TbTim11, TbTim12 and TbTim13 are novel trypanosomal members of the small Tim family.

<p>A) Sequence alignment of putative novel small Tims with known small Tims from trypanosomal mitochondria demonstrates conservation of twin CX3C motifs in all candidates except TbTim12. B) Total cells (T) were treated with 0.015% digitonin to separate a mitochondria enriched fraction (M) from the cytosol-containing supernatant (S). The tagged and endogenous small Tim-like proteins co-fractionate with the mitochondrial marker ATOM40, while the cytosolic protein elongation factor 1A (EF1a) stays in the cytosol. C) Alkaline carbonate extraction at pH 11.5 was performed on digitonin extracted crude mitochondria (M). The resulting pellet fraction (P) contains mitochondrial membrane proteins such as TbTim17 and ATOM40, while the soluble marker protein cytochrome c (Cyt C) is released to the supernatant (S). For this experiment, a cell line co-expressing TbTim11-HA and TbTim13-myc was used. D) Inducible expression of individual tagged small Tim candidates in the background of TbERV1 RNAi. Steady state levels of tagged candidates and endogenous small Tim9 are analyzed by immunoblotting. Cyt C is an IMS protein whose import is independent of the MIA pathway [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006550#ppat.1006550.ref030" target="_blank">30</a>] and the cytosolic protein EF1a serves as loading control. The lower panel depicts a densitometric quantification of the western blot results. Values were normalized to those of EF1a. For a characterization of the three ERV1-RNAi cell lines see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006550#ppat.1006550.s001" target="_blank">S1 Fig</a>.</p

Small Tims form soluble complexes of approximately 70 kDa.

<p>A) 2D BN-PAGE analysis of digitonin solubilized crude mitochondrial fractions. Lysates of cell lines expressing TbTim17-myc and one of the HA-tagged novel small Tims were combined and subjected to 6–16.5% BN PAGE in the first dimension, followed by 14% SDS PAGE in the second dimension and finally western blotting. The gels were aligned to the 66 kDa marker. The TIM complex components TbTim17-myc and TbTim42 were detected in all three analyses along with the respective HA-tagged small Tims. The control blots for the analyses of TbTim11-HA and TbTim13-HA can be found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006550#ppat.1006550.s002" target="_blank">S2 Fig</a>. Arrowheads indicate the approximate positions of the high molecular weight complexes containing small Tim proteins. B) 2D BN-PAGE analysis of submitochondrial fractions. The soluble content of the IMS (“soluble fraction”) was separated from the “membrane fraction” using 0.1% digitonin. Solubilized proteins were separately subjected to 2D-BN/SDS-PAGE as described above. Cyt C serves as a marker for soluble IMS proteins. Arrowheads indicate high molecular weight complexes containing small Tim proteins. C) Co-immunoprecipitation from submitochondrial fractions targeting myc-tagged TbTim13. A cell line co-expressing TbTim11-HA and TbTim13-myc was used to prepare a crude mitochondrial fraction or a “soluble fraction” containing IMS proteins by differential digitonin extraction. 5% of the initial crude mitochondrial fraction (M) and the same amounts of “Load”, “Unbound” and “Eluate” as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006550#ppat.1006550.g002" target="_blank">Fig 2A</a> were separated on SDS-PAGE and subjected to western blotting. The TIM complex component TbTim17 as well as the small Tims Tim9 and TbTim12 were detected by specific antibodies along with the HA- and myc-tagged other small Tims. The IMS protein Cyt C and the outer membrane protein VDAC were detected to confirm proper fractionation. D) Same experiment as in (C) targeting myc-tagged TbTim12 in a cell line co-expressing TbTim11-HA.</p

Small Tims are bound to the IM by association with the TIM complex.

<p>A) A cell line co-expressing TbTim11-HA and TbTim12-myc was subjected to co-immunoprecipitation targeting either the HA- (left panel) or the myc-tagged protein (right panel). 5% of the respective lysate (”Load”), 5% of the unbound proteins after IP (“Unbound”) and 100% of the final eluate (“Eluate”) were subjected to SDS-PAGE and western blotting. The blots were probed for the tagged small Tims and the TIM components Tim9, TbTim17, TbTim42 and TimRhom I. ATOM40, the central components of the OM translocase, and the cytochrome oxidase subunit IV (CoxIV) served as controls. The asterisk denotes the co-eluted heavy chain of the anti-myc antibody. B) Alkaline carbonate extraction at low (pH 10.7) and high stringency (pH 11.5) was performed on digitonin-extracted crude mitochondria (M). Mitochondrial transmembrane proteins such as TbTim17 and ATOM40 as well as tightly associated proteins are retained in the resulting pellet fraction (P), while the soluble marker protein cytochrome c (Cyt C) is released to the supernatant (S). Cell lines co-expressing TbTim11-HA and TbTim13-myc in either wildtype (WT) or TbTim17 RNAi background (2 days induced) were used to analyze small Tim fractionation in the presence (left panel) or absence of TbTim17 (right panel).</p

Novel small Tims are essential for TIM complex biogenesis.

<p>A) Growth curves of uninduced (-Tet) and induced (+Tet) procyclic RNAi cell lines ablating either, TbTim11, TbTim12 or TbTim13. The insets show Northern blots of total RNA extracts of uninduced (-Tet) and 2 days induced cells (+Tet). The respective mRNAs were detected by specific DNA probes, while ethidiumbromide-stained rRNAs (EtBr) serve as loading controls. Error bars correspond to standard deviation of three independent replicates. B) BN-PAGE analysis of the TIM complex in RNAi cell lines ablating either TbTim11, TbTim12 or TbTim13. Crude mitochondrial fractions were prepared after 0–4 days of RNAi induction and separated on a 4–13% BN PAGE. The TIM core component TbTim17 was in situ HA-tagged in the background of the respective RNAi cell line and detected by anti-HA antibodies. The Coomassie-stained gel serves as a loading control (Coom.). Numbers at the bottom indicate the percentage of TbTim17-HA present in the high molecular weight TIM complex. C) Immunoblots depicting steady state levels of TbTim17, TimRhom I and CoxIV in whole cell extracts of the same RNAi cell cultures as in (A). Precursor (p) and mature (m) variants of CoxIV are marked. The cytosolic protein EF1a serves as a loading control. Time of induction is indicated at the top. The graphs at the bottom show a quantification of the TbTim17 levels relative to EF1a from three to four independent experiments. The levels in uninduced cells were set to 1. Mean and standard errors of the means are indicated.</p

Global analysis of mitochondrial protein abundance changes upon ablation of TbTim13.

<p>A) Crude mitochondrial fractions of uninduced and induced (2.5 days) TbTim13 RNAi cells were subjected to quantitative MS using peptide stable isotope dimethyl labeling. For proteins exhibiting decreased abundance upon expression of TbTim13 RNAi, the mean log₂ of normalized ratios (induced/uninduced) was plotted against the corresponding–log₁₀ P value (two-sided t-test). The t-test significance level of 0.05 is indicated by a horizontal dashed line, while the vertical dashed line indicates a fold-reduction in protein abundance of 1.5. Mitochondrial proteins (dark grey) and TIM complex components (red) are highlighted. For a complete list of proteins see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006550#ppat.1006550.s007" target="_blank">S3 Table</a>. B) Alkaline carbonate extraction of crude mitochondrial extracts from a cell line expressing TbTim17-HA in the background of TbTim13 RNAi was performed after 0–3 days of induction. Equal cell equivalents of crude mitochondria (M), membrane pellets (P) and soluble fractions (S) were subjected to SDS-PAGE and western blotting. The single-spanning outer membrane protein ATOM14 and the IMS protein Cyt C serve as markers for the membrane and soluble fraction, respectively. The Coomassie-stained gel serves as a loading control (Coom.). C) Individual abundance ratios (induced/uninduced) of all MCPs and β-barrel proteins detected in the experiment shown in (A). Dashed horizontal line, 1.5 fold reduction. Standard deviations are indicated.</p

Electrophysiology of p34.

<p>(<b>A</b>) Current recordings of a bilayer containing purified p34 at indicated holding potential. Buffer conditions were symmetrical with 1.5 M KCl, 10 mM MOPS/Tris, pH 7.0 in <i>cis</i> and <i>trans</i> chamber. (<b>B</b>) Current-voltage relationship of p34. The conductance was calculated from the slope of the graph. (<b>C</b>) Current recordings of a bilayer containing p34(37–319) at holding potential as indicated. Buffer conditions were symmetrical with 1.5 M KCl, 10 mM Na-Acetat, pH 4.0 in <i>cis</i> and <i>trans</i> chamber. (<b>D</b>) Current-voltage relationship of p34(37–319). (<b>E</b>) Current recordings of a bilayer containing p34 at holding potential as indicated. The buffer conditions were the same as in (A). (<b>F</b>) Current recordings in the presence of 100 µM NPPB.</p

Complex formation of purified p34.

<p>(<b>A</b>) p34 was expressed in <i>Escherichia coli</i> and purified using conventional methods. Lane 1, inclusion bodies collected by centrifugation; lane 2, proteins dissolved in presence of 8 M urea and precipitated by ammonium sulfate (30% saturation); lane 3, eluate from Phenyl-Sepharose; lane 4, flow through from DEAE-Sephacel column. The proteins were separated by SDS-PAGE and the gel was stained by Coomassie. (<b>B</b>) CD spectra of p34. The purified protein was dissolved in 8 mM N-Decyl-β-D-Maltopyranosid, 10 mM KCl, 20 mM K₂HPO₄/KH₂PO₄, pH 7.0 and analyzed using a Jasco J-810 spectrapolarimeter. (<b>C</b>) Chemical cross-linking. Purified p34 was incubated with 50 µM DSS (lane 2) or 0.5 mM Sulfo-MBS (lanes 3 and 4) for 30 min at 0°C. The reaction was stopped by addition of 0.5 M Tris-HCl pH 7.4, the proteins were precipitated by TCA, separated by SDS-PAGE, transferred on nitrocellulose, and labelled using a polyclonal antiserum. (<b>D</b>) Analysis of p34 in BN-PAGE. Purified p34 was dissolved in 0.5% Triton X-100, 10% Glycerol, 50 mM NaCl, 0.1 mM EDTA, PMSF 1 mM, 20 mM Tris-HCl pH 7.0, and applied on a gel for BN-PAGE in the presence of 500 mM ε-aminocaproic acid (first dimension). A lane from the gel was excised and layered on top of a conventional SDS-PAGE for separation of the proteins under denaturing conditions (second dimension). The proteins were transferred on nitrocellulose and labelled using a polyclonal antiserum directed against p34 (upper panel). Truncated p34 comprising residues 37–319 was similarly analyzed (middle panel), or dissolved in the presence of 8 M urea and 1% dodecylmaltoside for separation in the first dimension (lower panel).</p

Import of p34 into isolated rat liver mitochondria.

<p>(<b>A</b>) Proteolysis of p34. Radiolabelled p34 was synthesized in reticulocyte lysate in the presence of ³⁵S-methionine and incubated with increasing concentrations of proteinase K (PK) as indicated. The samples were incubated for 10 min at 0°C, proteolysis was stopped by addition of PMSF. Aliquots were analyzed by SDS-PAGE. The radiolabelled protein was visualized and the relative amounts were determined using a phosphoimager. At 10 µg PK/ml, about 1% of p34 was retained, no p34 was detected at 20 µg PK/ml. (<b>B</b>) Import of p34 into mitochondria and digitonin fractionation. ³⁵S-labelled p34 and Tom70 (a subunit of 70 kDa of the protein translocase of the mitochondrial outer membrane) were synthesized in reticulocyte lysate and incubated with freshly isolated rat liver mitochondria for 10 min at 25°C. The mitochondria were reisolated by centrifugation and resuspended in 250 mM sucrose, 1 mM EDTA, 10 mM MOPS KOH, pH 7.2. As indicated, digitonin was added at increasing concentrations (up to 1% w/v), or proteinase K (PK) at a final concentration of 50 µg/ml, respectively. Proteolysis was stopped by addition of PMSF, and the mitochondria were collected by centrifugation. The proteins were separated by SDS-PAGE for subsequent analysis by digital autoradiography using a phosphoimager. (<b>C</b>) Import after exchange of residues in the p34 N-terminus. Reticulocyte lysate containing ³⁵S-labelled p34 was incubated with mitochondria for 10 min at 25°C (upper panel). Parallel samples contained mutant versions of p34 (P9A, G14A, K33A). Samples 3 and 4 contained valinomycin to dissipate the membrane potential, samples 2 and 4 were subsequently incubated with proteinase K (PK, final conc. 25 µg/ml). The mitochondria were reisolated and the proteins were separated by SDS-PAGE. (<b>D</b>) Proteolysis of p34(1–35)-DHFR. The experiment was carried out as in (A). (<b>E</b>) Function of the p34 N-terminus. Left panel (lanes 1 and 2), incubation of N-terminally truncated p34 (residues 37-319) with mitochondria. Reticulocyte lysate containing the radiolabelled protein was incubated with isolated rat liver mitochondria as in (C). 25 µg/ml proteinase K were subsequently added to sample 2 to degrade the protein outside the mitochondria. Right panel (lanes 3–5), import of a hybrid protein comprising the 35 N-terminal residues of p34 fused to the entire dihydrofolate (DHFR) of the mouse. Lane 3, sample of reticulocyte lysate containing the radiolabelled hybrid protein p34(1–35)-DHFR and DHFR. Lanes 4 and 5, incubation of the lysate with mitochondria and reisolation of the mitochondria. Proteinase K was added to sample 5 (+PK). Asterix (*), degradation product of the hybrid protein. (<b>F</b>) Import of p34 into trypsin-pretreated mitochondria. Rat liver mitochondria were pretreated with trypsin (20 µg/ml) for 10 min at 0°C, proteolysis was stopped by addition of soybean trypsin inhibitor and the mitochondria were reisolated. ³⁵S-labelled p34 was synthesized in reticulocyte lysate and incubated with the mitochondria at 25°C for different times as indicated. The trypsin pretreatment was omitted in parallel samples. The mitochondria were again reisolated and the proteins were separated by SDS-PAGE. The relative amounts of ³⁵S-labelled p34 were determined using a phosphoimager. The highest value was set to 100% (control). (<b>G</b>) Import of p34(1–35)-DHFR into trypsin-pretreated mitochondria. The experiment was carried out as in (F).</p

Import of p34 into isolated yeast mitochondria.

<p>(<b>A</b>) Protease-sensitivity of acid-pretreated p34. Reticulocyte lysate containg ³⁵S-labelled p34 was pretreated with HCl at pH 5 for 5 min, diluted twentyfold into 1 mM EDTA, 10 mM MOPS/KOH pH 7.4, and incubated with proteinase K at increasing concentrations. (<b>B</b>) Import of p34 into mitochondria. p34 was imported as described in legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000878#ppat-1000878-g002" target="_blank">Fig. 2</a>, using yeast mitochondria instead of mammalian mitochondria. The organelles were isolated from the wild type strain PK82 (lanes 1–7) or from the mutant strain <i>ssc1-3</i> (lane 8). No mitochondria were added in the sample of lane 4. (<b>C</b>) Import of p34 in protease-pretreated yeast mitochondria. The mitochondria were incubated with trypsin (20 µg/ml) for 10 min at 0°C, proteolysis was stopped by addition of soybean trypsin inhibitor and the mitochondria were reisolated. ³⁵S-labelled p34 was incubated with the mitochondria at 25°C for different times as indicated. (<b>D</b>) Import of p34 into mitochondria isolated from a strain lacking the outer membrane import receptor Tom20 (<i>tom20Δ</i>). Parallel samples contained mitochondria from the corresponding wildtype (WT). The standard deviation was calculated from 3 experiments, the highest value of each series was set to 100% (control). (<b>E</b>) Relative import efficiencies of p34(1–319), p34(1–35)-DHFR, and porin with mitochondria isolated from yeast strains lacking Tom20 or Tom70, respectively. The radiolabelled proteins were incubated with the mitochondria for 5 min at 25°C, reisolated, and treated with proteinase K. In parallel samples, the proteins were incubated with mitochondria isolated from the corresponding wildtype strains. The proteins were analyzed by SDS-PAGE and the relative amounts of the radiolabelled proteins were determined using the phosphoimager. The amounts detected in wildtype mitochondria were set to 100%. The standard deviations were calculated from 5 independent experiments. (<b>F</b>) Import of ³⁵S-labelled p34 into mitochondria isolated from a yeast strain containing a defect in Tom40 (<i>tom40-4</i>). The highest value of each experiment was set to 100% (control), n = 3. (<b>G</b>) Import of radiolabelled p34 into mitochondria and preparation of outer and inner membrane vesicles. ³⁵S-labelled p34 was imported into isolated yeast mitochondria, the mitochondria were reisolated and sonified. The membrane vesicles were layered on top of a sucrose step gradient and centrifuged for 16 h at 100.000 g. Fractions were collected for TCA-precipitation of proteins and subsequent analysis by SDS-PAGE, immuno blotting and visualization of radiolabelled p34 by digital autoradiography. Polyclonal antisera were used for labelling of Tom40 (outer membrane) and Tim23 (inner membrane), respectively. Fraction 1, upper part of the gradient (0.85 M sucrose); fraction 8, lower part of the gradient (1.6 M sucrose). Upper panel, radiolabelled p34 as visualized by digital autoradiography; lower panel, immuno blotting to visualize Tom40 and Tim23. (<b>H</b>) Quantification of the proteins shown in (G), the highest values were set to 100%.</p

Distribution of p34 in intact cells.

<p>(<b>A</b>) Hybrid proteins containing an EGFP domain (enhanced green fluorescent protein) fused to the complete p34 (residues 1–319), or parts of p34 comprising residues 37–319 or 1–36, respectively, were expressed in HeLa cells (green fluorescence) and their distribution was monitored by confocal microscopy. About 10% of the cells were found to be transfected. Mitochondria were visualized with a Tom20-specific labelling (red colour; Tom20 is a subunit of the protein translocase of the mitochondrial outer membrane), the DNA of the cells was stained with DAPI (blue). The overlay (right panel) shows the co-localization between the EGFP-labelled proteins and Tom20 (yellow). (<b>B</b>) Hydrophobicity plot of p34(1–319) and sequence of the 32 N-terminal residues.</p