Appendix 1—key resources table.
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Trypanosoma brucei brucei) | TREU 927/4 procyclic cells expressing T7 RNA polymerase and the tetracycline repressor to allow inducible expression | Poon et al., 2012 | SmOxP9 | Background strain used for derivation of cell lines described in Supplementary file 1 |
| Cell line (Trypanosoma brucei brucei) | TREU 927/4 expressing T7 RNA polymerase, tetracycline repressor, and the Cas9 nuclease | Beneke et al., 2017 | PCF1339 | Background strain used for derivation of cell lines described in Supplementary file 1 |
| Strain, strain background (Escherichia coli) | Rosetta 2(DE3)pLysS | Novagen | 71403 | |
| Strain, strain background (Escherichia coli) | BL21(DE3) | Novagen | 69450 | |
| Recombinant DNA reagent | pEnT5-Y, endogenous YFP tagging, hygromycin |
Kelly et al., 2007 | pEnT5-Y (pBA1) | See Supplementary file 1 for construction details on all plasmids used in this study |
| Recombinant DNA reagent | pBA148, endogenous tdTomato tagging, blasticidin |
Akiyoshi and Gull, 2014 | pBA148 | See Supplementary file 1 for construction details on all plasmids used in this study |
| Recombinant DNA reagent | pMig96 (pBA152), 256 LacO, integrate at rDNA |
Landeira and Navarro, 2007 | pMig96 (pBA152) | See Supplementary file 1 for construction details on all plasmids used in this study |
| Recombinant DNA reagent | pJ1339 (1173+Cas9), for CRISPR-Cas9 gene editing | Beneke et al., 2017 | pJ1339 | See Supplementary file 1 for construction details on all plasmids used in this study |
| Recombinant DNA reagent | p2T7-177, inducible expression of RNAi constructs (head to head), integrate at 177 bp | Wickstead et al., 2002 | p2T7-177 (pBA3) | See Supplementary file 1 for construction details on all plasmids used in this study |
| Recombinant DNA reagent | pRSFDuet-1, expression of one or two target proteins in bacteria | Novagen | pRSFDuet-1, catalog number 71341-3 | See Supplementary file 1 for construction details on all plasmids used in this study |
| Recombinant DNA reagent | pBA310, inducible expression vector, integrate at 177 bp | Nerusheva and Akiyoshi, 2016 | pBA310 | See Supplementary file 1 for construction details on all plasmids used in this study |
| Recombinant DNA reagent | pBA795, inducible GFP-NLS-LacI expression vector, integrate at 177 bp | Ishii and Akiyoshi, 2020 | pBA795 | See Supplementary file 1 for construction details on all plasmids used in this study |
| Recombinant DNA reagent | pPOTv7 (pBA1919), (eYFP, Hygromycin) vector for PCR only tagging (POT) of target genes in Trypanosoma brucei | Dean et al., 2015 | pPOTv7 (pBA1919) | See Supplementary file 1 for construction details on all plasmids used in this study |
| Recombinant DNA reagent | pEnT6-Y (pBA191), endogenous YFP tagging, G418 |
Kind gift from Dehua Lai | pEnT6-Y (pBA191) | See Supplementary file 1 for construction details on all plasmids used in this study |
| Recombinant DNA reagent | pMig75, Tet inducible GFP-LacI, SAT, ClonNAT | Kind gift from Miguel Navarro | pMig75 (pBA150) | See Supplementary file 1 for construction details on all plasmids used in this study |
| Recombinant DNA reagent | pMig96, rDNA targeting, 256LacO, Phleo | Kind gift from Miguel Navarro | pMig96 (pBA152) | See Supplementary file 1 for construction details on all plasmids used in this study |
| Recombinant DNA reagent | BAG164 (shRNA against KIN-A 5’ UTR) in pMK-RQ plasmid | Life Technologies Ltd (Invitrogen Division) | NA | See Supplementary file 1 for sequence information |
| Recombinant DNA reagent | BAG165 (shRNA against KIN-A 3’UTR) in pMK-RQ plasmid | Life Technologies Ltd (Invitrogen Division) | NA | See Supplementary file 1 for sequence information |
| Recombinant DNA reagent | BAG157 (shRNA against CPC1 5’ UTR) in pMK-RQ plasmid | Life Technologies Ltd (Invitrogen Division) | NA | See Supplementary file 1 for sequence information |
| Recombinant DNA reagent | BAG158 (shRNA against CPC1 3’ UTR) in pMK-RQ plasmid | Life Technologies Ltd (Invitrogen Division) | NA | See Supplementary file 1 for sequence information |
| Recombinant DNA reagent | BAG159 (shRNA against CPC2 5’ UTR) in pMK-RQ plasmid | Life Technologies Ltd (Invitrogen Division) | NA | See Supplementary file 1 for sequence information |
| Recombinant DNA reagent | BAG160 (shRNA against CPC2 3’ UTR) in pMK-RQ plasmid | Life Technologies Ltd (Invitrogen Division) | NA | See Supplementary file 1 for sequence information |
| Recombinant DNA reagent | BAG62 (shRNA against KKT16 CDS) in pMK-RQ plasmid | Life Technologies Ltd (Invitrogen Division) | NA | See Supplementary file 1 for sequence information |
| Recombinant DNA reagent | BAG80 (shRNA against KKT12 3’UTR) in pMK-RQ plasmid | Life Technologies Ltd (Invitrogen Division) | NA | See Supplementary file 1 for sequence information |
| Antibody | Mouse monoclonal anti-GFP | Roche | 11814460001 | For immunoprecipitation experiments: 12 μg of antibodies preconjugated with 60 μl slurry of Protein-G magnetic beads |
| Antibody | Rabbit polyclonal anti-GFP | OriGene | TP401 | Dilution for western blot 1:5000 |
| Antibody | Mouse monoclonal TAT1 (anti-trypanosomal-alpha-tubulin) | kind gift from Keith Gull Woods et al., 1989 | TAT1 | Dilution for western blot 1:5000 |
| Antibody | IRDye 680RD goat anti-mouse | LI-COR | 926-68070 | Dilution for western blot 1:20,000 |
| Antibody | IRDye 800CW goat anti-rabbit | LI-COR | 926-32211 | Dilution for western blot 1:20,000 |
| Commercial assay, kit | Protein-G magnetic beads | Thermo Fisher Scientific | 10004D | |
| Chemical compound, drug | RapiGest | Waters | 186001860 | |
| Peptide, recombinant protein | Trypsin | Promega | V5111 | |
| Chemical compound, drug | BS3 (bis(sulfosuccinimidyl)suberate) | Thermo Fisher Scientific | 21580 | |
| Commercial assay, kit | TALON metal affinity resin | TAKARA BIO EUROPE | 635503 | |
| Commercial assay, kit | Zeba spin desalting columns | Thermo Fisher Scientific | 89883 | |
| Commercial assay, kit | SimplyBlue SafeStain | Life Technologies Ltd (Invitrogen Division) |
LC6060 | |
| Peptide, recombinant protein | Porcine tubulin | Cytoskeleton | T-240 | |
| Software, algorithm | TriTrypDB | http://tritrypdb.org/tritrypdb/ | RRID:SCR_007043 | |
| Software, algorithm | MaxQuant (version 2.0.1) | Cox and Mann, 2008 | RRID:SCR_014485 | |
| Software, algorithm | pLink2 | Chen et al., 2019 | RRID:SCR_000084 | |
| Software, algorithm | AlphaFold2-Multimer-v2 | Evans et al., 2022; Jumper et al., 2021 | ||
| Software, algorithm | ColabFold | Mirdita et al., 2022 | ||
| Software, algorithm | hmmsearch | https://www.ebi.ac.uk/Tools/hmmer/search/hmmsearch | ||
| Software, algorithm | MAFFT | https://mafft.cbrc.jp/alignment/server/index.html | RRID:SCR_011811 | |
| Software, algorithm | Fiji | Schneider et al., 2012 | RRID:SCR_002285 | |
| Software, algorithm | Python | https://www.python.org/ | RRID:SCR_008394 | |
| Software, algorithm | xiView | Graham et al., 2019, https://xiview.org/index.php |