A novel connection between Trypanosoma brucei mitochondrial proteins TbTim17 and TbTRAP1 is discovered using Biotinylation Identification (BioID)

Figure 1. Expression and mitochondrial localization of the BirA∗-TbTim17 fusion protein in T. brucei. A, a schematic showing the cloning strategy of the TbTim17 ORF into the modified pLew100_Myc_BirA∗ plasmid. Restriction sites were indicated at the top. B, growth curve for BirA∗-TbTim17 cell line in the absence (uninduced) and presence (induced) of doxycycline for 10 days. The log of the cumulative cell numbers was plotted versus time. C, analysis of the subcellular fractions obtained from the parental control and BirA∗-TbTim17 T. brucei cells grown in the presence of doxycycline for 2 days. Proteins from the total (T), cytosolic (C), and mitochondrial (M) fractions were subjected to SDS-PAGE, transferred to membrane, and probed with BirA and TbTim17 antibodies. VDAC and TbPP5 were used as the mitochondrial and cytosolic protein markers, respectively. D, sodium carbonate extraction was followed by immunoblot analysis of mitochondrial proteins from BirA∗-TbTim17 T. brucei cells grown in the presence of doxycycline. The supernatant (S) and the pellet (P) fractions were analyzed using BirA∗, TbTim17, VDAC, and RBP16 antibodies. E, mitochondria isolated from uninduced (Control) and induced…

Figure 2. Biotinylation pattern of T. brucei proteins by BirA∗-TbTim17. BirA∗-TbTim17 cells were induced or kept uninduced and grown either in the absence of biotin (I) or regular (II) medium. After 12 h postinduction, cells grown in regular medium were divided into two parts, and one part was supplemented with excess biotin (III). The parental control cells also were grown in parallel under similar conditions. At 24 h postinduction, cells were harvested and subcellular fractions were separated. Immunoblots of total (A), cytosol (B), and mitochondrial (C) fractions were probed with HRP-conjugated streptavidin. TbPP5 and VDAC were used as the total, cytosolic, and mitochondrial protein markers, respectively. Red asterisks represent unique biotinylated bands compared to control and uninduced BirA∗-TbTim17 samples. HRP, horseradish peroxidase; VDAC, voltage-dependent anion channel.

Figure 3. Purification of biotinylated proteins from BirA∗-TbTim17 cells. The BirA∗-TbTim17 cells were grown in the presence of doxycycline for 12 h in regular medium. Next, excess biotin (50 μM) was added to the medium, and cells were grown for an additional 12 h. Uninduced (Control) cells were grown in parallel under similar conditions. Cells were harvested, and crude mitochondrial fractions were isolated. Mitochondrial proteins were solubilized with NP-40 (1%) and used for affinity purification using Streptavidin-conjugated Dynabeads. Samples from each step of the purification process were collected for further analysis. Immunoblot analysis of the input (I), flow-through (Ft), washed (W1-W3), and bound (B) proteins were performed using streptavidin-HRP (Top panel) as the probe. Blots were reprobed with BirA∗, TbTim17, VDAC, and mHsp70 antibodies. HRP, horseradish peroxidase; VDAC, voltage-dependent anion channel.

Figure 4. Analysis of the mass-spectrometry results. Data files produced from duplicate sets of MS-MS were parsed into Scaffold (version 5.0.1) for filtering and validation. Proteins identified with high confidence and enriched 3- to 12-fold in the sample from induced BirA∗-TbTim17 cells were enlisted. GO annotation was conducted for cellular component of the biotinylated proteins to identify their subcellular localization. The proteins were submitted to the UniProtKB mapping tools for annotation. The percentage of proteins with a given GO term is indicated in the corresponding slices with different colors as indicated in the key in the pie chart. In the mitochondrial protein layer of the chart, well-characterized proteins were annotated as matrix, inner membrane, outer membrane, and ribosomal subunits. Others include proteins for which submitochondrial locations were not determined. GO, Gene Ontology; MS, mass spectrometry.

Figure 5. Expression and subcellular localization of in situ tagged TbTRAP1-3xHA and 6XMyc-TbTim17 in T. brucei. A, schematics of the in situ tagging strategy. Targeting loci of TbTRAP1 and TbTIM17 in chromosomes are shown. Under each targeting locus, the corresponding in situ tag constructs are depicted. Crossovers for homologous recombination are shown by dashed lines. Red arrows indicate the linearization sites in the construct. B, mid-log phase T. brucei expressed both 6xMyc-TbTim17 and TbTRAP1-3xHA, only 6xMyc-TbTim17, and the parental control cells were harvested. The cytosolic (C) and mitochondrial fractions were isolated by digitonin extraction as described in the Experimental procedures. Proteins from these fractions were separated on SDS-PAGE, transferred to nitrocellulose membrane, and probed with antibodies for Myc and HA epitopes. VDAC and TbPP5 were used as mitochondrial and cytosolic markers. C, in situ immunofluorescence analysis of T. brucei procyclic form expressed both TbTRAP1-3xHA and 6xMyc-TbTim17. The parental Pro427 cells were used in parallel as control. Anti-Myc polyclonal (rabbit) and anti-HA monoclonal (mouse) were used as primary antibodies, and anti-rabbit IgG…

Figure 6. Coimmunoprecipitation analysis of TbTim17 and TbTRAP1 from T. brucei mitochondrial lysate. A and B, mitochondrial proteins from T. brucei expressing both 6xMyc-TbTim17 and TbTRAP1-3xHA were solubilized with digitonin (1.0%) and subjected for immunoprecipitation with anti-Myc (A) and anti-HA (B) antibody-coupled agarose beads. Mitochondrial lysate from the parental control cells was used in parallel. Input (I), flowthrough (Ft), and bound (B) proteins were analyzed by immunoblot analyses probing with HA, Myc, and VDAC antibodies. C and D, mitochondrial proteins from T. brucei expressing both 6xMyc-TbTim17 and TbTRAP1-3xHA were solubilized first with 1% SDS and diluted 20-fold with buffer containing 0.2% Triton X-100, as described in the Experimental procedures section. Solubilized proteins were subjected for immunoprecipitation with anti-Myc (C) and anti-HA (D) antibody-coupled agarose beads. Input (I), flowthrough (Ft), and bound (B) proteins were analyzed by immunoblot analysis probing with HA, Myc, mHsp70, VDAC, and RBP16 antibodies. HA, hemagglutinin; VDAC, voltage-dependent anion channel.

Figure 7. Effect of TbTRAP1 RNAi on TbTIM17 protein complex. A, TbTRAP1 RNAi cells were grown in the presence and absence of doxycycline for induction of TbTRAP1 dsRNA expression. Cell numbers were counted daily for 6 days, and a log of cumulative cell numbers was plotted versus time post-induction. B, BN-PAGE analyses of mitochondrial protein complexes. T. brucei cells expressed in situ tagged TbTRAP1-3xHA and tetracycline-inducible TbTRAP1 dsRNA (TbTRAP1-3xHA/TbTRAP1-RNAi) were grown for 2 days in the absence (−) and presence (+) of doxycycline for isolation of mitochondria. Mitochondrial proteins were solubilized with 1% digitonin and subjected to BN-PAGE, followed by immunoblot analyses using HA (TbTRAP1), TbTim17, VDAC, mHsp70, Atom69, and TbATPase antibodies for detection of the respective complexes. Coomassie blue (CB)–stained gel image are shown as the loading control. C, TbTRAP1-3xHA/TbTRAP1-RNAi cells were grown for 2 days in the absence (−) and presence (+) of doxycycline for isolation of mitochondria. Total mitochondrial proteins were analyzed by immunoblot analyses using antibodies for HA, TbTim17, TbATPase, TbTim62, RBP16, and Atom69. D, quantitation of the levels…

Figure 8. Assembly of TbTim17-2xMyc into the TbTIM complex in TbTRP1-depleted T. brucei.T. brucei cells express TbTim17-2xMyc alone and with TbTRAP1 dsRNAs in the background of in situ tagged TbTRAP1-3xHA upon induction with doxycycline were used to analyze the import and assembly of the TbTim17-2xMyc. A, schematics for the in situ tagged TbTRAP1-3xHA in the T. brucei genome and the constructs for the expression of TbTim17-2xMyc and TbTRAP1 dsRNA. Tetracycline operator (TetO) and the direction of transcription by T7 polymerase are shown. B, immunoblot analysis of the mitochondrial proteins from the TbTRAP1-3xHA/TbTRAP1-RNAi/TbTim17-2xMyc cells uninduced (-) and induced for 4 days with doxycycline were probed with different antibodies as indicated. Mitochondrial proteins from TbTim17-2xMyc cells induced for the same time were analyzed in parallel as the control. C, BN-PAGE analysis of the mitochondrial proteins from the TbTRAP1-3xHA/TbTRAP1-RNAi/TbTim17-2xMyc uninduced (−) and induced (+) for 4 days with doxycycline were probed with anti-myc and Anti-TbTim17 antibodies. Mitochondrial proteins from the TbTim17-2xMyc cells, induced for the same time, were analyzed in parallel as the control.…

Figure 9. A schematic of the role of TbTRAP1 on TbTIM17 complex biogenesis. The simplified form of the mitochondrial outer and inner membrane translocases, ATOM and TbTIM17, respectively, are shown. The translocation pathway of TbTim17, a nuclear-encoded mitochondrial protein, is indicated by red arrows. The left panel represent the translocation of TbTim17 and its assembly in the TbTIM17 complex in the inner membrane in the WT parasite. Whereas, the right panel shows the pathway that is hampered due to depletion of TbTRAP1 (TbTRAP1 KD). Abbreviations used are MOM; mitochondrial outer membrane, MIM; mitochondrial inner membrane, IMS; intermembrane space. TbTim17 represents the protein TbTim17, TbTIM17 represents the TbTim17 complex. TbTRAP1 shown as homodimer.