, transfection of procyclic cells
 and RNA isolation
 were performed using previously published protocols. Oligonucleotides used to prepare clones and probes for Northern blots are listed in Additional file
2: Figure S9.
The 987 transcripts were translated using the getorf program of the European Molecular Biology Open Software Suite setting a lower limit of 25 aa and including only ORFs that contained a start and stop codon
We used the NCBI BLAST suite (BLAST 2.2.28,
) for our protein searches. The predicted 4,699 T. brucei ORFs were used as queries for blastp to search all non-redundant kinetoplastid protein sequences (taxid: 5653), using an e-value cutoff of 0.1. T. brucei (taxid: 5691). T. b. gambiense (taxid: 31285) and T. b. brucei (taxid: 5702) sequences were excluded from the search. Similarly, tblastn was used to search the kinetoplastid translated nucleotide database with an e-value cutoff of 0.1. The annotated proteins of S. cerevisiae (taxid: 4932), C. elegans (taxid: 6239), A. thaliana (taxid: 3702), D. melanogaster (taxid: 7227), M. musculus (taxid: 10090) and H. sapiens (taxid: 9606) were queried with the same strategy. All alignments were manually inspected and verified to exclude false positives due to the relaxed threshold.
The predicted T. brucei proteins were scanned for domains using the NCBI CD-Search Tool (cdsearch/cdd v3.10;
). Transmembrane helices in proteins were predicted using the TMHMM Server v. 2.0
[38, 58] and the presence and location of signal peptide cleavage sites were scanned at the SignalP 4.1 Server
[37, 59]; both servers are at the Technical University of Denmark.
Mass spectrometry analysis
Procyclic-form trypanosomes (MiTat 1.4) were lysed in 50 mM Tris (pH 7.3) with 4% SDS. To decrease sample complexity, the samples were filtered directly using different (3 kDa, 10 kDa, 30 kDa and 50 kDa molecular weight cut off (MWCO)) Amicon Ultracel centrifugal filter units (Millipore, Billerica, MA, USA) and under strong denaturing conditions with 8 M urea. The filtrate was precipitated using 4 volumes of ethanol and subsequently resuspended in 20 μl 8 M urea. The samples were reduced with 1 mM dithiothreitol (DTT) and alkylated using 5 mM iodoacetamide, prior to digestion with 0.2 μg Lys-C (Wako, Richmond, VA, USA) for three hours followed by digestion with 0.2 μg trypsin (Promega, Madison, WI, USA) overnight. The peptides were desalted using a StageTip
. For MS analysis, peptides were separated by a nanoflow liquid chromatography EASY-nLC system on a capillary packed with Reprosil-C18 (Dr. Maisch) with an acetonitrile gradient from 2% to 60% at a flow rate of 250 nl/minute for 230 minutes. The Orbitrap XL mass spectrometer was operated in a data-dependent acquisition mode performing Top10 MS/MS per full cycle. Data analysis was done with MaxQuant version 126.96.36.199
 using a concatenated database of TREU 927 v.2.3 (10,533 entries, tritrypDB.org) and the hits generated by the RNA-Seq analysis (987 entries, Additional file
1). Enzyme search specificity was set for tryptic peptides. Up to two miscleavages for each peptide were allowed. Carbamidomethylation on cysteines was set as fixed modification, while methionine oxidation and protein N-acetylation were considered as variable modifications. The search was performed with an initial mass tolerance of 6 ppm mass accuracy for the precursor ion and 0.5 Da. False discovery rate was fixed at one percent on peptide and protein level. For the second data set
, we reanalyzed previously generated MS data using MaxQuant standard settings with the above described concatenated database. The MS proteomics data have been deposited to the ProteomeXchange Consortium
 via the PRIDE partner repository
 with the dataset identifier PXD000711
 and PXD000712 (this study).
T. brucei 29.13.6 Lister 427 procyclic cells
 were cultured at 28°C with 5% CO2 in Cunningham’s media supplemented with 10% Tet-system approved heat inactivated fetal bovine serum (FBS, Clontech, Mountain View, CA, USA), 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml gentamicin, 15 μg/mL G418 and 50 μg/ml hygromycin B. A total of 1 × 107 29.13.6 cells were used for each procyclic-form transfection. Cells were spun down, washed in Cytomix (20 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4, 25 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (Hepes), 2 mM ethylenediaminetetraacetic acid (EDTA) and 5 mM MgCl2, pH7.6), then resuspended in 500 μl Cytomix. Then, 25 μg of linearized plasmid DNA was added to the solution and cells were pulsed twice at 1,600 V with a time constant of 0.6 ms on a GenePulser Xcell (BioRad, Hercules, CA, USA). Cells were allowed to rescue for 24 hours before selective drug was added. Phleomycin and blasticidin were added to a final concentration of 2.5 μg/ml and 10 μg/ml, respectively. Transfected cells were cloned at least 24 hours after transfection. Serial dilutions of the cells were then made in media with 20% serum and the presence of 3 × 107 29.13.6 cells that had not been transfected. A total of 200 μl of transfected cells were added to 1.8 ml of the cloning media and further diluted in six, five-fold dilutions. Each dilution was plated in a 96-well plate and clones were selected from dilutions where fewer than 30% of wells had growth. T. brucei SM Lister 427 bloodstream-form cells
 were maintained at 37 °C with 5% CO2 in HMI-9 media supplemented with 10% Tet-system approved heat inactivated FBS (Clontech), 100 units/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml gentamicin, and 2.5 μg/ml G418. For bloodstream form transfections, 3 × 107 cells were centrifuged, washed quickly with Tb-BSF buffer
, resuspended in 100 μl Tb-BSF buffer containing 10 μg of plasmid DNA and transfected with protocol X-01 in an AMAXA Nucleofector®. Transfected cells were placed in 30 ml pre-warmed HMI-9 media and two 10-fold serial dilutions were plated in a 24-well plate. Six hours after transfection, pre-warmed medium supplemented with appropriate selectable drugs was added. The final concentration of selectable markers was 2.5 μg/ml phleomycin and 5 μg/ml blasticidin. For induction of hairpin or RNAi-resistant construct expression, 10 μg/ml and 1 μg/ml of doxycycline was added to procyclic- and bloodstream-form cells, respectively.
Constructs encoding hairpin RNAs
We followed the Gateway®-adapted cloning scheme developed by Margaret Phillips and colleagues
. Briefly, a 300 to 400 base pair region was PCR amplified and TA-cloned (deoxythymidine, T, deoxyadenosine, A) into plasmid pCR/8GW/TOPO (Invitrogen, Grand Island, NY, USA) to generate an entry clone. The entry clone was then recombined with the destination vector pTrypRNAiGate. Final constructs were verified by restriction enzyme digestions and DNA sequencing.
To rescue the lethal RNAi phenotype, RNAi-resistant versions of the CDS were synthesized by GeneWiz, Inc, Cambridge, MA, USA. A silent mutation was introduced every twelfth nucleotide
, a C-terminal HA-TEV-FLAG tag was added and the CDS was flanked at the 5’ and 3’ end by a Hind III and Bam HI restriction site, respectively. The RNAi-resistant construct was cloned into the inducible pLew100v5 BSR plasmid flanked by the GPEET procyclin 5’ UTR and an aldolase 3’UTR. In addition, inducible GFP-tagged RNAi-resistant constructs were generated in pLew100v5 BSR. The plasmids containing the RNAi-resistant constructs were digested with Not I to allow for recombination into a T. brucei ribosomal RNA spacer region following transfection. For each knock-down, ten clones were tested for a phenotype.
Total RNA was separated on 1.5% agarose gels in the presence of 6.3% formaldehyde in 40 mM 3-morpholinopropane-1-sulfonic acid (MOPS) and 2 mM EDTA. RNA ladder 0.5 to 10 kb (Invitrogen) was used as marker. The RNA was transferred overnight to a Hybond-N nylon membrane (GE Healthcare, Little Chalfont, United Kingdom) by capillary transfer with 10× SSC (0.15 M sodium citrate, 1.5 M sodium chloride), UV cross-linked to the membrane and stained with methylene blue. The membrane was pre-hybridized for one hour in 5× SET (0.75 M sodium chloride, 5 mm EDTA, 0.15 M Tris-HCl, pH 7.4), 10× Denhardt’s solution, 1% SDS and 100 μg/ml yeast RNA, and then hybridized overnight in the same solution. DNA probes were internally labeled by synthesis with specific dsDNA templates, sense and antisense primers and Pfx DNA polymerase (Invitrogen) in the presence of [α-32P]dCTP. The membrane was washed two to three times (10 minutes each) with 2× SSC, 0.1% SDS and hybridization signals were detected by PhosphorImager.
Semi-quantitative reverse transcriptase (RT)-PCR
For each sample, 5 μg of total RNA was treated with 2 units of DNase RQ (Promega), phenol extracted and ethanol precipitated. DNase-treated RNA was reverse transcribed using random primers (Promega) and Superscript II enzyme (Invitrogen) according to the manufacturer’s protocol. Twenty-two cycles of PCR were then performed using Platinum Pfx (Invitrogen), according to the manufacturer’s instructions, for each knockdown with histone 4 used as a control. An annealing temperature of 50°C was used for all oligonucleotides with an extension time of 30 seconds.
A total of 5 to 8 × 106 cells were spun down and washed twice with cell wash (20 mM Tris–HCl (pH 7.5), 100 mM NaCl and 3 mM MgCl2) before being placed on slides coated with poly-L-lysine and settling for five minutes. Then, 4% paraformaldehyde (PFA) was added and the cells were fixed for 30 minutes at 4°C. Cells were washed twice and then exposed to 0.1% NP-40 detergent in a solution of 2% goat serum. Slides were washed again and blocked with 10% goat serum for 10 minutes. Upon removal of blocking solution, primary antibody, diluted in 2% goat serum, was administered for one hour. Anti-GFP (Roche, Basel, Switzerland), anti-HA (Covance, Princeton, NJ, USA), and α-GPEET procyclin (Cedarlane labs, Burlington, Ontario, Canada) antibodies were obtained commercially. The antibodies to BiP were generously provided by Jay Bangs. Cells were washed five times, before the addition of secondary antibody and 5 μg/ml Hoechst (Cell Signaling Technology, Inc, Beverly, MA, USA). Cells were exposed to secondary antibody, diluted in 2% goat serum, for one hour. All secondary antibodies (Alexa Fluor 488-conjugated goat anti-mouse, 594-conjugated goat anti-mouse, 488-conjugated goat anti-rabbit and 594-conjugated goat anti-rabbit (Invitrogen)) were used at a 1:1,000 dilution. Samples were washed five times for 10 minutes total. Wash solution was removed and FluorSave (Calbiochem, Darmstadt, Germany) was added. Next, the coverslip was placed on the slide, and FluorSave reagent dried for two hours to overnight before cells were imaged on a Zeiss (Jena, Germany) Axioplan 2 fluorescence microscope. For kDNA size estimation, cells were fixed and stained with Hoechst, following the protocol outlined above. Cells were imaged and kDNA size was compared visually between un-induced cells and induced cells.
A total of 1 × 107 cells was spun down, washed once in cell wash (20 mM Tris–HCl (pH 7.5), 100 mM NaCl and 3 mM MgCl2) and resuspended in Cunningham’s media with no added serum. MitoTracker Red CM-H2xRos (Invitrogen) was added to a final concentration of 1 μM. Cells were incubated at 28°C and 5% CO2 for 10 to 15 minutes, centrifuged, washed and placed in fresh media without serum. Cells were rescued in media without MitoTracker for 25 minutes, then Hoechst (5 μg/ml) was added; cells were spun down and finally resuspended in 20 to 50 μl of PBSG.
Live cell imaging
A total of 5 to 8 × 106 cells was collected, Hoechst (5 μg/ml) was added and the cells were incubated in the dark for two minutes. Cells were centrifuged, washed once in phosphate-buffered saline with glucose (PBSG), resuspended in 20 to 50 μl of PBSG, and imaged on a Zeiss Axioplan 2 fluorescence microscope.
Cell compartment qproteome kit (Qiagen)
Each fractionation used 1 × 109 cells expressing GFP-tagged Tb11.NT.29 and the manufacturer’s (Qiagen, Venlo, Limburg, The Netherlands) instructions were followed. The anti-HSP70 and anti-BiP antibodies were generously provided by Jay Bangs.
Mitochondrial isolation qproteome kit (Qiagen)
Each fractionation used 1 × 109 cells expressing GFP-tagged Tb10.NT.87 or Tb11.NT.28 and the manufacturer’s (Qiagen) instructions were followed. The antibodies to REAP and TbMP63 were generously provided by Steve Hajduk and Ken Stuart, respectively.
Digitonin solubilization assay
Cells (1 × 108) expressing GFP-tagged Tb10.NT.87 or HA-tagged Tb11.NT.28 were spun down for each assay, washed twice with 20 mM sodium phosphate (pH 7.9), 20 mM glucose and 0.15 M NaCl, and then resuspended in 500 μl SoTE buffer (20 mM Tris–HCl (pH 7.5), 0.6 M sorbitol, 2 mM EDTA). Next, 500 μl of SoTE buffer with varying digitonin amounts was added to each sample to a final concentration of detergent of 0.015%, 0.025%, 0.04%, 0.05% or 0.1%
[48, 49]. Samples were incubated for five minutes at 4°C followed by centrifugation for three minutes at 5,000 g at 4°C. SDS-sample buffer was added to the supernatants and samples were analyzed by SDS-PAGE and Western blotting. The antibodies to TAO and mtHSP70 were generously provided by Minu Chaudhuri and Jay Bangs, respectively.
Samples were fixed in 4% PFA/0.1% gluteraldehyde in PBS for 30 minutes followed by further fixation in 4% PFA for one hour, rinsed in PBS, scraped and re-suspended in 10% gelatin. Chilled blocks were trimmed, placed in 2.3 M sucrose overnight on a rotor at 4°C, transferred to aluminum pins and frozen rapidly in liquid nitrogen. The frozen blocks were cut on a Leica Cryo-EMUC6 UltraCut and 65 nm thick sections were collected using the Tokoyasu method
 and placed on carbon/formvar coated grids and floated in a dish of PBS for immunolabeling. Grids were placed section side down on drops of 0.1 M ammonium chloride to quench untreated aldehyde groups, then blocked for nonspecific binding on 1% fish skin gelatin in PBS. Single labeled grids were incubated on a primary antibody mouse anti-HA (Covance) 1:50 dilution, which required a rabbit anti-mouse bridge (JacksonImmuno, West Grove, PA, USA). The secondary antibody was 10 nm Protein A gold (Utrecht Medical Center). All grids were rinsed in PBS, fixed using 1% gluteraldehyde for five minutes, rinsed again and transferred to a UA/methylcellulose drop before being collected and dried. Samples were viewed using a FEI Tencai Biotwin TEM at 80 Kv. Images were taken using Morada CCD and iTEM (Olympus) software.