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

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

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

Steady-state SPLIFF measurements.

<p>Each point represents a single cell measurement. The expressions of the N_ub -fusion proteins were induced by100 μM Cu²⁺. The calculated medians and SEMs are shown in red. N_ub-fusions are grouped according to their interaction signal. (<b>A</b>) Strong interaction (SPLIFF signal ≥ 70%). (<b>B</b>) Weak interaction (SPLIFF signal ≥ 30%). (<b>C)</b> No interaction (SPLIFF signal ≤ 30%). The negative value for Nap1 might result from a weaker expression of Nap1 than the control N_ub. Note a subpopulation of Glc7 cells that have a SPLIFF signal >50%. grey squares: negative control N_ub-empty; grey circle: positive control N_ub-Cdc11. (<b>D</b>) Comparison between stable collar and split septin rings SPLIFF signals induced by N_ub- and -Bud4 with 0 (left) or 100 μM Cu²⁺ (right).</p

Timing of the interaction of Bud3 and Bud4 with the septins.

<p><b>(A)</b> Time lapse microscopy of Bud3-GFP or Bud4-GFP and Shs1-mCherry expressing cells. The time of the first appearance of the GFP fusion protein after the appearance of Shs1-mCherry at the bud neck was recorded. <b>(B)</b> Pulldown of Bud3-TAP and Bud4-TAP on immobilized septin rods and detection with an anti-Protein A antibody. The asterisk marks the hardly visible Bud3-TAP signal. A cross reaction of the antibody with one of the septins is marked with +. <b>(C)</b> Time resolved SPLIFF analysis (N_ub-Bud3 or N_ub-Bud4 vs. Shs1-CCG). Imaging time frame 3 min, 100 μM Cu²⁺. N = 10 for Bud3 and N = 9 for Bud4. The start of the N_ub induced conversion of Shs1-CCG is marked with an arrow.</p

Workflow for SILAC-AP-MS.

<p>Yeast strains expressing Cdc11-TAP and Cdc11-GFP (metabolically labelled with ¹³C₆,¹⁵N₄-L-arginine and ¹³C₆,¹⁵N₂-L-lysine) are blocked with the indicated methods and subjected separately to affinity purification. The eluates are mixed and subjected to separation by SDS-PAGE followed by nano HPLC-ESI-MS/MS analysis and statistical data evaluation.</p

Overview of the SILAC-AP-MS datasets.

<p><b>(A)</b> Overlap of the specific hits in alpha-factor arrested cells, S-phase (HU) and late anaphase (<i>cdc15-1</i>). <b>(B)</b> Summary and overlap of the candidate proteins selected for further validation.</p

Validation of selected candidate proteins by fluorescence microscopy.

<p>(<b>A</b>) Live cell imaging of cells expressing Shs1-mCherry and Bni4-GFP at indicated time points of the cell cycle. Scale bar 5 μm. (<b>B</b>) Fluorescence microscopy images of blocked cells expressing Shs1-mCherry and Nip1-, Pno1-or Tub1-GFP. Scale bar 6 μm.</p