Asexual blood stages

In 2005 we published a comparative proteome of the life stages of P. berghei in which a ‘reference’ preparation of asexual blood stages was derived from mice infected with a gametocyte-less clone 2·33 (Hall et al. Reference Hall, Karras, Raine, Carlton, Kooij, Berriman, Florens, Janssen, Pain, Christophides, James, Rutherford, Harris, Harris, Churcher, Quail, Ormond, Doggett, Trueman, Mendoza, Bidwell, Rajandream, Carucci, Yates, Kafatos, Janse, Barrell, Turner, Waters and Sinden2005). We now have reason to doubt the original phenotypic characterization of this clone. Briefly; for some 20 years we had observed clone 2·33 and never found any evidence for the presence of mature gametocytes in the blood of infected mice by light or electron microscopy, nor by molecular analysis of late stage gene products (e.g. mRNA of P28). Whilst recognising the identical morphologies and similar metabolic profiles of the immature gametocytes and the asexual trophozoite, it was not until the publication of Deligianni et al. (Reference Deligianni, Morgan, Bertuccini, Kooij, Laforge, Nahar, Poulakakis, Schüler, Louis, Matuschewski and Siden-Kiamos2011) that evidence emerged that clone 2·33 may form immature gametocytes (which then fail to develop). Thus the proteome for the asexual blood stages reported by us (Hall et al. Reference Hall, Karras, Raine, Carlton, Kooij, Berriman, Florens, Janssen, Pain, Christophides, James, Rutherford, Harris, Harris, Churcher, Quail, Ormond, Doggett, Trueman, Mendoza, Bidwell, Rajandream, Carucci, Yates, Kafatos, Janse, Barrell, Turner, Waters and Sinden2005), must now carry the caveat ‘the asexual, and immature sexual blood stages’! For us this is a lesson well learnt, and in retrospect represents a ‘Rumsfeldian’ unknown-unknown.

The calculated 81% coverage of the genome achieved in the proteomic analysis of the P. berghei life cycle (Hall et al. Reference Hall, Karras, Raine, Carlton, Kooij, Berriman, Florens, Janssen, Pain, Christophides, James, Rutherford, Harris, Harris, Churcher, Quail, Ormond, Doggett, Trueman, Mendoza, Bidwell, Rajandream, Carucci, Yates, Kafatos, Janse, Barrell, Turner, Waters and Sinden2005), permitted interesting global comparisons to be made between the different life strategies (invasion, replication and sex) and stages (e.g. within the invasion strategy – merozoite; sporozoite; ookinete). Parasite stages within a ‘strategy’ were found to share a greater proportion of proteins than are committed to the unique activities distinguishing individual stages (see Fig. 1). Lasonder et al. (Reference Lasonder, Janse, van Gemert, Mair, Vermunt, Douradinha, van Noort, Huynen, Luty, Kroeze, Khan, Sauerwein, Waters, Mann and Stunnenberg2008) similarly found stage-specific protein expression to represent just ∼12 to ∼28% of all proteins expressed by that stage. An unexpected finding was that the male gamete, despite being entirely derived from the microgametocyte in a process lasting just 15 minutes, nonetheless 34% of the proteins found in the gamete have not yet been described in the proteome of the un-activated gametocyte. These proteins are therefore either synthesised de novo during the process of exflagellation, or they represent a group of molecules ‘lost’ from the gametocyte proteome for technical reasons (low abundance or recovery).

Fig. 1.

Venn diagrams to illustrate the distribution of proteins detected in individual life stages that found uniquely in one life stage, or shared between stages within strategy classes (invasion, replication and sex). Proteins subject to translation control were identified as those found uniquely as mRNA in the gametocyte but as protein in the ookinete. (Modified from Hall et al. Reference Hall, Karras, Raine, Carlton, Kooij, Berriman, Florens, Janssen, Pain, Christophides, James, Rutherford, Harris, Harris, Churcher, Quail, Ormond, Doggett, Trueman, Mendoza, Bidwell, Rajandream, Carucci, Yates, Kafatos, Janse, Barrell, Turner, Waters and Sinden2005)

Where significant proteomes exist for any life stage, we have often found the apparent absence/presence of individual proteins to be in error. Conversely, we have found the presence or absence of functionally related groups of proteins in replicate experiments a compelling entrée to understand their biology. Thus, in the case of the ookinete, in which both micronemes and rhoptries had been identified by electron microscopy (Sinden, Reference Sinden, Killick-Kendrick and Peters1978), the absence of the known rhoptry proteins paralleled by the presence of a large and broadly representative group of known microneme proteins prompted our conclusion that the ookinete lacked rhoptries (Lal et al. Reference Lal, Prieto, Bromley, Sanderson, Yates, Wastling, Tomley and Sinden2009). The ability to test this hypothesis using protein tagging studies (Tufet-Bayona et al. Reference Tufet-Bayona, Janse, Khan, Waters, Sinden and Franke-Fayard2009) validated this hypothesis. This re-analysis then provided a rational molecular understanding for the contrasting biological observations that midgut epithelial cells invaded by ookinetes are lysed and die, expressing vATPase (Shahabuddin and Pimenta, Reference Shahabuddin and Pimenta1998; Cociancich et al. Reference Cociancich, Park, Fidock and Shahabuddin1999; Han and Barillas-Mury, Reference Han and Barillas-Mury2002) whereas host cell invasion by merozoites and sporozoites (both of which possess rhoptries) permits their respective host cells to survive invasion and support subsequent vegetative growth of the parasite. Sporozoites which transit multiple host cells prior to beginning vegetative growth in the liver may therefore be hypothesised to employ phased/regulated secretion of the micronemes (traversal and residency) and rhoptries (residency).

A second example of the utility of observations on molecular groups has been the integration of transcriptomic and proteomic data to understand protein expression in the mature female gametocyte. The identification of 9 proteins in the ookinete that were present as transcripts but not proteins in the mature female gametocyte (Hall et al. Reference Hall, Karras, Raine, Carlton, Kooij, Berriman, Florens, Janssen, Pain, Christophides, James, Rutherford, Harris, Harris, Churcher, Quail, Ormond, Doggett, Trueman, Mendoza, Bidwell, Rajandream, Carucci, Yates, Kafatos, Janse, Barrell, Turner, Waters and Sinden2005), combined with the knowledge that for one of these proteins (P28/Pbs21) convincing data existed for the presence in the macrogametocyte of the message in discrete cytoplasmic structures (Thompson and Sinden, Reference Thompson and Sinden1994) provided provocative evidence for the wider use of translational control mechanisms in Plasmodium biology (Fig. 1). Subsequent elegant molecular dissection of this concept followed, leading to the recognition that as many as 370 proteins may be suppressed at the level of translation in the gametocyte (Mair et al. Reference Mair, Braks, Garver, Wiegant, Hall, Dirks, Khan, Dimopoulos, Janse and Waters2006). Noting the similar strategic positions of the gametocyte and the sporozoite in the parasite life cycle – both are terminally differentiated cells that must survive extended periods in one host before having to respond rapidly to the environment of the new host following transmission – it is interesting to note translational control mechanisms are employed by both (Gomes-Santos et al. Reference Gomes-Santos, Braks, Prudencio, Carret, Gomes, Pain, Feltwell, Khan, Waters, Janse, Mair and Mota2011). It would be fascinating to understand whether similar mechanisms also regulate the remaining ‘latent’ stage, the hypnozoite.

The microgamete

One of the most recent, and currently unpublished, proteomes we have produced is from the intact microgamete. Our reasons for undertaking the challenge of preparing this very rare and physically small stage of the life cycle are manifold. First, it occupies a critical point in the parasite life cycle and one that has become the focus of much attention since the recognition of the key roles of gametogenesis, fertilisation and ookinete development in the development of new and powerful transmission-blocking intervention strategies (Wells et al. Reference Wells, Alonso and Gutteridge2009). Second, the preparation of the parasite material required no subcellular fractionation steps that might change the protein composition of the preparation. Third, our previous electron microscopic studies (Sinden et al. Reference Sinden, Canning and Spain1976; Reference Sinden, Canning, Bray and Smalley1978, Sinden, Reference Sinden1983), supported by later genetic analyses (Creasey et al. Reference Creasey, Ranford Cartwright, Moore, Williamson, Wilson, Walliker and Carter1993; Okamoto et al. Reference Okamoto, Spurck, Goodman and McFadden2008) suggested the microgamete has essentially just 4 cellular compartments: a nucleus in which lies a condensed genome; an axoneme; cytoplasm and cell membrane. This we anticipated might offer a simplified dataset for the development of improved methods for the bioinformatic analysis of data (and its validation by genetic manipulation techniques) as described below. The publication of these data has been delayed by the ‘inconvenient’ publication of a revised P. berghei genome, which required a complete re-analysis of all the spectra against the new gene models, a substantial exercise that changed 152 of 624 protein identifications!

Recognising prior conclusive evidence that the microgamete lacked a mitochondrion, it was surprising that despite strong evidence that the microgamete preparations lacked significant cellular contamination, the proteome contained a few mitochondrial proteins (see below). Whilst it has been long established that microgamete motility is dependent upon extracellular glucose (Nijout and Carter, Reference Nijhout and Carter1978), nonetheless the abundance of all 11 enzymes of the glycolytic pathway, and the hexose transporter (PbHT) in the proteome was surprising, as was our corresponding failure to detect a putative lactate transporter (Martin et al. Reference Martin, Ginsburg and Kirk2009). The presence/activity of a lactate transporter may be a key regulator of both the fast/slow flagellar activity and the brief (∼40 min.) life of the gamete. Validation of the importance of glycolysis was achieved in two ways first by location of a C-terminus myc-tagged PbHT to the gamete surface (see Fig. 2), and second by demonstrating the impact of inhibitors of glycolysis upon motility. The outstanding question still to be resolved is to ask whether/how lactate is removed from the cell?

Whilst the axoneme is the most prominent structure in the microgamete, cytoskeletal (axoneme-related) proteins represented just 6% of all proteins identified by our techniques. Downstream analysis of location and ‘functions’ of those identified has nonetheless proved a fertile area of study (see Fig. 2) that will have an important role to play in unravelling the molecular organisation of the spectacularly rapid process of exflagellation, notably in the spatial organisation of the nucleating centromeres/basal bodies (Straschil et al. Reference Straschil, Talman, Ferguson, Bunting, Xu, Bailes, Sinden, Holder, Smith, Coates and Tewari2010; Marques, unpublished data) and the intracellular assembly of the 8 axonemes (Sinden et al. Reference Sinden, Talman, Marques, Wass and Sternberg2010).

Fig. 2.

All proteome-predicted male gamete proteins we have attempted to localize thus far have been found in situ in the male gamete: PF16 (Straschil et al. Reference Straschil, Talman, Ferguson, Bunting, Xu, Bailes, Sinden, Holder, Smith, Coates and Tewari2010), Kinesin13 (Marques, unpublished), PbGEST (Talman et al. Reference Talman, Lacroix, Marques, Blagborough, Carzaniga, Ménard and Sinden2011) and PbHT (Slavic et al. Reference Slavic, Delves, Prudencio, Talman, Straschil, Derbyshire, Xu, Sinden, Mota, Morin, Tewari, Krishna and Staines2011).

Perhaps the most important contribution of the microgamete proteome in the longer term may be the identification of 23 putative surface molecules, each of which must now be pursued to question their roles in fertilization and their potential for development as targets for transmission-blocking antibodies. Amongst these proteins were some that prior evidence has suggested very strongly to be synthesised exclusively in the female gamete cell lineage e.g. the LAP/CCCP proteins and p47 (Khan et al. Reference Khan, Franke-Fayard, Mair, Lasonder, Janse, Mann and Waters2005; van Schaijk et al. Reference van Schaijk, van Dijk, van de Vegte-Bolmer, van Gemert, van Dooren, Eksi, Roeffen, Janse, Waters and Sauerwein2006; Scholz et al. Reference Scholz, Simon, Lavazec, Dude, Templeton and Pradel2007). Recognising that we were unable to detect any female gametes in the originating cell preparation we are led to question the mechanism explaining their presence in the proteome. Amongst many others, one interesting possibility we are pursuing, surmises that these female proteins may specifically bind to the male extracellular surface (should they be released into the medium). Thus these female ‘contaminants’ have become priority candidates in our search for additional fertilisation ligands.

The ookinete surface

Three broad methodologies exist for the generation of surface proteomes. One of the most commonly used techniques has been the combination of cell lysis and differential centrifugation and/or sucrose gradient flotation to purify the plasma membrane. Whilst fractionation has proved successful for proteomic analysis of Plasmodium rhoptries and of the micronemes of P. berghei ookinetes (see below), we anticipated that the close association of the plasma membrane and underlying inner membrane complex would be likely to lead to co-purification of this entire alveolar structure, the subsequent rigorous detergent lysis required to disrupt this complex could dissociate some surface-exposed proteins. Trypsin ‘shaving’, a method successfully employed to analyse the merozoite/RBC interactions (Hadley and Miller, Reference Hadley and Miller1988), was discounted because the ookinete naturally lives in an environment rich in this enzyme (Gass, Reference Gass1977; Gass and Yeates, Reference Gass and Yeates1979; Muller et al. Reference Muller, Crampton, Della Torre, Sinden and Frisanti1993). We therefore chose to label and then affinity-purify surface-exposed proteins from cultured, purified, intact ookinetes. Preliminary comparative analysis of WT, P28ko, P25ko and P25/P28 double ko ookinetes showed these key ookinete surface proteins could be labelled readily with sulfo-NHS-LC-biotin. Biotinylation identified 518 proteins, of which 67 were predicted to harbour a signal peptide and/or transmembrane domain. Of the 16 known ookinete surface proteins, 15 were identified in the dataset. To compare the data with the whole ookinete proteome (Hall et al. Reference Hall, Karras, Raine, Carlton, Kooij, Berriman, Florens, Janssen, Pain, Christophides, James, Rutherford, Harris, Harris, Churcher, Quail, Ormond, Doggett, Trueman, Mendoza, Bidwell, Rajandream, Carucci, Yates, Kafatos, Janse, Barrell, Turner, Waters and Sinden2005), the Relative Spectral Abundance (RSA) was calculated for each of the 320 proteins detected in both the surface and whole ookinete proteomes. The proportion represented by each protein of all spectra in either dataset was calculated, giving two spectral proportion values SP_surface and SP_whole. When (SP_surface – SP_whole) >1 proteins were considered to be enriched in the surface proteome. Of the 50 proteins most enriched in the surface proteome (Table 2), six are known to be surface associated or secreted (P28, P25, CTRP, LAP1, LAP2 and chitinase). Whilst the ookinete surface may still hold some surprises, as recently suggested by the descriptions of enolase and actin on the ookinete plasma membrane (Hernandez-Romano et al. Reference Hernandez-Romano, Rodriguez, Pando, Torres-Monzom, Alvarado-Delgado, Lecona Valera, Ramos, Martinez-Barnetche and Rodriguez2011), many of these ‘enriched proteins’ are highly unlikely to be found naturally on the ookinete surface e.g. histones H2A and H2B (previously surface located (Watson et al. Reference Watson, Edwards, Shaunak, Parmelee, Sarraf, Gooderham and Davies1995)), dynein and ATP-dependent RNA helicase. Amongst those proteins with an RSA <1, i.e. depleted, were some such as von Willibrand factor A domain Related Protein (WARP), previously found on the ookinete surface. In trying to understand this ‘depletion’, we are reminded that the methodology does not report the final /functional location of a protein, but rather reports the fraction of that protein that is resident in a sample at the time of labelling, thus proteins whose trafficking to the surface is highly regulated (i.e. a high proportion of the total is in cytoplasmic vesicles) might appear to be surface depleted by this analysis.

Table 2.

The 50 proteins detected with the most spectra in the ookinete surface proteome. Proteins shown in Bold are those known to be on the ookinete surface and those in italics have a predicted surface localisation.

We were challenged by the presence of so many intracellular proteins in these ‘surface’ fractions. We noted many of the proteins belonged to the actin-myosin motor complex, which is known to be located immediately beneath the plasma membrane and associated with the inner membrane complex (see Fig. 3). In retrospect we then examined, during the surface biotinylation procedure, the permeability of ookinetes by exposing them to propidium iodide – which reportedly only enters cells whose plasma membrane is compromised. Following affinity purification; washing in PBS pH 8·0; and biotinylation, we saw 6%; 21%; and 75% respectively of the ookinetes were permeant to the dye. It was then unsurprising that we had biotinylated intracellular proteins. We nonetheless recognise other factors may also contribute to cytoplasmic contamination of these proteomes e.g. failure to quench the biotinylation reagent following labelling, and non-specific binding of proteins to streptavidin.

Fig. 3.

Diagrammatic representation of molecules detected in the putative surface proteomes of the ookinete, noting how different labelling methods identified proteins from different reported locations. (R.Stanway, 2009)

We attempted to validate the biotinylated surface proteome by both surface iodination and CyDye technologies. Surface iodination, followed by SDS-PAGE and autoradiography suggested the ookinete surface is dominated by two proteins, identified as P25 and P28 in Western blots. CyDye methods similarly identified both P25 and P28, but additionally labelled actin and protein disulphide isomerase (PDI). PDI has been described on the surface of Toxoplasma gondii tachyzoites (Meek et al. Reference Meek, Back, Klaren, Speijer and Peek2002) and in Neospora caninum has been shown to localise to the micronemes and to be involved in interaction with the host cell (Naguleswaran et al. Reference Naguleswaran, Alaeddine, Guionaud, Vonlaufen, Sonda, Jenoe, Mevissen and Hemphill2005).

A microneme proteome

Micronemes, together with rhoptries and mononemes, are regulated secretory vesicles that may be found at the anterior of the invasive stages of apicomplexan parasites (Singh et al. Reference Singh, Plassmeyer, Gaur and Miller2007). Proteins secreted from these organelles have roles in motility and host cell invasion. The physical composition of our microneme preparations from the ookinete was compelling; the initial ookinete preparation was 97% pure, analysis of the density gradients by Western blotting revealed significant enrichment of known micronemal proteins in fractions of the anticipated density (1·1 g/ml). EM observations confirmed the abundance of micronemes but clearly identified other unspecified cell debris (Lal et al. Reference Lal, Prieto, Bromley, Sanderson, Yates, Wastling, Tomley and Sinden2009). This enrichment was paralleled by the simultaneous depletion of known surface proteins and proteins predicted to locate at the ER, mitochondria, apicoplast and nucleus. We had anticipated that by determining the RSA, we would uniquely identify micronemal proteins. Micronemal proteins were amongst the most abundant in these fractions and the most enriched (1·6% of whole ookinete vs. 18·3% of fraction spectra), but other protein classes were also enriched e.g. ribosomal proteins (8·4% of whole ookinete vs. 9·7% of fraction spectra), suggesting in this instance RSA may be less informative. The abundance of micronemal proteins ranged from 1094 spectra (SOAP) to 4 (PPLP4), but some proteins previously reported in this organelle were not found (e.g. Sub-2, PPLP3) (Table 3).

Table 3.

Proteins with known or putative functions detected in the microneme proteome are shown with the associated number of spectra. Cargo proteins are released from micronemes into the extracellular space with a potential role in invasion. Microneme proteins not detected in this proteome are shown in italics. Vesicle trafficking proteins that may be important to microneme biogenesis, trafficking and secretion are shown. Proteins possibly linked with microneme delivery are termed ‘motility’ proteins.

Quantitative proteomic methods have been examined in detail by others (Nirmalan et al. Reference Nirmalan, Sims and Hyde2004; Sims and Hyde, Reference Sims and Hyde2006; Foth et al. Reference Foth, Zhang, Chaal, Sze, Preiser and Bozdech2011; Southworth et al. Reference Southworth, Hyde and Sims2011). It has been argued that spectral count correlates with protein abundance, but without knowing the individual behaviours of proteins during the preparative steps, we must be cautious in extrapolating these values to the originating cell. Nonetheless, for our purposes the most highly abundant proteins are certainly worthy of attention, thus the high position of PDI in the microneme proteome suggests this chaperone is carried through into the mature secretory vesicle from its presumed initial interaction with cargo proteins in the lumen of the endoplasmic reticulum. Recalling that PDI was also present in the surface proteome we suggest it may be carried from the ER through the micronemes to the parasite surface. Others have suggested extracellular roles for PDI e.g. manipulating glycoprotein interactions (Bi et al. 2011) and Chlamydia cell invasion (Abromatis and Stephens, Reference Abromaitis and Stephens2009). Whilst the greatest attention has been placed on the identification of protein cargo of the micronemes, it was not unexpected that this enriched vesicle preparation contained many cytoplasmic proteins known to mediate the trafficking of secretory vesicles between the Golgi apparatus and the cell surface. Amongst these, the relative abundance of actin (39 spectra) is entirely consistent with the interesting observation of high concentrations of GFP-tagged actin at the anterior pole of the ookinete where the micronemes ducts fuse with the plasma membrane (Vontas et al. Reference Vontas, Siden-Kiamos, Papagiannakis, Karras, Waters and Louis2005).

Location

Table 4 shows the overall results for subcellular localization prediction. The largest fraction of proteins was predicted to be cytoplasmic (189) or nuclear (69). We were able to assign locations to all but 43 of the microgamete proteins, for 12 of these weak predictions could be made but with unacceptably low confidence, for others multiple locations were predicted. Twenty nine of the microgamete proteins in this extended analysis appear to be mitochondrial, which suggested cellular contamination in the sample (see above).

Table 4.

Predicted subcellular localisation of Plasmodium berghei microgamete proteome. The number of proteins predicted to be present in each category is shown for whole proteome and also for those proteins that are unannotated.

Using 3 different location criteria (intracellular, transmembrane or extracellular/membrane associated/nuclear envelope) the majority (393) of the proteins are intracellular, 103 are transmembrane and 129 extracellular or membrane associated.

We have predicted subcellular localizations for all but 30 of the currently unannotated proteins. The locations predicted are spread throughout the different regions of the gamete (Table 4). The largest fraction of the proteins is predicted to be transmembrane (34 either in the plasma or nuclear membranes). Of the non-transmembrane proteins 27 are predicted to be localized to the nucleus, which is nearly 40% of all the proteins predicted to be present in the nucleus. Many of the uncharacterized proteins are predicted to be either extracellular or associated with the plasma-membrane. Due to their location it is possible that some of the proteins have roles in fertilisation, potentially including recognition of the female gamete.

Function

A summary of the function predictions for the microgamete proteins is shown in Table 5. In this summary the proteins have been assigned to one of three different functional types. Those for which we have made the most informative predictions have been assigned to a process (e.g. translation). Where it has not been possible to predict the biological process, it has often been possible to predict a function (e.g. protease or DNA binding). For some proteins just a domain has been identified (e.g. armadillo-like domain), but in these ‘extreme’ cases we appreciate this may have little relevance to the true protein function.

Table 5.

Predicted functions of the Plasmodium berghei microgamete proteome. The predicted functions generated by the bioinformatics analysis of the proteome are displayed. Numbers are shown for the complete proteome and also for currently uncharacterized proteins in the proteome. The Enzyme* category includes general enzyme functions which not considered highly informative. The Other† category includes functions that are only predicted for a single protein and are not present in the unannotated set of proteins

Function predictions for the unannotated proteins is more challenging than predicting their subcellular localizations and we have predicted functions for 98 of the 182 proteins. The predicted functions are spread throughout many different areas from transport (including transporters), and signalling to processes regulating DNA and RNA. These predictions in combination with the subcellular localization predictions have been used to identify targets for experimental characterization.

Functions for flagellar proteins

Here we use the proteins predicted to be part of the flagellum to provide an example of how the predictions were made. Eighteen uncharacterized proteins were predicted to be potential components (see Table 6). Recognising the axoneme is the sole and dominant cytoskeletal structure in the microgamete most proteins predicted to have cytoskeletal roles (e.g. actin binding) are likely to be associated with this structure (especially if they are not expressed in the proteome of other life cycle stages). Predictions were often based on the combination of individually weak results from different methods. The main features used were the presence of domains that are widely present in, though not exclusive to, flagellar proteins (e.g. WD40 and coiled coils). This was often supported by homologies to actin interacting proteins identified by searches of the Phyre2 protein structure library. A further source of evidence was the GO predictions made by both ConFunc and PFP, many examples predicted functions such as cytoskeleton-binding, actin (or actin-filament) binding, motor activity, microtubule motor activity.

Table 6.

Unannotated Plasmodium berghei proteins predicted to form part of the microgamete flagellum. The features used to make the predictions are displayed. Structural library hits – are structures in the Phyre2 structural library to which microgamete proteins show homology. Coil and LC indicate if the protein is predicted to have coils and low complexity regions respectively. Predicted GO functions are those predicted by PFP, ConFunc, FFPRED and the mapping of Interpro hits to GO. In this table the main relevant functions predicted from combining the results are displayed. LRR – Leucine rich region

Overall the predictions of flagellar proteins rely heavily on the prediction of a role in the cytoskeleton. While this may be an effective way to predict proteins that may be part of the flagellum, it is also possible that some of these proteins do in fact have functions within the cytoskeleton. This may be more likely for proteins that have predictions that suggest other functions, such as PBANKA_132600 which also has Interpro domain and structural hits to a guanylate binding protein and PBANKA_135880 which also has predictions for a kinetochore function (Table 6).