Novel Binding Partners of the Vacuolar Transporter Chaperone (VTC) complex in Acidocalcisomes of Leishmania tarentolae

Novel Binding Partners of the Vacuolar Transporter Chaperone (VTC) complex in Acidocalcisomes of Leishmania tarentolae | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Novel Binding Partners of the Vacuolar Transporter Chaperone (VTC) complex in Acidocalcisomes of Leishmania tarentolae View ORCID Profile Paulina Królak , View ORCID Profile Orquidea Ribeiro , Bernadette Gehl-Väisänen , View ORCID Profile Mimmu K. Hiltunen , View ORCID Profile Adrian Goldman , View ORCID Profile Keni Vidilaseris doi: https://doi.org/10.1101/2025.09.23.677757 Paulina Królak 1 Faculty of Biological and Environmental Sciences, Molecular and Integrative Biosciences Research Programme, University of Helsinki , Helsinki, Finland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Paulina Królak Orquidea Ribeiro 1 Faculty of Biological and Environmental Sciences, Molecular and Integrative Biosciences Research Programme, University of Helsinki , Helsinki, Finland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Orquidea Ribeiro Bernadette Gehl-Väisänen 1 Faculty of Biological and Environmental Sciences, Molecular and Integrative Biosciences Research Programme, University of Helsinki , Helsinki, Finland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mimmu K. Hiltunen 1 Faculty of Biological and Environmental Sciences, Molecular and Integrative Biosciences Research Programme, University of Helsinki , Helsinki, Finland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mimmu K. Hiltunen Adrian Goldman 1 Faculty of Biological and Environmental Sciences, Molecular and Integrative Biosciences Research Programme, University of Helsinki , Helsinki, Finland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Adrian Goldman Keni Vidilaseris 1 Faculty of Biological and Environmental Sciences, Molecular and Integrative Biosciences Research Programme, University of Helsinki , Helsinki, Finland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Keni Vidilaseris For correspondence: keni.vidilaseris{at}helsinki.fi Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Acidocalcisomes 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 (Ca 2+ -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. Author summary Protozoan parasites such as Leishmania and Trypanosoma cause serious diseases affecting millions of people worldwide. To better understand how these parasites survive environmental changes during transmission between hosts, we studied a specialised organelle called the acidocalcisome, which stores polyphosphates and helps regulate stress responses. In this work, we used the non-pathogenic Leishmania tarentolae as a safe and cost-effective model that shares key cellular features with disease-causing species. Using a combination of CRISPR-Cas9 genome editing, proximity-based labelling (BioID), confocal microscopy, pulldown assays and AlphaFold 3 structure prediction, we investigated the vacuolar transporter chaperone (VTC) complex, which synthesises and transports polyphosphate into the acidocalcisome lumen. Proximity proteomics identified several known proteins located near the VTC complex, and importantly, led us to discover three novel proteins that interact with it. These findings open new directions for exploring the organisation and regulation of the VTC complex in protozoan parasites. By revealing novel protein interactions, our study contributes to a deeper understanding of parasite biology and may help identify therapeutic targets for treating neglected tropical diseases. Introduction Neglected tropical diseases, caused by Leishmania spp. and Trypanosoma spp., pose significant health, social, and economic challenges worldwide [ 1 ]. These diseases affect millions of people annually: each year there are 700,000 to 1 million cases of leishmaniasis, 6–7 millions of Chagas disease, and up to 1,000 cases of African trypanosomiasis [ 2 ]. While these infections are primarily endemic to tropical regions, global warming is predicted to alter the distribution and seasonality of their insect vectors, expanding transmission to previously unaffected areas and populations [ 3 ]. Kinetoplastid parasites have a unique organelle, the acidocalcisome, which is essential for environmental adaptation and regulation of stress response. Acidocalcisomes play a role in the evasion of host immune defences, ensuring parasite survival during host switching and facilitating the transition between insect and mammalian hosts by maintaining pH homeostasis and osmotic balance [ 4 ]. They also function as reservoirs of cations and inorganic phosphate (P i ), and take part in calcium signalling [ 5 – 7 ]. In Trypanosoma brucei, acidocalcisomes are also involved in autophagy [ 8 ] and contribute to macromolecular degradation via autophagosomes [ 9 ]. P i , essential for cellular metabolism, is stored primarily as polyphosphate (polyP), a linear anionic polymer composed of three to thousands of P i residues linked by high-energy phosphoanhydride bonds [ 7 ]. PolyP plays a crucial role in osmoregulation and supports parasite survival during changes in environmental conditions associated with host transitions [ 10 , 11 ]. In kinetoplastids, polyP is synthesised by the vacuolar transporter chaperone (VTC) complex, which simultaneously translocates it into acidocalcisomes to prevent cytosolic accumulation, as excess polyP can be toxic [ 12 ]. In Saccharomyces cerevisiae, the VTC complex consists of five subunits (Vtc1–Vtc5) [ 11 – 13 ]. These subunits share a common structural feature: three transmembrane helices (TMHs) at the C-terminus, which are essential for substrate translocation. However, they differ in their additional domains. For example, Vtc1 only contains TMHs, whereas Vtc4 contains, in addition to TMHs, a cytoplasmic catalytic tunnel domain responsible for polyP synthesis and an N-terminal SYG1/Pho81/XPR1 (SPX) domain involved in phosphate sensing and regulation [ 11 – 14 ]. Recent cryo-electron microscopy (cryo-EM) studies have shown that the yeast VTC structure is a heteropentameric complex composed of three Vtc1, one Vtc3, and one Vtc4 subunit [ 15 , 16 ]. By contrast, only two VTC subunits, Vtc1 and Vtc4 ( Fig 1A ), have been identified by sequence similarity in kinetoplastids [ 17 – 20 ], though both are important in parasite survival. In T. brucei , Vtc1 knockdown disrupts acidocalcisome morphology, impairs osmoregulation, and causes cytokinesis defects, whereas Vtc4 depletion results in impaired growth, virulence, and infectivity [ 17 , 18 ]. In Leishmania major , Vtc4 is not required for promastigote viability and proliferation, but its loss delays lesion formation in mice and reduces survival at elevated temperatures [ 19 ]. Download figure Open in new tab Figure 1. CRISPR/Cas9 tagging and expression of LtVtc1 and LtVtc4. (A) Schematic representation of LtVtc1 and LtVtc4. SPX: SYG1/Pho81/XPR domain, CD: Catalytic domain, TMH: Transmembrane helix domain. (B) PCR verification of mNG-tagged LtVtc1 and LtVtc4 showing a band at ∼2200 bp, absent in WT. (C) Fluorescent detection of mNG-LtLtVtc1, and LtVtc4 expressed in L. tarentolae ; Coomassie blue staining of WT L. tarentolae , mNG-LtVtc1, mNG-LtVtc4 confirms comparable total protein levels, ensuring consistent sample loading for fluorescent detection. (D) Fluorescence microscopy micrographs of mNG-LtLtVtc1 and mNG-LtVtc4 with mPPase in L.tarentolae (Pearson’s r = 0.85 and 0.83, respectively), scale bar: 5 µm. Since kinetoplastids have only two VTC components, we speculated that additional, yet unidentified, subunits might contribute to the complex composition and function. We therefore tagged Leishmania tarentolae Vtc1 and Vtc4 (LtVtc1 and LtVtc4) with the mutant biotin ligase (BirA*) to map their protein interaction networks via proximity-dependent biotinylation (BioID) [ 21 , 22 ]. BioID is an established technique that identifies proteins within a ∼10 nm radius of the bait protein in live cells, offering insights into potential protein-protein interactions [ 22 , 23 ]. Our results demonstrate that the VTC complex is closely associated with several acidocalcisomal proteins, including membrane-bound pyrophosphatase (mPPase), vacuolar-type H⁺-ATPase (V-H + -ATPase), Ca²⁺-transporting P-type ATPase (Ca 2+ -ATPase), zinc transporter (ZnT), and palmitoyl acyltransferase 2 (PAT2), as well as three proteins of unknown function, which we named ‘VTC Binding Partners’ (VBPs). Immunofluorescence microscopy confirmed the acidocalcisomal localisation of these proteins, encompassing both previously characterised acidocalcisomal components and the newly identified VBPs. Additionally, pulldown assays revealed that VBPs interact with the VTC complex, which is further supported by AlphaFold 3 structural predictions. Together, the identification of novel VTC binding partners lays the groundwork for investigating their contributions to polyphosphate metabolism and acidocalcisome homeostasis in L. tarentolae . Results Localisation of L. tarentolae Vtc1 and Vtc4 to acidocalcisomes To investigate the localisation of LtVtc1 and LtVtc4 in L. tarentolae , we tagged both proteins with mNeonGreen (mNG) using CRISPR-Cas9, following the method of Beneke et al . [ 24 ], which has been successfully applied in L. tarentolae [ 25 ]. PCR analysis showed clear bands at ∼2200 bp corresponding to mNG-tagged VTC subunits, with no amplification in the wild-type (WT) control, confirming successful chromosomal tagging ( Fig 1B ). Expression of mNG-LtVtc1 and mNG-LtVtc4 was confirmed by SDS-PAGE followed by fluorescent imaging ( Fig 1C ). Immunofluorescence microscopy showed that both fusion proteins colocalised with the acidocalcisomal marker mPPase, confirming their acidocalcisomal localisation ( Fig 1D ). Identification of proteins in close proximity to the VTC complex After confirming the acidocalcisomal localisation of LtVtc1 and LtVtc4, both genes were N-terminally tagged with BirA* to enable proximity labelling of potential VTC interactors from the cytoplasmic side. Successful integration of the BirA* cassette into the chromosomes and expression of the fusion proteins were verified by PCR and western blot, respectively ( S1 Fig panel A-B ). In the presence of biotin, L. tarentolae cells expressing BirA*-LtVtc1 and BirA*-Vtc4 showed strong enrichment of biotinylated proteins ( S1 Fig panel C ). Following cell lysis, iodixanol-gradient ultracentrifugation ( S1 Fig panel D ) yielded acidocalcisome-enriched fractions ( S1 Fig panel E ), with BirA*-LtVtc1 and BirA*-LtVtc4 predominantly localising to fractions 4 and 5, respectively ( S1 Fig panel F-G ). The slight difference in fractionation is due to variations in the collected gradient volumes. Solubilisation of acidocalcisome fractions from BirA*-LtVtc1 and BirA*-LtVtc4 cell lines in 2% DDM or 2% FC-12 efficiently extracted more than 50% of the VTC complex ( S1 Fig panel H ). 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 ( Fig 2A ). 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 ( Fig 2A ). As expected, a substantial overlap was observed across conditions, with 414 proteins detected in all datasets ( Fig 2A ). Download figure Open in new tab Figure 2. Number of proteins observed in the proximity-dependent biotinylation experiment. (A) Venn diagram showing overlap of proteins observed in the four datasets (BirA*-LtVtc1, FC-12; BirA*-LtVtc4, FC-12; BirA*-LtVtc1, DDM; BirA*-LtVtc4, DDM). (B) Protein observed localised to small cytoplasmic organelles based on the TrypTag database ( http://www.tryptag.org/ ) for the ortholog proteins in T. brucei . To further characterise the overlapping proteins, we performed gene ontology (GO) analysis, which revealed significant enrichment across multiple GO categories ( S2 Fig ). Among these, ATP metabolic process (GO 0046034), intracellular organelle (GO 0043229) and proton channel activity (GO 0015252) were notably overrepresented in the biological processes, cell components and molecular functions categories, respectively. Although many identified proteins localised to acidocalcisomes, others were associated with glycosomes and RNA granules ( Fig 2B , S1 and S2 Table) . While some of these may reflect contamination during acidocalcisome isolation, the high number of non-acidocalcisomal interactors, combined with the known presence of polyP in other organelles [ 26 ], prompted further analysis. We identified 50 polyP-binding proteins, distributed across acidocalcisomes (11 %), glycosomes (12 %), nucleoplasm (12 %), other compartments (20 %), and the cytoplasm (45 %), with ribosomal subunits highly represented ( S3 Table ). Based on gene ontology (GO) analysis, performed with the TriTrypDB GO analysis tool ( www.tritrypdb.org ) [ 27 , 28 ], translation initiators and ribosomal proteins are overrepresented among the BioID datasets. Additionally, 36 proteins from the BioID datasets ( S2 Table ) were predicted to localise to RNA granules, cytosolic, non-membrane-bound structures involved in RNA storage, processing, and degradation. Among these, Alba domain-containing protein 3 (LtALBA3, LtaPh_3424900), Poly(A) binding protein 2 (LtPABP2, LtaPh_2500900) and Zinc finger protein family member (ZC3H41, LtaPh_2713900) were consistently detected across all VTC BioID datasets. LtALBA3 and LtPABP2 also showed high peptide coverage ( S2 Table ), and, furthermore, their orthologs occur in the acidocalcisome proteome of T. brucei [ 6 ]. Proximal acidocalcisome proteins and novel interactors of the VTC Complex in Leishmania tarentolae To identify relevant protein interactions within acidocalcisomes, we manually filtered the 1240 unique hits by examining the localisation of their T. brucei orthologs to small cytoplasmic organelles (acidocalcisomes, glycosomes, lipid droplets, and RNA granules) [ 29 ] using the TrypTag database ( http://www.tryptag.org/ ) [ 30 ], a genome-wide protein localisation resource for T. brucei , a close relative of Leishmania ( S3 Fig ). This analysis yielded 103 homologous proteins localised to these organelles ( Fig 2B and S4 Table ). Among them, 14 proteins were previously shown to localise to acidocalcisomes in T. brucei [ 7 , 31 , 32 ], with eight consistently present across all BioID datasets: mPPase, Ca 2+ -ATPase, ZnT, PAT2, and two V-H + -ATPase subunits (-A, -c putative subunit), as well as Vtc1 and Vtc4. V-H + -ATPase is a multi-subunit proton pump consisting of two sub-complexes, the peripheral V1 complex (composed of eight subunits: A-H), responsible for ATP synthesis/hydrolysis, and the integral-membrane V0 complex (composed of five subunits: a, c, c’, c”, d, and e), responsible for proton translocation [ 33 ]. In addition to the two V-H + -ATPase subunits present in all datasets, subunits D and a occurred in one and three datasets, respectively ( Table 1 ). These findings suggest proximity between the VTC complex and the V-H + -ATPase complex, although not all subunits may be accessible to BirA* biotinylation. Supporting this, VTC1 deletion in yeast decreased the levels of several V-H + -ATPase subunits and impaired proton uptake activity, indicating that Vtc1 is important in maintaining V-ATPase stability [ 34 ]. View this table: View inline View popup Download powerpoint Table 1. Acidocalcisome proteins in close proximity to the VTC complex. We also detected a magnesium transporter (LtMgT) and an inward rectifier potassium channel (LtIRK) in two datasets, while phosphate transporter 91 (LtPho91) and acid phosphatase (LtAP) appeared only in one ( Table 1 ). The limited detection of these proteins may reflect spatial separation from the VTC complex within the acidocalcisome membrane; for instance, acid phosphatase is known to localise to the acidocalcisome lumen in T. brucei [ 7 ], making it unlikely to be directly biotinylated by BirA*. Further, we assessed the colocalisation of mNG-tagged LtVtc4 with several of these proteins (mCherry (mCh)-tagged LtV-H + -ATPase_A, LtPho91, LtCa 2+ -ATPase, LtZnT and LtVtc1) in the acidocalcisome of L. tarentolae using immunofluorescence microscopy; LtVtc1 was the positive control. As expected, LtVtc1 colocalised strongly with LtVtc4, with a Pearson’s correlation coefficient of 0.87 ( Fig 3 ). LtCa 2+ -ATPase, LtV-H + -ATPase_A, LtZnT, and LtPho91 also colocalised with LtVtc4 with Pearson’s correlation coefficients of 0.83, 0.87, 0.84, and 0.87, respectively ( Fig 3 ), suggesting similar kinds of proximities. Download figure Open in new tab Figure 3. Colocalisation analysis of L. tarentolae expressing mNG-LtVtc4 and mCh-tagged by immunofluorescence microscopy. mNG-LtVtc4 colocalises with mCh-LtVtc1 (Pearson’s r = 0.83) as well as with mCh-LtPho91, mCh-LtCa²⁺-ATPase, mCh-LtV-H⁺-ATPase_A, and mCh-LtZnT, showing Pearson’s correlation coefficients of 0.87, 0.83, 0.87, and 0.84, respectively. A lower degree of colocalisation is observed with mCh-IP₃R (r = 0.67). Scale bar: 5 µm. To test whether these proteins directly interact with the VTC complex, we performed a pulldown assay using ChromoTek mNeonGreen-Trap Agarose beads to capture mNG-tagged LtVtc4. As a positive control, mCh-LtVtc1 was successfully captured with mNG-LtVtc4, as indicated by two strong bands in the bead fraction after washing ( Fig 4A and S4 Fig ). Inositol 1,4,5-triphosphate receptor (IP 3 R), which was not identified in the BioID datasets, served as a negative control. Although it colocalised with LtVtc4 in the acidocalcisome ( Fig 3 ), IP 3 R was not captured by mNG-Vtc4, confirming the absence of a direct interaction ( Fig 4B and S5 Fig ). However, none of the acidocalcisome proteins that colocalised with LtVtc4 were captured in the pulldown assay ( Fig 4B and S5 Fig ), suggesting no direct interaction with LtVtc4. By contrast, LtVtc1 and LtVtc4 appear to form a stable, direct complex interaction ( Fig 4A and S4 Fig ). Download figure Open in new tab Figure 4. Pulldown assay of acidocalcisome proteins with mNG-LtVtc4. (A) mCh-LtVtc1 is captured by mNG-LtVtc4, indicated by two distinct bands in both input and bound fractions. (B) No capture is detected for negative control mCh-IP₃R or for mCh-LtV-H⁺-ATPase_A, mCh-LtPho91, mCh-LtCa²⁺-ATPase, mCh-LtZnT and mCh-LtmPPase, as indicated by the absence of mCh signal in the bound fraction. Lane labels: I – input; FT – flow-through; W3 – third wash; B – bound proteins. Green bands correspond to mNG-LtVtc4; some I and B lines appear yellowish due to partial mNG detection in the 532 nm channel (see S6 Fig ); red bands correspond to mCh-tagged proteins. The fluorescence signal marked by a white asterisk in the gel represents free mCh tag, visible only in the input due to partial degradation. In addition to the previously identified acidocalcisome proteins, all datasets also contained two novel, putative membrane proteins, LtaPh_2928300 (13.6 kDa) and LtaPh_3617400 (60 kDa), which we named VTC Binding Partner 1 (VBP1) and VTC Binding Partner 2 (VBP2). Further analysis revealed a third hypothetical protein (LtaPh_2928300, 34.4 kDa), present in three out of four datasets ( Table 2 ), which we named VTC Binding Partner 3 (VBP3). In T. brucei , they are homologous to Tb927.3.3140 (hypothetical protein), Tb927.10.6180 (Fla1-like protein), and Tb927.4.860 (hypothetical protein), respectively. View this table: View inline View popup Download powerpoint Table 2. Novel VTC binding partners identified by BioID of L. tarentolae . These proteins are unique to kinetoplastids and lack orthologs in other organisms ( S6 Fig ). While the TrypTag database suggests that each ortholog may localise to the acidocalcisome ( S3 Fig ), only the LtVPB2 ortholog was found in the T. brucei acidocalcisome proteome [ 7 ], with experimental studies supporting its acidocalcisomal localisation [ 31 ]. We used immunofluorescence microscopy to confirm colocalisation of mCh-tagged LtVBP1-3 with mNG-LtVtc4 in acidocalcisomes ( Fig 5 ). Download figure Open in new tab Figure 5. Colocalisation analysis of L. tarentolae expressing mNG-LtVtc4 and mCh-tagged LtVBP1, LtVBP2, and LtVBP3 by immunofluorescence microscopy. All three proteins show colocalisation with LtVtc4 in acidocalcisomes. Pearson’s correlation coefficients with LtVtc4 are 0.83 for LtVBP1, 0.85 for LtVBP2, and 0.82 for LtVBP3. Scale bar: 5 µm. To evaluate direct binding between these proteins and the VTC complex, we conducted pulldown assays as described above, using mNG-LtVtc4 as bait. SDS-PAGE fluorescence visualisation detected mNG-LtVtc4 together with each mCh-LtVBP in the bound fraction, suggesting interaction ( Fig 6A-C and S8 Fig panel A-C ). However, LtVBP3 bound LtVtc4 weaker than LtVBP1 or LtVBP2 did, with a fraction detected in the flowthrough ( Fig 6C and S8 Fig panel C ). To further validate this interaction, we performed a pulldown assay using mCh-specific beads with mCh-LtVBP3 as bait and confirmed that mNG-LtVtc4 was captured by mCh-LtVBP3 ( Fig 6D and S8 Fig panel D ), although a significant portion of mNG-LtVtc4 was also detected in the flowthrough. Download figure Open in new tab Figure 6. Pulldown assay of novel VTC binding partners with mNG-LtVtc4 in L. tarentolae. (A-C) SDS-PAGE with fluorescence detection shows capture of mCh-LtVBP1, mCh-LtVBP2, and mCh-LtVBP3 by mNG-LtVtc4 using mNeonGreen-Trap agarose beads, as indicated by signal in the bound fraction. (D) Pulldown of mNG-LtVtc4 with mCh-LtVBP3 using ChromoTek RFP-Trap beads confirms interaction. Lane labels: I – input; FT – flow-through; W3 – third wash; B – bound fraction. Green bands correspond to mNG-LtVtc4; some I and B lines appear as yellowish due to partial detection of the mNG tag in the 532 nm channel ( S6 Fig ). Red bands correspond to mCherry-tagged proteins. The fluorescence signal marked by a white asterisk in the gel represents free mCh tag, visible only in the input due to partial degradation. Predicted structures of the VTC complex components and their binding partners support complex assembly Following confirmation of direct interaction by pulldown assays, we used AlphaFold 3 [ 35 ] to predict the structures of LtVtc1, LtVtc4, and their binding partners, and assessed the potential for complex formation. The transmembrane topology and orientation of each protein were predicted using MEMSAT-SVM via the PSIPRED server [ 36 , 37 ]. AlphaFold 3 predicted well-folded structures for all proteins, with most residues showing high-confidence pLDDT scores (pLDDT > 70), indicating good confidence in the model ( Fig 7A ). The C-terminal TMHs of both LtVtc1 and LtVtc4 have an average pLDDT above 70. In contrast, the N-terminal region of LtVtc1 (aa1-59) is unstructured, with pLDDT scores below 50, indicating very low confidence in the model. The N-terminal cytoplasmic region of LtVtc4, containing an SPX domain and a CD domain, is well structured with high pLDDT scores ( Fig 7A ), except in the loop connecting domains. Unlike LtVtc1 and LtVtc4, all VBPs have only a single-pass TMH, at the N-terminus for LtVBP1 and LtVBP2 and at the C-terminus for LtVBP3 ( Fig 7A ). On the lumenal side, LtVBP1 has two β-hairpin motifs; LtVBP2 contains a β-propeller domain composed of six four-stranded β-sheets; and LtVBP3 consists of a C-terminal domain structurally similar to LtVtc4-CD with an RMSD of 5.85 Å ( Fig 7B ) and to the inorganic polyphosphatase from Escherichia coli (EcygiF) [ 38 ] with an RMSD of 4.13 Å ( Fig 7C ), despite very low sequence identity – 10.6% with LtVtc4-CD and 15.4% with EcygiF-NTD, respectively ( S9 Fig) . Download figure Open in new tab Figure 7. Novel VTC complex interactors. (A) AlphaFold 3 model of LtVtc1, LtVtc4, LtVBP1, LtVBP2 (signal peptide not shown), and LtVBP3. Transmembrane topology and orientation were predicted using MEMSAT-SVM [ 36 ]. The colour is based on the pLDDT colouring scheme. N: N-terminal residue, C: C-terminal residue. Red square: CD domain of Vtc4 and dashed-red square: SPX domain of Vtc4. (B) The predicted structure of the C-terminal domain (CTD) of LtVBP3 (magenta) superimposes on the catalytic domain of LtVtc4 (wheat) with an RMSD of 5.85 Å. (C) The predicted structure of the C-terminus domain of LtVBP3 (magenta) on the structure of inorganic polyphosphatase from Escherichia coli (EcygiF) (PDB: 5a60) (green) with an RMSD of 4.13 Å. To analyse complex formation between the VTC components with LtVBPs, we used AlphaFold 3 to predict all pairwise interactions among the five proteins and evaluated the inter-chain predicted TM-score (ipTM) [ 39 ] for each combination ( S10 Fig panel A ). The LtVtc1-LtVtc4 complex was used as a positive control, as this interaction has been structurally confirmed by the yeast VTC complex [ 15 ]. Although the ipTM score for the LtVtc1-LtVtc4 is only 0.62, relatively low for a high-confidence prediction, the predicted aligned error (PAE) plot reveals consistent low-error regions between subunits, indicating a good intermolecular structural correlation ( S10 Fig panel B ). Among the predicted complexes, LtVtc4-LtVBP1, LtVtc4-LtVBP2, and LtVBP1-LtVBP3 have ipTM scores of 0.50, 0.52, and 0.66, respectively ( S10 Fig panel A ). To further assess these interactions, we calculated the actifpTM (actual interface pTM) scores, which focus specifically at predicted interfacial residues [ 40 ]. The results show that the actifpTM scores of LtVtc4 in complexes with LtVtc1, LtVBP1, LtVBP2, and LtVBP1 with LtVBP3 are 0.93, 0.91, 0.92, and 0.96, respectively ( S10 Fig panel A ), which indicates strong confidence in complex formation. Interestingly, the interacting sites of the complexes align with the transmembrane orientation of each protein predicted by MEMSAT-SVM ( Fig 7A ). Based on the predicted structures, and consistent with the yeast VTC complex structure [ 15 ], LtVtc4 forms a complex with two LtVtc1 subunits via their transmembrane helices (TMHs) at two distinct interfaces: interface 1 pairs TMH1/TMH3 of the first LtVtc1 with TMH1/TMH2 of LtVtc4, whereas interface 2 couples TMH1/TMH2 of the second LtVtc1 to TMH1/TMH3 of LtVtc4 ( Fig 8A ; S10 Fig panel C) . Although the yeast complex also forms Vtc1-Vtc1 interactions, the predicted LtVtc1–LtVtc1 homodimer scores were low (ipTM = 0.41; actifpTM = 0.40), suggesting a possible false-negative prediction (S10 Fig panel A) . Despite that limitation, the model indicates two additional contact sites on LtVtc4. A cytoplasmic helix-turn-helix of LtVtc4-CD domain engages a short helix in LtVBP1, while the lumenal end of TMH3 contacts the junction between the β-propeller and single TMH of LtVBP2, respectively ( Fig 8B-C ; S10 Fig panel 8D-E) . Both interfaces are dominated by hydrophobic contacts with a few hydrogen bonds and salt bridges. No direct interface is predicted for LtVBP3; its low ipTM/actifpTM values agree with a model in which LtVBP3 binds LtVBP1 via its single TMH and adjacent loop, rather than LtVtc4 itself, forming a β-sheet with the first β-hairpin of LtVBP1 ( Fig 8D ; S10 Fig panel F) . This suggests that, unlike LtVBP1 and LtVBP2, which bind directly to Vtc4, LtVBP3 binds indirectly to the VTC complex via its interaction with LtVBP1. Download figure Open in new tab Figure 8. AlphaFold 3 prediction of the complex formed by VTC components and VBPs. (A) Complex prediction of two LtVtc1 (green) with one LtVtc4 (wheat). The zoomed-in inset shows the arrangement of TMHs of each LtVtc1 and LtVtc4 viewed from the lumenal side. H1, 2, 3 = TMH1, 2, and 3, respectively. Red-dashed lines are interfaces separating TMH of LtVtc1 and LtVtc4. (B) Complex prediction of LtVtc4 (wheat) with LtVBP1 (cyan). Red rectangle: CD domain of LtVtc4. Black rectangle: Interaction region between LtVtc4 and LtVBP1. (C) Complex prediction of LtVtc4 (wheat) with LtVBP2 (light blue). Black rectangle: Interaction region between the TMH3 of LtVtc4 with LtVBP2. (D) Complex prediction of LtVBP1 (cyan) with LtVBP3 (magenta). Discussion To date, over 30 proteins have been identified in kinetoplastid acidocalcisomes, primarily through proteomic studies in T. brucei [ 6 ], with more limited data from T. cruzi [ 41 ] and L. donovani [ 10 ], as well as localisation data from the TrypTag.org project [ 30 , 31 , 42 ]. Among these proteins, the VTC complex is one of the key components of kinetoplastid acidocalcisome function, but its composition remains elusive. In yeast, the complex consists of five subunits [ 15 ], but only Vtc1 and Vtc4 are conserved and are found in kinetoplastids. This highlights a major gap in our understanding of how the VTC complex has been remodelled in kinetoplastids and whether its function is supported by additional lineage-specific protein components. To address this gap, we used BioID method in L. tarentolae to identify novel VTC-interacting proteins in the acidocalcisomes. In yeast, the VTC complex comprises three Vtc1, one Vtc4, and one Vtc2/3 subunit [ 15 ]. Here, we initially sought VTC subunits in L. tarentolae with structural similarity to Vtc2 or Vtc3, despite low or no sequence identity. However, we did not observe any Vtc2- or Vtc3-like subunits in our BioID data. Instead, we identified three unique single-pass TMH proteins (LtVBP1-3) conserved only in kinetoplastids ( S7 Fig ) and confirmed their colocalisation with LtVtc4 to acidocalcisomes ( Fig 5 ). Pulldown assays ( Fig 6A-D ) and AlphaFold 3 predictions ( Fig 8 ) further validated direct interactions among them ( Fig 9 ). Download figure Open in new tab Figure 9. Schematic interaction map of the VTC complex with the three novel VTC-binding proteins (VBP1-3). Red lines mark the regions where physical interactions between VTC subunits and VBPs were predicted based on AlphaFold 3. The cytosolic face is oriented upward and the acidocalcisome lumen downward. Based on the AlphaFold 3 prediction, the C-terminal region of LtVBP1 interacts with LtVtc4-CD ( Fig 8B ) in the cytoplasmic space, while its single-pass TMH and N-terminal β-hairpin motifs interact with the single-pass TMH of LtVBP3 ( Fig 8D ). No high-confidence interactions were predicted between LtVBP3 and either LtVtc1 or LtVtc4 ( S9 Fig panel A ), although this could represent a false negative, as seen with the Vtc1-Vtc1 complex ( S10 Fig panel A) . Interestingly, the LtVBP3-CTD is structurally similar to LtVtc4-CD ( Fig 7B ) and EcygiF ( Fig 7C ) despite having very low sequence identity and a currently unknown function. LtVBP2, on the other hand, is predicted to interact with the C-terminal region of LtVtc4 via the neck that links its single-pass transmembrane helix (TMH) to the β-propeller domain ( Fig 8C ). The presence of such a domain in the putative VTC complex is a surprise. However, β-propeller domains support diverse cellular functions: four-bladed propellers frequently mediate transport; five-bladed variants can act as transferases or hydrolases; and larger, six-to eight-bladed propellers are often associated with structural and signaling roles [ 43 ]. Among parasites, such β–propeller–containing proteins participate in a range of processes. For example, in T. brucei , oligopeptidase B uses a β-propeller for substrate gating and virulence [ 44 ]; in Plasmodium falciparum , the β-propeller domain of K13 is linked to artemisinin resistance [ 45 ]; and in L. major , WD-repeat β-propeller proteins contribute to cell cycle control and signaling [ 46 ]. Given that LtVBP2 directly interacts with the VTC complex and contains a six-bladed β-propeller, it may be involved in structural or signaling functions within the complex, consistent with roles attributed to larger β-propellers [ 43 ]. In summary, our study identifies VBP1–3 as the first VTC-associated proteins beyond Vtc1 and Vtc4 in kinetoplastids, exemplified by L. tarentolae . The data suggest that LtVBP1 may contribute to the stabilization of the complex, whereas LtVBP2, with its predicted six-bladed β-propeller fold, could provide structural support or mediate signaling functions that influence phosphate homeostasis. LtVBP3 appears to interact more weakly, which may indicate a transient or regulated association. These findings clearly suggest a kinetoplastid-specific adaptation of the VTC complex, distinct from the architecture described in yeast. Nevertheless, the stoichiometry and overall arrangement of the kinetoplastid VTC complex remain unresolved. High-resolution structural approaches, such as cryo-electron microscopy, in combination with targeted genetic and biochemical experiments, will be essential to clarify the role of VBPs and to determine whether they represent integral subunits of the VTC machinery or context-dependent interactors. Materials and methods L.tarentolae culture L. tarentolae P10 strain promastigotes (LEXSY host P10, Jena Biosciences) were cultured at 27 °C in ventilated tissue culture flasks in 10 ml of brain heart infusion (BHI) medium containing 37 mg/mL BHI powder (Lexsy Broth BHI, Jena Biosciences) supplemented with 5 μg/mL hemin as described by the manufacturer (Jena Biosciences). Cultures were diluted in 1:10 to 1:20 with fresh medium to maintain growth in the mid-log phase at 5 x 10 7 cells/ml. Generation of L. tarentolae CRISPR-Cas9 cell lines An L. tarentolae cell line transiently expressing Cas9 and T7 RNA polymerase was generated using the method reported previously [ 24 , 25 ]. Briefly, L. tarentolae P10 in the mid-log phase were placed in BHI media and mixed with plasmid pTB007 (∼2-5 µg), which encodes for the humanised Streptococcus pyogenes Cas9 nuclease gene and T7 RNA polymerase, in a pre-chilled 4 mm electroporation cuvette (BioRad) on ice. Electroporation was performed using the BTX electroporator ECM630 at 1500 V, 25 μF with a double pulse (10 s intervals) and the cuvette was kept on ice for 10 min before being transferred to 10 mL LEXSY Broth BHI (containing 5 μg/mL hemin and 50 μg/mL Pen-Strep) and incubated at 27 °C. After 24h incubation, 20 μg/mL hygromycin B was added to the culture for polyclonal selection. Generation of chromosomally tagged L. tarentolae cell lines Tagged strains were generated according to the CRISPR-Cas9 method developed by Beneke, Madden (24). Constructs for chromosomal tagging of LtVtc1 and LtVtc4 were amplified by PCR using the pPLOT-mNG-Blast, pPLOT-mCherry-Puro, and pPLOT Puro BirA*plasmids [ 24 ]. The generic primer pairs used for LtVtc4 and LtVtc1 were obtained from the LeishGEdit database ( www.leishgedit.net , S5 Table). These primers are designed to include ∼30 bp homology flanks (HFs), which match sequences flanking the target gene locus. As a result, the PCR product contains a fluorescent tag (with a Myc epitope), an antibiotic resistance cassette, and the necessary homology arms to guide recombination at the intended genomic site. PCR amplification was performed using a two-step protocol. The sgRNA was expressed in vivo from a double-stranded DNA (dsDNA) template containing a T7 promoter, the target-specific sgRNA sequence, and a scaffold region. For transfection, this dsDNA template was delivered alongside the CRISPR repair cassette. N-terminal tagging required a 5′ sgRNA and a matching repair cassette, both of which were used in our experiments to tag LtVtc1 and LtVtc4 at their N-termini. The sgRNA templates for this CRISPR system were synthesised by PCR from a forward primer that consisted of a T7 promoter sequence followed by the gene-specific target sequence and an overlapping region with the sgRNA scaffold. A generic reverse primer with a 20 bp overlap to the forward primer was then provided, which encoded for the remainder of the scaffold. These short PCR products ( ca . 120 bp) were also not purified for transfection into L. tarentolae cells. Transfection and selection of L. tarentolae L. tarentolae cells were transfected as described in the LEXSY protocol (Jena Bioscience). Briefly, the L. tarentolae P10 strain was cultured with hygromycin selection over several passages in LEXSY BHI medium and diluted at 1:10 dilution one day before transfection with the pT007 plasmid. We used the BTX electroporator ECM630 at 1500 V, 25 μF with a double pulse in 10 s intervals to transfect the cells with 1-10 μg tagging cassette and sgRNA. The cells were then incubated for 20 h in fresh media at 27 °C. Next, 32 μg/mL hygromycin B (Invitrogen, cat 10687010) and Pen-Strep (50 μg/mL) and blasticidin (10 μg/mL, Gibco cat 46-1120) or puromycin (10 μg/mL,Gibco cat A11130-03) were added and the culture was continued until the resistant cells emerged. Successful transfection was confirmed by PCR (primers listed in S5 Table ) and by sequencing (Eurofins, GE). Acidocalcisome isolation/preparation L. tarentolae BirA*-LtVtc1 and BirA*-LtVtc4 cell lines, as well as control cells lacking BirA*, were grown in 500 ml of BHI medium supplemented with 50 μM biotin (Sigma-Aldrich, B4501). Biotin was added 24 h after inoculation, and cells were harvested 30 h later. Cells were collected by centrifugation at 2,000 × g for 3 min, and the pellet (∼2 g wet weight) was washed with cold isolation buffer (125 mM sucrose, 50 mM KCl, 4 mM MgCl₂, 0.5 mM EDTA, 5 mM DTT, 20 mM Hepes-KOH, pH 7.2) supplemented with Pierce™ Protease Inhibitor Mini Tablets, EDTA-free (Thermo Scientific, A32955). Lysis was performed by grinding for 60 s with silicon carbide (stored at –20 °C before use). The mixture was resuspended in isolation buffer and centrifuged sequentially at 100 × g for 5 min, 300 × g for 10 min, and 1,200 × g for 10 min at 4 °C) to remove silicon carbide and cell debris. The clarified lysate was then centrifuged at 15,000 x g for 10 min at 4 °C to pellet the organelles. The pellet was resuspended in isolation buffer and applied to the 34% step of the discontinuous density gradients containing 20, 24, 28, 34, 37, and 40% (w/V) iodixanol in isolation buffer. Following the published protocol in [ 6 ] with one modification ( S1 Figure panel D ), we performed single gradient ultracentrifugation instead of two in order to recover not only the acidocalcisome-enriched fraction but also biotinylated proteins from other cellular compartments. The gradient was centrifuged at at 50,000 × g for 1 h at 4 °C in an SW Ti rotor. Fractions were collected from the top of the gradient based on their colour and analysed by SDS–PAGE followed by western blotting with anti-mPPase to identify the acidocalcisome-containing fractions (see detailed method below). Western blot analysis The cells (washed twice in 1x PBS) or the acidocalcisome fractions were lysed on ice for 30 min in RIPA buffer (150 mM NaCl, 20 mM Tris/HCl, pH 7.5, 1 mM EDTA, 1% SDS, and 0.1% Triton X-100) containing a protease inhibitor tablet (Thermo Scientific A32955). Protein concentration was determined via Bradford assay (M172-L, VWR Life Sciences). Cell lysates were mixed with 4x Laemmli sample buffer for separation by SDS-PAGE. After SDS-PAGE, the gels were transferred onto nitrocellulose membranes, blocked with 3% bovine serum albumin (BSA) in 1x TBS buffer with 0.5% Tween for 1h, RT. The blots were incubated with primary rabbit antibodies against TmPPase (1:10,000) and secondary antibodies against anti-rabbit IgG antibody (1:10,000) (sc-2004 by Santa Cruz Biotechnology, INC) or HRP-conjugated avidin (BioRad cat 170-6628) for 1 h. After washing three times with 1xTBS-T, the immunoblots were visualised using Pierce ECL Western blotting substrate according to the manufacturer’s protocol. Affinity capture of biotinylated proteins and mass spectrometry BioID 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. The 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. Pulldown assays L. tarentolae cultures (10 mL) expressing mNG-tagged LtVtc4 and mCh-tagged binding partner candidates were pelleted by centrifugation (4000 × g, 10 min at 4 °C) and washed with 1x PBS. The cell pellets were lysed by seven freeze-thaw cycles in 100 µL of lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM IP 6 , 1mM DTT, 1 mM PMSF, 2 µg/mL Pepstatin A, and Pierce™ Protease Inhibitor Mini Tablets, EDTA-free (Thermo Scientific A32955)). Membrane proteins were solubilised by adding 1% DDM, followed by incubation on a roller (1.5 h, 4 °C). After centrifugation (25,000 × g, 1 h at 4 °C) to remove cell debris, the supernatants were incubated on a roller (o/n, 4 °C) with 25 µL of ChromoTek mNeonGreen-Trap Agarose beads (Proteintech, AB_2827593). Additionaly, for mNG-LtVtc4 + mCh-LtVBP3, ChromoTek RFP-Trap® Magnetic Particles M-270 (Proteintech, AB_2861253) were used. Beads were collected by centrifugation (1000 × g, 1 min) using a spin column, washed three times with 1 mL of lysis buffer containing 0.05% DDM (centrifugation at 1000 × g, 1 min per wash). For magnetic beads, a magnetic stand was used for collection and washing. Finally, beads were resuspended in 50 µl of lysis buffer containing 0.05% DDM. Samples from each purification step were resolved by SDS-PAGE and analysed by in-gel fluorescence using the Sapphire FL Biomolecular Imager (Azure Biosystems). Fluorescence was detected using 488 nm excitation for mNG and 532 nm for mCh, which also partially excites the mNG emision signal due to spectral overlap. As a result, some mNG signals may appear yellowish in the 532 nm channel ( S10 Fig ). A 638 nm channel was used to detect the fluorescent protein marker. Immunofluorescence microscopy Cells were attached to coverslips by centrifugation (3,000 × g , 5 s) and fixed with 4% (w/v) paraformaldehyde (20 min, RT). After fixation, cells were washed with 1x PBS, permeabilised with 0.25% Triton X-100 in 1x PBS (V/V) for 5 min at RT, washed again with 1x PBS, and blocked with 3% BSA in 1x PBS (w/v) (30 min, RT). The coverslips were incubated with primary antibodies (ChromoTek mouse anti-mNeonGreen (1:250, 32F6, Proteintech), Rabbit Polyclonal anti-mCh (1:400, 26765-1-AP, Proteintech)) diluted in 1x PBS in a humidified chamber (1 h, RT), washed three times with 1x PBS, and then incubated with secondary antibodies (CoraLite®488-Conjugated Goat Anti-Mouse IgG(H+L) (1:750, SA00013-1, Proteintech), Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (1:1000, A-11012, ThermoFisher Scientific)) diluted in 1x PBS (1 h, RT). After three washes with 1x PBS, the coverslips were rinsed with MilliQ water and mounted on glass slides using Fluoromount G + DAPI (Southern Biotech). Cells were imaged using a Leica Stellaris 8 confocal microscope with a DMI8 base, operated using LAS X 3.5.2 acquisition software. The objective used was an HC PL APO CS2 40×/1.25 GLYC (Glycine Immersion Medium). Fluorescence detection was performed using hybrid detectors (HyD) and solid-state lasers, with the following settings: mNG detected using Alexa 488 (HyD X2; emission range: 510–540 nm), DAPI detected using Alexa 405 (HyD S1; emission range: 420–490 nm), and mCh detected using Alexa 568 (HyD S3; emission range: 590–740 nm). The pinhole size was set to Airy 1, and the scan speed was 400 Hz. Images were acquired with these settings by capturing Z-stacks ranging from 8 to 18 slices, with slice spacing between 2.16 and 6.13 µm and pixel size ranging from 56.78 to 116.44 nm. Images were processed using FIJI (ImageJ) [ 54 ], and deconvolution was performed for 10 cycles using the DeconvolutionLab2 plugin [ 55 ]. Pearson’s correlation coefficients were calculated using the Coloc2 plugin [ 54 ] by analysing Z-stacks. A total of 20 cells were randomly selected for analysis. Gene ontology analysis GO analysis of biological processes, cell components, and molecular functions was performed on the 412 overlapping LtVtc1 and LtVtc4 interactors using the TriTrypDB GO analysis tool [ 56 ] with the complete L. tarentolae parrot tar II 2019 (ATCC 30267) genome as the reference. Fisher’s exact test was applied, with a p-value cutoff of 0.01, and significance was defined as Bonferroni-adjusted p-value < 0.01 and Benjamini-Hochberg FDR < 0.01. Protein structure prediction and analysis Protein structure predictions were performed using AlphaFold 3 [ 35 ] and ColabFold for actifpTM analysis [ 40 , 57 ]. Protein transmembrane topology and orientation were predicted using MEMSAT-SVM via the PSIPRED server ( https://bioinf.cs.ucl.ac.uk/psipred/ ) [ 36 , 37 ]. Molecular analysis and molecular graphics were produced in PyMOL ( https://pymol.org ). Protein sequence similarity searches were performed using BLAST via the UniProt database [ 58 ]. Sequence alignment was performed using Clustal Omega [ 59 ] and displayed using ESPript 3.0 [ 60 ]. Author contributions Conceptualisation, KV, AG; Methodology, KV, OR, BG, PK, MH; Investigation, PK, OR, MH, KV; Resources, KV, AG; Writing – Original draft, PK, OR, MH, KV; Writing – Review and Editing, PK, OR, MH, BG, KV, AG; Visualisation, PK, KV; Supervision and Project administration, KV, AG; Funding Acquisition, KV, AG. Competing interests The authors declare no competing financial interests. Acknowledgements This work was supported by the Research Council of Finland, awarded to KV (No. 308105 and No. 1355187) and AG (No. 1322609 and 13364501), by Sigrid Juselius to KV (No. 250258), and by the BBSRC to AG (No. BB/M021610/1). We thank Eva Gluenz and Tom Beneke for providing plasmids for CRISPR/Cas9 editing in L. tarentolae . We acknowledge Rabah Soliymani and the Meilahti Clinical Proteomics Core facility, supported by Biocenter Finland, for technical assistance and mass spectrometry analyses. Imaging was performed at the Light Microscopy Unit, Institute of Biotechnology, supported by HiLIFE and Biocenter Finland. Funder Information Declared Research Council of Finland , 308105 , 1355187 , 1322609 , 13364501 Sigrid JusM-CM-)lius Foundation , 250258 Biotechnology and Biological Sciences Research Council , BB/M021610/1 References 1. ↵ Lozano R , Naghavi M , Foreman K , Lim S , Shibuya K , Aboyans V , et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010 . Lancet . 2012 ; 380 (9859): 2095 -128. doi: 10.1016/S0140-6736(12)61728-0 . PubMed PMID: 23245604 . 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