A paradigm shift: The mitoproteomes of procyclic and bloodstream <i>Trypanosoma brucei</i> are comparably complex

A paradigm shift: The mitoproteomes of procyclic and bloodstream <i>Trypanosoma brucei</i> are comparably comple

Pie charts showing distribution of mass spectrometry–identified mitochondrial proteins in PCF (left) and BSF (right) trypanosomes in terms of molecular functions.

<p>A total number of 1,195 and 956 proteins were assigned to PCF and BSF mitoproteome, respectively. Different colors show different metabolic pathways and categories. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006679#ppat.1006679.s001" target="_blank">S1 Table</a>. IFA-mito in PCF (left) and in BSF cell (right). BSF, bloodstream form; Hsp70, heat shock protein 70; IFA-mito, immunofluorescence analysis of a mitochondrial Hsp70; mitoproteome, mitochondrial proteome; PCF, procyclic form.</p

Schematic representation of carbon source metabolism in the bloodstream form of <i>T</i>. <i>brucei</i>.

<p>Red arrows represent enzymatic steps that were experimentally shown to be active in BSF. Green arrows represent enzymatic steps that might be active in BSF because the enzymes (in green) were identified in BSF proteomic data. Glucose-derived metabolites (acetate, pyruvate, succinate, alanine, aspartate) are on a blue background. NADH molecules are on a pink background. Dashed arrows indicate enzymatic steps for which no experimental proof exists. The glycosomal and mitochondrial compartments are indicated. 2-OGDH, 2-oxoglutarate dehydrogenase; AAC, ADP/ATP carrier; AAT, amino acid transporter; ACH, acetyl-CoA thioesterase; AKCT, 2-amino-3-ketobutyrate coenzyme A ligase; Ala TR, alanine transaminase; AOX, alternative oxidase; ASCT, acetate:succinate CoA-transferase; Asp TR, aspartate transaminase; BSF, bloodstream form; cI, complex I (NADH:ubiquinone oxidoreductase); cII, complex II (succinate dehydrogenase); cIII, complex III (cytochrome bc1 complex); cIV, complex IV (cytochrome c oxidase); cV, complex V (F_oF₁ ATPase); cyt, cytosolic; DHAP, dihydroxyacetone phosphate; FH, fumarate hydratase (i.e., fumarase); FR, fumarate reductase; G3P, glyceraldehyde 3-phosphate; GluDH, glutamate dehydrogenase; gly, glycosomal; Gly3P, glycerol 3-phosphate; Gly-3-PDH, glycerol-3-phosphate dehydrogenase; m, mitochondrial; MDH, malate dehydrogenase; PDH, pyruvate dehydrogenase; PEP, phosphoenolpyruvate; PEPCK, phosphoenolpyruvate carboxykinase; PiC, phosphate carrier; ProDH, proline dehydrogenase.</p

Ultrastructural alterations induced in <i>T</i>. <i>brucei</i> BSF by ATG24 silencing.

<p>Electron microscopy of BSF wild-type cells (<b>A, B</b>) and BSF ATG24 RNAi cells after 72 h of RNAi induction (<b>C</b>, <b>D</b>). The two characteristic effects of ATG24 silencing are (i) <b>enlargement of the flagellar pocket</b> (FP) shown in <b>C</b>, <b>D</b>; compare <b>B</b> with <b>C</b> (notice different scale bars); insert in <b>C</b> better shows a cross-section of the flagellum; and (ii) <b>autophagy</b> [<b>D</b>]. The large vesicle, painted in green, has a single limiting membrane and heterogeneous content including two well-recognizable entrapped glycosomes (G’), undistinguishable from free glycosomes (G) in the cytosol. This structure thus demonstrates active autophagy of glycosomes. The two arrows indicate CV (clathrin-coated vesicles): notice the contrast, thickness and indentation regularity typical of clathrin coat indicated by the black arrow, while the white arrow points to a suggestive additional CV profile. These structures suggest (some) preservation of endocytic entry. All scale bars: 0.5 μm.</p

Growth curve for ATG24 RNAi cell lines.

<p>Minor growth inhibition by ATG24 silencing in <i>T</i>. <i>brucei</i> BSF (<b>A</b>) and PCF (<b>B</b>). Comparison of WT <i>vs</i> non-induced (NI) or induced (I) ATG24 RNAi cell line. WT–squares, NI–circles, I–triangles. Results are from one representative experiment out of three, giving the following population doubling times (PDT; mean ± SD): bloodstream-form wild-type cells 7.45 ± 0.61 h; ATG24 RNAi NI: 7.66 ± 0.48 h; ATG24 RNAi Ind: 11.51 ± 2.0 h. At 120 h the differences in PDT between BSF WT and ATG24 Ind and between ATG24 NI and ATG24 Ind are statistically significant with p-values of 0.012 in both cases, whereas the value for WT <i>versus</i> ATG24 NI is 0.708 and the difference thus not statistically significant. The PDT for procyclic trypanosomes are: WT, 9.16 ± 1.74 h; ATG24 RNAi NI, 10.17 ± 1.62 h; ATG24 RNAi Ind, 13.65 ± 3.6 h. At 120 h the p-value for differences in PDT of PCF WT <i>versus</i> ATG24 Ind is 0.204, ATG24 NI <i>versus</i> ATG24 Ind is 0.315, and WT <i>versus</i> ATG24 NI is 0.201; none of these differences is statistically significant.</p

Dual localization of ATG24 in <i>T</i>. <i>brucei</i> cytosol and endosomes.

<p><b>A</b>) ATG24 subcellular localization by immunofluorescence. Notice that ATG24 immunolabeling (in red) produces a combined dotty pattern (arrowheads) with diffuse cytosolic signal, both in BSF (left panel) and PCF wild-type cells (right panel). Blue is DAPI, N = nucleus, k = kinetoplast. <b>B</b>) Relation with endosomes in wild-type BSF cells expressing ATG24-GFP (green) incubated with the receptor-mediated endocytic tracer, Alexa568-transferrin (red), at 37°C for 30 min. Yellow in the merge at right indicates extensive colocalization. DAPI is shown in blue. <b>C</b>) Relation with endosomes by triple imaging with concanavalin A and DAPI. Wild-type BSF cells expressing ATG24-GFP (green) were incubated with the adsorptive endocytosis tracer, Alexa594-concanavalin A (red) at 37°C for 30 min. All scale bars: 5 μm. In two independent experiments the mean Pearson's correlation of colocalization observed between ATG24 and transferrin was 0.24 and with concanavalin A was 0.21. As positive and negative controls, respectively, endosomal RAB11 had a Pearson's coefficient of 0.34 and DAPI 0.13.</p

ATG24 silencing accelerates <i>T</i>. <i>brucei</i> differentiation.

<p><b>A</b>) Morphological evidence. Immunofluorescence image at the upper left compares the typical appearance of a BSF (at left) and a PCF trypanosome (at right) labeled with antiserum against phosphoglycerate kinase (PGK). Quantification of differentiation for wild-type (WT) cells and ATG24 RNAi trypanosomes first induced for 48 h, then transferred to <i>in vitro</i> differentiation medium for 48 h. PCF-like morphology is seen in 31% ± 14% of the wild-type cells, but 93% ± 9% of ATG24 RNAi cells. 50 to 400 cells were counted for each sample from three independent experiments (p value = 0.002). <b>B</b>) PPDK expression was assayed by western blot analysis after 72 h in differentiation medium for wild-type and ATG24 RNAi non-induced (NI) or induced (Ind) trypanosomes expressing GFP-ATG8.1 or GFP-ATG8.2 as indicated. Partial depletion of ATG24 can be observed in these cells. ZFP3 is a loading marker, i.e. a protein known to remain rather constant during differentiation. <b>C</b>) An illustration of mitochondrial morphology, as revealed by ASCT staining, in wild-type BSF and PCF cells (as controls), BSF cells expressing GFP-ATG8.1 or GFP-ATG8.2 with and without ATG24 RNAi 48 h induced before <i>in vitro</i> differentiation (72 h). ASCT (red), DNA (blue, DAPI). Note the developed mitochondrion of the PCF cells in contrast to the BSF trypanosomes. After 72 h in differentiation conditions, the cells expressing GFP-ATG8.2 have a more developed mitochondrion than cells expressing GFP-ATG8.1, while in cells depleted for ATG24 there is no significant difference between those expressing tagged ATG8.1 and ATG8.2. All scale bars: 0.5 μm.</p

ATG24 depletion in <i>T</i>. <i>brucei</i> results in an increased number of ATG8-associated puncta.

<p><b>A</b>) Validation of autophagosome labeling in <i>T</i>. <i>brucei</i>. Starvation-induced dotty labeling with GFP-ATG8.1 or GFP-ATG8.2 upon transfer of transfected PCF cells from control medium (left) to PBS for 15 min (right). All scale bars: 5 μm. <b>B</b>) Quantification of autophagy upon ATG24 silencing in PCF trypanosomes. Comparison of the percentage of increase in number of cells displaying puncta labeled for GFP-ATG8.1 or GFP-ATG8.2 before and after transfer to PBS of non-induced (NI) and induced (I) cells in two separate experiments (Exp1 with 15 min incubation in PBS and Exp2 with 30 min incubation in PBS); 50 to 200 cells were counted in each condition. All cells were fixed in the same manner in order to minimize different effects caused by fixation. Selected were only cells that had retained their structure and in which there was visible fluorescence. The fluorescence distribution in whole cells was analyzed and cells displaying fluorescent puncta versus cells in which GFP-ATG8 had only a cytosolic distribution were counted. <b>C</b>) Quantification of autophagy upon ATG24 silencing in BSF cells. Comparison of the percentage of increase in number of cells displaying puncta associated to GFP-ATG8.1 or GFP-ATG8.2 before and after 24 h in DTM <i>in vitro</i> differentiation medium in wild-type (WT), non-induced (NI) and induced (Ind) cells in two separate experiments (Exp1 and Exp2); at least 100 cells were counted in each condition. After harvesting, the cells were treated and analyzed as in the experiments shown in panel B.</p

SCA of a low-conductance channel.

<p>(<b>A</b>) Current traces of two low-conductance channels. The bath solution (<b>A–C</b>) contained 3 M KCl at both sides of the membrane. See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g004" target="_blank">Figure 4<b>A</b></a> for other details. Note that two types of channels were registered. Most of them showed a larger current amplitude at +10 mV than at −10 mV (upper trace). In contrast, some channels displayed an opposite trend (lower trace). (<b>B</b> and <b>C</b>) Current traces of the channels in response to the indicated voltage-ramp (<b>B</b>) and voltage-step (<b>C</b>) protocols. Most detected channels displayed a current rectification at negative holding potentials (upper panels). However, in a few cases the rectification was observed at positive holding potentials (lower panels). (<b>D</b>) Dependence of the low-conductance channel activity on the electrolyte concentration. The channel insertion was registered at a holding potential of +10 mV using 3 M KCl as a bath solution. After confirming that the channel shows current rectification at negative voltages by application of a voltage-ramp protocol, the electrolyte in the chambers was diluted to 2.0 M or 1.0 M KCl and the current amplitudes were measured at +10 mV. Data are mean±SD, n = 4–5. (<b>E</b>) Ion selectivity of the low-conductance channel. See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g004" target="_blank">Figure 4<b>F</b></a> for details. The current-voltage relationship of channels (rectification at negative voltages) was validated using a voltage-ramp protocol.</p

ATG24 membrane recruitment in <i>T</i>. <i>brucei</i> depends on VPS34.

<p><b>A</b>) Pharmacological inhibition. ATG24 immunofluorescence was determined in wild-type PCF cells in the absence of wortmannin and after 1 h of incubation with 0.3 to 30 μM of this compound. Shown here are (in black and white for better contrast and resolution) non-treated trypanosomes (in the left panel) and trypanosomes treated with 3 μM wortmannin for 1 h (in the panel at the right). Quantification of the effect of wortmannin was performed in two experiments with 60 and 150 cells, respectively. <b>B</b>) VPS34 silencing. ATG24 immunofluorescence (red) in wild-type (WT) and VPS34 RNAi BSF at 24 h after induction. Blue shows DAPI. The intensity value assigned to ATG24 fluorescence in endosomes was 3.4 times higher in WT BSF than in VPS34 RNAi BSF 24-h induced cells. Results are from one representative experiment out of two where 45 WT and 10 VPS34 RNAi cells and 18 WT and 21 VPS34 RNAi cells were counted, respectively. All scale bars: 5 μm.</p