Fig 1.
Characterisation of the Myc-BirA*-TAC102 cell line.
(A) Immunofluorescence microscopy pictures of Myc-BirA*-TAC102 PCF cells. A monoclonal antibody was used to visualise TAC102 (magenta). An anti-Myc antibody was used to visualise Myc-BirA*-TAC102 (green). DAPI stained nuclei and kDNA (cyan). Around 100 cells were observed before choosing the example pictures. (B) Western blot analysis of digitonin fractionated cell extracts from the Myc-BirA*-TAC102 cell line (PCF) after 24 hours of induction with tetracycline. Myc-BirA*-TAC102 (red arrow) and the endogenous TAC102 (blue arrow) were detected using the monoclonal anti-TAC102 antibody. Molecular weights (MW) are in kDa. As control, we probed for the mitochondrial membrane protein ATOM40. The digitonin fractionation was done twice and both experiments showed the same results.
Fig 2.
Biotinylation by Myc-BirA*-TAC102 in PCF cells.
(A) Western blot analysis of biotinylated proteins from the Myc-BirA*-TAC102 cell line (PCF) with (“+ tet”) or without (“- tet”) induction with tetracycline overnight and with (“+ bio”) or without (“- bio”) addition of biotin in the medium. Left lane indicates protein size in kDa. Arrows indicate examples of proteins only detected in the condition “+ tet, + bio”. The two upper arrows indicate the expected size for Myc-BirA*-TAC102 and for the endogenous TAC102 protein. Data were obtained from one experiment. (B) Immunofluorescence microscopy pictures of Myc-BirA*-TAC102 cell line (PCF) with or without induction with tetracycline for five hours. A monoclonal antibody was used to visualise TAC102. Streptavidin-Alexa 488 recognised the biotinylated proteins. DAPI stained nuclei and kDNA. Around 100 cells were observed before choosing the example pictures.
Fig 3.
Purification of TAC102 binding partners and near neighbours using BioID.
(A) Schematic of purification protocol. (B) Coomassie staining of the SDS-PAGE gel from BioID fractions. The experiment was done in four independent replicates and all showed the same results. (C) Western blot analysis of the BioID fractions. Streptavidin-HRP was used to detect the biotinylated proteins. Total cells extracts (E1), cleared supernatants (S1), first pellet (P1) and streptavidin bead bound (B1, B2) fractions were loaded on the gel. For E1, S1 and P1, the equivalence of 7.2x106 cells was loaded. For B1 and B2, the equivalence of 137x106 cells was loaded. MW: molecular weight. (D) TAC102 BioID enriched proteins (n = 4). The dashed lines represent the thresholds used for enrichment (> 3) and p-value (p < 0.05). Mitochondrial proteins identified with an enrichment > 3 (p < 0.05) are shown in red. IF: initiation factor; MIP: mitochondrial intermediate peptidase; NDUFS1: NADH-ubiquinone oxidoreductase complex I subunit; PIP5K: phosphatidylinositol-4-phosphate 5-kinase related; POLID: mitochondrial DNA polymerase I D; SSU: small subunit; sub.: subunit; tet: tetracycline.
Fig 4.
Characterisation of MIP RNAi cell line (BSF) (gene: Tb927.10.9820).
(A) Growth curves of MIP RNAi cell line with induction (red) and without induction (blue) with tetracycline (n = 1). MIP was C-terminally Myc-tagged in the RNAi cell line. The fusion protein was expected at 80 kDa. The RNAi efficiency was assessed by western blot using an anti-Myc antibody (rabbit). The upper band corresponded to MIP-Myc. The lower band was an unspecific band detected by the antibody. (B) Cell cycle status of MIP RNAi cells (BSF) with (red) and without (blue) induction of RNAi for 24 h. Around 100 cells were observed per condition (non-induced and induced, n = 1). Exemplary composite pictures are shown on top of the columns for “1K2N small K”, “1K1N tiny K” and “2K1N misseg” categories. The kDNAs are indicated with white arrows. (C) Western blot of MIP RNAi cell line probed for TAC102. A 3.5% gel was used. (D) Western blot of MIP RNAi cell line probed for TAO. A 12% gel was used. (E) Western blot of MIP RNAi cell line probed for MRP2. A 12% gel was used. kDa: kilodalton; MW: molecular weight; NI: RNAi non-induced; i28h: RNAi induced during 28 h. n = 1 for all experiments.
Fig 5.
TAC102 enrichment and immunoprecipitation.
The experiments were done in three independent replicates and all showed similar results. (A) Western blot analysis of the digitonin fractionation and subsequent lysis of the pellet fraction with 1% (v/v) Nonidet P-40 of whole cell protein from procyclic cells. T: total; S: supernatant; P: pellet. In orange: the pellet from the digitonin fractionation was used for the subsequent Nonidet P-40 lysis. The elongation factor EF1α served as a control. (B) Western blot analysis of the elutions (E1 and E2) of TAC102 immunoprecipitation replicates. (C) Silver stained SDS PAGE of TAC102 immunoprecipitation fractions. On the left are the molecular weights in kDa. Under each lane are written the equivalence of cells loaded. TAC102, indicated by a white arrow, was detected in E1. FT: flow-through; W1-W2-W3: washes; E1-E2: elutions. (D) Volcano plot representing the significance versus enrichment of the results from mass spectrometry analysis of TAC102 immunoprecipitation. A total of 775 proteins were identified from which 100 proteins were significantly enriched in the TAC102 immunoprecipitation (p ≤ 0.01). The two dotted lines represent the cut-off used (p ≤ 0.01 and enrichment > 3). The protein the most enriched in the condition was TAC102. Three other TAC components were also highly enriched (p166, TAC40 and TAC60).
Fig 6.
Localisations of the hypothetical proteins enriched significantly in both TAC102 BioID and TAC102 immunoprecipitation.
Immunofluorescence microscopy was done in PCF. TAC102 was recognised by a monoclonal antibody. The candidate was HA tagged (Tb927.10.900, Tb927.7.850 and Tb927.8.3160) or MYC tagged (Tb927.9.6410) and was recognised by an anti-HA antibody or an anti-MYC antibody. DAPI stained the DNA content (nuclei and kinetoplasts). Around 100 cells were observed before choosing the example pictures.
Table 1.
Nine proteins were enriched significantly in both TAC102 BioID and TAC102 immunoprecipitation (enrichment > 3; p ≤ 0.01).
Fig 7.
Depiction of the TAC102 interactions identified from BioID, immunoprecipitation and a yeast two hybrid screen.
Gene identities of the protein of interest are shown here as chromosome and location coordinates only. The common interactors (BioID/CoIP) are connected with a dotted line (——). The 15 most enriched proteins of the TAC102 IP or BirA*-TAC102 (BioID) are connected with a solid line (___). The five most enriched proteins are in bold. The two proteins identified by the yeast two hybrid screen are circled in grey (⬭). BC1: cytochrome b-c1 subunit; IM: inner mitochondrial membrane; NOP: nucleolar protein; NUP: nucleoporin; OM: outer mitochondrial membrane; PIP5K: phosphatidylinositol-4-phosphate 5-kinase related; POLID: DNA polymerase I D; Pol beta PAK: Polymerase beta with PAK domain.