Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD075501/20211215_BioID_VTC1_FC12_1_TryTryp_2019.xlsxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD075501/20211215_BioID_VTC1_DDM_1_TryTryp_2019.xlsxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD075501/20211215_BioID_VTC4_DDM_1_TryTryp_2019.xlsxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD075501/20211215_BioID_VTC4_FC12_1_TryTryp_2019.xlsxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD075501/checksum.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD075501/20211215_BioID_VTC1_DDM_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD075501/20211215_BioID_VTC1_FC12_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD075501/20211215_BioID_VTC4_FC12_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD075501/20211215_BioID_VTC4_DDM_1.rawprimaryOK200keni.vidilaseris@helsinki.fiKeni VidilaserisAffinity purification coupled with mass spectrometry proteomicsMass SpectrometryNot availableLc-msLeishmania tarentolaeBioidVtc binding partnersAcidocalcisomesVtc complexProteome discoverer 2.5https://www.ebi.ac.uk/pride/archive/projects/PXD075501BioID pulldown was done as previously described [21]. The acidocalcisome fraction (BirA*-LtVtc1 or BirA*-LtVtc4) was resuspended in 1 mL of lysis buffer (50 mM Tris pH 7.5, 500 mM NaCl, 0.4% SDS, 1 mM DTT). The samples were solubilised in 2% DDM or 2% FC-12 on ice for 30 min. The samples were centrifuged at 16,000 × g for 10 min at 4 °C, and 100 μL of StrepTactin Sepharose (Iba, 2-1201-025) beads were added to the supernatant and incubated overnight at 4 °C with gentle rotation. The beads were washed three times for 5 min with 1 mL wash buffer (50 mM Tris-HCl pH 7.4, 8 M Urea). The biotinylated proteins bound to the StrepTactin beads were washed eight times with 8 M urea/0.2 M ammonium bicarbonate. Lys-C peptidase was added and incubated o/n at RT in ∼4 M urea/0.1 M ammonium bicarbonate. The peptide digests were centrifuged and recovered. Then, trypsin solution was added and the mixture incubated for 4 hrs at 37 °C. The resulting peptide mixtures were desalted using reverse-phase C18 tips (Pierce). Peptides were then vacuum-dried and reconstituted in 1% trifluoroacetic acid (TFA) with brief sonication to ensure they were completely resuspended. Samples were analyzed by liquid chromatography–mass spectrometry (LC-MS). The acquired MS data were searched against TriTrypDB databases (www.tritrypdb.org) [27] for L. tarentolae Parrot Tar II 2019 [47], using Thermo Proteome Discoverer 2.5.PrideNot availableThe protein list from the mass spectrometry data was screened for overlap and manually checked for homology to T. brucei proteins using the TriTrypDB-8.1 TREU 927 database (https://tritrypdb.org/tritrypdb/app). Their potential subcellular localisation was analysed using the TrypTag database (http://www.tryptag.org/) [30], a trypanosome genome-wide protein localisation resource. Hits localised to small cytoplasmic organelles [29] were further examined using published proteomes of the acidocalcisome [6], glycosome [48, 49], and RNA-granule/polysomes [50] from Leishmania and Trypanosoma. Additional checks were performed to assess potential localisation in the mitochondria [51, 52], nucleus [53], and for polyP binding capacity [26], based on available proteomic datasets. Affinity purification of the biotinylated proteins followed by liquid chromatography-mass spectrometry (LC-MS) identified 3,160 proteins, corresponding to 1,240 unique hits, across four datasets. From the BirA*-LtVtc1 samples, 912 proteins were identified in the DDM-solubilised and 752 in the FC-12-solubilised fractions, while BirA*-LtVtc4 samples yielded 929 and 567 proteins, respectively. As expected, a substantial overlap was observed across conditions, with 414 proteins detected in all datasets.ProteomicsKeni VidilaserisDepartment of Molecular and Integrative Biosciences University of HelsinkiPARTIALLeishmania Tarentolaekeni.vidilaseris@helsinki.fiNot availableUniversity of HelsinkiFinlandfalseLC-MS of the VTC Complex BioID from Leishmania tarentolaeAcidocalcisomes are evolutionarily conserved acidic organelles that are rich in cations and inorganic phosphate, primarily polyphosphates. In kinetoplastid parasites, acidocalcisomes and their polyphosphate content are essential for osmoregulation and environmental adaptation during host switching. In this organelle, polyphosphate is synthesised and transported to the lumen by the vacuolar transporter chaperone (VTC) complex. Interestingly, unlike yeast VTC, which has five components, only two have been observed in kinetoplastids: Vtc1, which contains only a transmembrane domain and Vtc4, which, in addition to a transmembrane domain, also consists of SPX and catalytic domains. In this study, we used proximity-dependent biotinylation (BioID) in Leishmania tarentolae to identify proteins located close to the VTC complex. The complex was found near several known acidocalcisomal proteins, including membrane-bound pyrophosphatase (mPPase), vacuolar-type H⁺-ATPase (V-H+-ATPase), Ca²⁺-transporting P-type ATPase (Ca2+-ATPase), zinc transporter (ZnT), and palmitoyl acyltransferase 2 (PAT2). Importantly, this approach revealed three novel VTC binding partners (VBPs) that colocalise and interact with the complex in acidocalcisomes, as confirmed by confocal microscopy, pulldown assays, and AlphaFold 3 structural predictions. Together, our results expand the acidocalcisome interactome and suggest that the newly identified VBPs may contribute to the structural organisation and regulatory function of the VTC complex in phosphate homeostasis of kinetoplastid parasites.2026-05-212026-03-11PXD075501NEWT:6945NEWT:3555NEWT:241368NEWT:2NEWT:157546NEWT:190802NEWT:35554NEWT:150475NEWT:9417NEWT:347515NEWT:1216979NEWT:307972NEWT:544496NEWT:5180NEWT:256737NEWT:115104NEWT:1081927NEWT:67825NEWT:13076NEWT:1249668NEWT:317NEWT:1736309NEWT:7227NEWT:7469NEWT:885318NEWT:4081NEWT:876138NEWT:554NEWT:98334NEWT:237561NEWT:10036NEWT:7574NEWT:1351NEWT:7215NEWT:272563NEWT:507601NCBITaxon:79857NCBITaxon:6157NEWT:95648NEWT:746360NEWT:6239NEWT:1589NEWT:470150NEWT:135622NEWT:216257NEWT:6915NEWT:9986NEWT:101510NEWT:4054NEWT:3880NEWT:8782NEWT:1000589NEWT:1902NEWT:85962NEWT:160488NEWT:28104NEWT:317447NEWT:7955NCBITaxon:2NEWT:985076NEWT:7959NEWT:2261NEWT:4565NEWT:1264690NEWT:6192NEWT:28532NCBITaxon:38727NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:626528NEWT:139927NEWT:4558NEWT:209285NEWT:216595NEWT:243230NEWT:8355NEWT:931281NEWT:7029NEWT:1283300NEWT:334747NEWT:61235NCBITaxon:79824NCBITaxon:4563NEWT:5755NEWT:3218NEWT:5759NEWT:1736231NEWT:436486NEWT:6287NEWT:2242NEWT:300641NEWT:4784NEWT:727NEWT:9796NEWT:725NEWT:360106NEWT:260707NEWT:287NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1836NEWT:1735272NEWT:83334NEWT:83332NEWT:29760NEWT:703612NEWT:260705NEWT:80863NEWT:44685NEWT:2697049NEWT:1148NEWT:11676NEWT:55571NEWT:100226NCBITaxon:6073NEWT:4530NEWT:4896NEWT:6279NEWT:7370NEWT:6282NEWT:1134506NEWT:575584NEWT:1773NEWT:38783NEWT:8727NEWT:1895NEWT:1182590NEWT:8726NEWT:10090NEWT:935293NEWT:749200NEWT:4120NEWT:5693NEWT:8724NEWT:51511NEWT:92867NEWT:8723NEWT:990346NEWT:5334NEWT:145953NEWT:257309NEWT:230741NEWT:284812NCBITaxon:10359NCBITaxon:1313NEWT:43330NEWT:242619NEWT:44544NEWT:373995NEWT:544404NEWT:3702NEWT:129249NEWT:8839NEWT:4232NEWT:990119NEWT:2758385NEWT:4113NEWT:837NEWT:11298NEWT:171101NEWT:196627NEWT:408172NEWT:5691NEWT:408170NEWT:493760NEWT:260710NEWT:627025NEWT:400772NEWT:1097677NEWT:3708NEWT:106592NEWT:9913NEWT:1432138NEWT:10312NEWT:4100NEWT:1076NEWT:6763NEWT:803NEWT:8030NEWT:29722NEWT:380394NEWT:1692259NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT:135588NEWT:1843183NEWT:95486NEWT:58002NEWT:9103NEWT:4577NEWT:5664NEWT:2157NEWT:146479NEWT:10306NEWT:1911079NEWT:8022NEWT:145943NEWT:3635NEWT:5811NEWT:235443NEWT:1480154NEWT:1274414NEWT:3197NEWT:9615NEWT:10299NEWT:860688NEWT:884019NEWT:169963NEWT:36329NEWT:9606NEWT:367830NEWT:157295NEWT:410289NEWT:373153NEWT:915099NEWT:74940NEWT:1450511NEWT:470NEWT:84023NEWT:9838NCBITaxon:9615NEWT:1193501NEWT:3055NEWT:6326NEWT:6689NEWT:2762NEWT:5476NEWT:1174673NEWT:562NEWT:1274432NEWT:1274426NEWT:1423NEWT:4932NEWT:70448NEWT:9825NEWT:1274423NEWT:3603NEWT:698936NEWT:2759NEWT:3847NEWT:39946NEWT:9823NEWT:9940NEWT:573NEWT:9031NEWT:1274420NEWT:70910000-0002-6453-6600