in time are shown. The original movies are part of the supplementary data.</p

Expression of HaloTagged proteins in <i>G. intestinalis</i> and <i>T. vaginalis</i>.

<p>Western blot analyses of cellular fractions of <i>G. intestinalis</i> and <i>T. vaginalis</i> transformants expressing GiIscU-Halo and TvFtx-Halo fusions, respectively. A) GiIscU-Halo was detected by specific anti-IscU polyclonal antibodies in cell lysate and high-speed pellet (HSP). Two bands in these fractions represent the nuclear encoded (GiIscU) and episomally encoded HaloTag fusion (GiIscU-Halo). B) TvFtx-Halo product was detected by anti-HA monoclonal antibodies in <i>T. vaginalis</i> cellular fractions. The fusion protein was found exclusively in cell lysate and in hydrogenosomes. The upper panels demonstrate the protein profile on the coomassie stained SDS-PAGE gel. Lys-lysate, Cyt-cytosol, HSP-high-speed pellet, Hyd-hydrogenosomes.</p