Live Imaging of Mitosomes and Hydrogenosomes by HaloTag Technology

Hydrogenosomes and mitosomes represent remarkable mitochondrial adaptations in the anaerobic parasitic protists such as Trichomonas vaginalis and Giardia intestinalis, respectively. In order to provide a tool to study these organelles in the live cells, the HaloTag was fused to G. intestinalis IscU and T. vaginalis frataxin and expressed in the mitosomes and hydrogenosomes, respectively. The incubation of the parasites with the fluorescent Halo-ligand resulted in highly specific organellar labeling, allowing live imaging of the organelles. With the array of available ligands the HaloTag technology offers a new tool to study the dynamics of mitochondria-related compartments as well as other cellular components in these intriguing unicellular eukaryotes

The Minimal Proteome in the Reduced Mitochondrion of the Parasitic Protist Giardia intestinalis

The mitosomes of Giardia intestinalis are thought to be mitochondria highly-reduced in response to the oxygen-poor niche. We performed a quantitative proteomic assessment of Giardia mitosomes to increase understanding of the function and evolutionary origin of these enigmatic organelles. Mitosome-enriched fractions were obtained from cell homogenate using Optiprep gradient centrifugation. To distinguish mitosomal proteins from contamination, we used a quantitative shot-gun strategy based on isobaric tagging of peptides with iTRAQ and tandem mass spectrometry. Altogether, 638 proteins were identified in mitosome-enriched fractions. Of these, 139 proteins had iTRAQ ratio similar to that of the six known mitosomal markers. Proteins were selected for expression in Giardia to verify their cellular localizations and the mitosomal localization of 20 proteins was confirmed. These proteins include nine components of the FeS cluster assembly machinery, a novel diflavo-protein with NADPH reductase activity, a novel VAMP-associated protein, and a key component of the outer membrane protein translocase. None of the novel mitosomal proteins was predicted by previous genome analyses. The small proteome of the Giardia mitosome reflects the reduction in mitochondrial metabolism, which is limited to the FeS cluster assembly pathway, and a simplicity in the protein import pathway required for organelle biogenesis

Iron-Induced Changes in the Proteome of <i>Trichomonas vaginalis</i> Hydrogenosomes

<div><p>Iron plays a crucial role in metabolism as a key component of catalytic and redox cofactors, such as heme or iron-sulfur clusters in enzymes and electron-transporting or regulatory proteins. Limitation of iron availability by the host is also one of the mechanisms involved in immunity. Pathogens must regulate their protein expression according to the iron concentration in their environment and optimize their metabolic pathways in cases of limitation through the availability of respective cofactors. <i>Trichomonas vaginalis</i>, a sexually transmitted pathogen of humans, requires high iron levels for optimal growth. It is an anaerobe that possesses hydrogenosomes, mitochondrion-related organelles that harbor pathways of energy metabolism and iron-sulfur cluster assembly. We analyzed the proteomes of hydrogenosomes obtained from cells cultivated under iron-rich and iron-deficient conditions employing two-dimensional peptide separation combining IEF and nano-HPLC with quantitative MALDI-MS/MS. We identified 179 proteins, of which 58 were differentially expressed. Iron deficiency led to the upregulation of proteins involved in iron-sulfur cluster assembly and the downregulation of enzymes involved in carbohydrate metabolism. Interestingly, iron affected the expression of only some of multiple protein paralogues, whereas the expression of others was iron independent. This finding indicates a stringent regulation of differentially expressed multiple gene copies in response to changes in the availability of exogenous iron.</p></div

Significantly regulated proteins in iron depleted conditions.

<p>Significantly regulated proteins in iron depleted conditions.</p

Proteins of hydrogenosomal energy metabolism identified in this study.

<p>Gene IDs marked with an asterisk denote proteins that were significantly regulated in response to iron availability.</p

Comparison between iron-regulated proteins determined with the proteomic approach and the expression of corresponding genes studied by DNA microarrays and comparative EST analysis [<b>18</b>]<b>.</b>

<p>Red square, significant upregulation under high iron; pink square, insignificant upregulation under high iron; green square; significant upregulation under low iron; light green square, insignificant upregulation under low iron; empty square, no change in the transcript level; black square, a gene that was not included in the analysis.</p

Proteins of the iron-sulfur cluster assembly machinery identified in this study.

<p>Gene IDs marked with an asterisk denote proteins that were significantly regulated in response to iron availability.</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

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