Thousands of Rab GTPases for the Cell Biologist
[['Intracellular compartmentalisation is found in all cellular lifeforms , yet eukaryotes have evolved extensive membranous compartments unique to this domain of life .', 'Protein trafficking pathways accomplish the movement of cellular components like proteins and lipids between the cellular compartments .', 'These essential pathways play house-keeping roles , such as transport of proteins destined for secretion to the plasma membrane via the secretory pathway , or recycling of membrane receptors via the endocytic pathway .', 'In addition , they play a variety of specialised roles , such as bone resorption in osteoclasts , pigmentation in melanocytes and antigen presentation in immune cells .', 'Malfunction of protein trafficking components leads to a large number of human diseases , ranging from hemorrhagic disorders and immunodeficiencies to mental retardation and blindness [1]–[4] , as well as cancer [5]–[9] .', 'Furthermore , protein trafficking pathways are frequently exploited by human pathogens to gain entry and survive within host cells [10]–[13] .', 'The endomembrane system accounts for a large fraction of the protein coding sequences in eukaryotic genomes [14] , and a plethora of data on molecules and interactions in different model organisms is available .', 'However , it is unclear how these data map across organisms , and how general the mechanisms characterised in single species are .', 'To answer these question we need to understand the evolution of the protein trafficking pathways and organelles .', 'An evolutionary framework for protein trafficking is particularly important given the overwhelming accumulation of genomes , many from pathogenic organisms .', 'Their comparative analysis can distinguish conserved from taxon-specific machineries , with clear practical applications .', 'For example , conservation of genes led to the discovery of novel components and mechanisms in ciliogenesis [15] , whereas the presence of taxon-specific pathways allowed the identification of Fosmidomycin as a potential antimalarial drug [16] .', 'Studying the evolution of protein trafficking is essential to understand the origins of eukaryotes .', 'Comparative genomics and phylogenetics have established that the Last Eukaryotic Common Ancestor ( LECA ) already had a complex membrane trafficking system [17] including most types of extant molecular components [18] .', 'These are believed to have expanded by duplication and specialisation giving rise to the full diversity of organelles and trafficking pathways observed today ( see [17] for a detailed description of this evolutionary scenario ) .', 'Rabs are central regulators of protein trafficking .', 'They are small GTPases that work as molecular switches to regulate vesicle budding , motility , tethering and fusion steps in vesicular transport [19] .', 'Most recently the authors of [20] also linked Rabs to membrane fission .', 'They recruit molecular motors to organelles and transport-vesicles , coordinate intracellular signalling with membrane trafficking , organise distinct sub-domains within membranous organelles and play a critical role in the definition of organelle identity ( recently reviewed in [21] ) .', 'Rab subfamilies localise to distinct cellular locations , and regulate trafficking in a pathway- , organelle- and tissue-specific manner .', 'This makes them ideal markers for the majority of trafficking-processes and compartments .', 'Among trafficking-associated proteins , the Rab family expanded most in evolution [17] , [22] , suggesting that it provided the primary diversification element in the evolution of trafficking [22] .', 'An important feature of the Rab family is that Rab orthologs tend to perform similar functions even in divergent taxa .', 'For example , the mouse Rab1 has been shown to be able to functionally replace its ortholog YPT1 in yeast [23] .', 'Hence assigning a Rab to a known and functionally described subfamily , e . g . Rab1 , is a strong functional prediction , i . e . functioning in the early secretory pathway in the case of Rab1 .', 'Together with the ability to classify them into subfamilies based on sequence alone , this allows to establish the presence or loss of pathways and organelles solely based on the annotation of the Rab repertoire—a procedure we subsequently refer to as Rab profiling .', 'Previously , we defined criteria to identify and classify Rab proteins [24] , which have been used as a basis for detailed manual analysis of the Rab families in a variety of organisms [25]–[33] .', 'However , manual identification of Rab repertoires is tedious and time-consuming and not compatible with the deluge of fully sequenced eukaryotic genomes that new sequencing technologies are generating .', 'We thus need to develop methods that enable the automated annotation of Rab proteins .', 'Several characteristics of the Rab family make this a challenging bioinformatics problem .', 'First , there is a strong non-specific signal from GTPase motifs spread throughout the protein sequence [34] , which makes it hard to distinguish Rabs from other small GTPases .', 'Second , the Rab family is large due to extensive duplication in several branches of the eukaryotic tree ( e . g . [28] , [29] ) .', 'Together with high sequence similarity amongst Rabs this causes difficulties to correctly classify Rabs into subfamilies and to further discern yet unseen subfamilies .', 'Lastly , any automated scheme has to respect and perpetuate as much as possible the current naming conventions , despite any inconsistencies stemming from the decentralised nature of scientific discovery and the huge bias of existing annotations towards Ophistokonts .', 'This requires a flexible , learning scheme both able to cope with the contingency of the field and to easily incorporate new naming consensuses .', 'Here , we overcame these problems and developed an automated bioinformatic pipeline for the identification and classification of Rabs .', 'We termed our pipeline the ‘Rabifier’ , which we describe , validate and benchmark .', 'Using our tool , we cataloged nearly 8 . 000 Rabs from 247 genomes covering the major taxa of the eukaryotic tree , which we make available along with our pipeline at www . RabDB . org .', 'Based on this comprehensive dataset of Rab proteins , we describe and analyse the evolution of Rabs .', 'We found a highly dynamic family undergoing frequent taxon-specific expansions and losses .', 'We extend the Rab repertoire previously reported to have been present in the LECA , identify the changes in the Rab family that accompanied the emergence of multicellularity and show that neo-functionalisation best explains the emergence of new human Rab subfamilies .'], ['We implemented a bioinformatics pipeline to identify and classify Rab GTPases in any set of protein sequences independently of taxonomical information , which we term ‘Rabifier’ .', 'The Rabifier proceeds in two major phases , which are schematised in Figure 1 .', 'First , it decides whether a protein sequence belongs to the Rab family , i . e . that it is not a Ras , a Rho , etc . , and in the second phase it classifies the predicted Rab sequence into a Rab subfamily ( e . g . Rab1 ) .', 'We describe the rationale for this procedure below—technical details are given in Materials and Methods and Text S1 .', 'Phase 1 ( Figure 1A ) , which classifies protein sequences to the Rab family , proceeds in three stages .', 'First , we check that the protein has a G-protein family domain .', 'As the presence of such a domain can be decided with near certainty , this step drastically reduces the number of candidate Rabs while not excluding any real Rab .', 'In order to do so , we align the sequence against a profile Hidden Markov Model ( HMMs ) [35] describing the known GTPase structures , as provided by the Superfamily database [36] .', 'Secondly , we search for local sequence similarity by performing a BLASTp [37] query against an internal reference set of manually curated GTPases and discard the protein if it is most similar to a GTPase other than a Rab .', 'At this stage of the workflow , the majority of non-Rab sequences has already been rejected ( see Figure 1C , where the number of sequences that transition between these phases is shown for M . brevicollis and for a database of 247 genomes described below ) .', 'However , small GTPases are so similar to each other that a residual amount of false positives still remains undetected .', 'We remove them in the third stage , where we scan the sequence for the presence of at least one of five characteristic RabF motifs defined in [24] .', 'If no motif is found , it is concluded that the protein cannot be a Rab and rejected .', 'Remaining sequences are all assigned to the Rab family at an individual confidence level computed for each Rab .', 'The confidence score is derived from the combination of the individual statistics generated by the three stages according to a procedure described in Text S1 .', 'The second phase ( Figure 1B ) proposes a classification into one of the Rab subfamilies present in our internal reference set , or suggests no similarity to any of those .', 'It proceeds in two stages .', 'First , we test whether the Rab respects a 40% identity cut-off to its BH that prevents assignment of too disparate sequences to any of the pre-defined subfamilies .', 'If the cut-off is met , a classification is proposed , if not , the Rab is classified as belonging to the undetermined subfamily RabX .', 'The use of a 40% threshold is supported in Figure S1 , and has previously been employed for example in [30] .', 'The actual subfamily classification is based on the computation of a likelihood score for each of the subfamilies in our reference set .', 'Intuitively , the protein is classified as belonging to the highest scoring subfamily , however , all scores are kept and thus provide an estimate of the relative uncertainty associated with each call .', 'Like the Rab family score generated in the first phase of the Rabifier , the computation integrates output statistics from different tools , namely from local alignments via BLAST and from alignments using reverse Ψ-BLAST ( RPS-BLAST [38] ) .', 'Similar to HMMs , RPS-BLAST compares a sequence against a summary of a set of sequences , in our case summaries of all sequences in our reference set belonging to a single Rab subfamily , and measures how likely the input belongs to any the subfamilies .', 'This way we take information from all sequences in the internal reference set into account .', 'For details on the procedure check Materials and Methods and Supplementary Methods Text S1 .', 'Any new methodology has to be validated .', 'Ideally this is based on a test data set fulfilling three requirements: the test data is correctly and comprehensively annotated with those features the tool automatically detects , it is large enough to provide robust statistics , and it covers the entire range of possible inputs the tool might encounter in its real-world application , at best even respecting the expected proportions of worst- to best-case inputs .', 'In our case , no dataset is available which fulfils the three requirements simultaneously: Rab repertoires are only available for a limited number of organisms which are not evenly distributed across eukaryotic phylogeny , and whose annotation was manually performed by different groups , hence may be inconsistent or even incorrect ( in some cases a ‘correct’ , i . e . consensual , classification might not even exist ) .', 'In the absence of a suitable validation dataset , we opted to validate the Rabifier against the manually curated Rab families of three organisms representing distinct worst case scenarios for the Rabifier ( Figure 2A–C , see Table S1 for a list of all sequences used ) .', 'This ensures that the validation is meaningful , as it provides a strict lower bound on the expected performance in everyday use .', 'First , we chose the Excavate Trypanosoma brucei [32] , which is one of the most distantly related organism to our reference sequences , which are dominated by Ophistokonts ( an unranked scientific classification sometimes also called ‘Fungi/Metazoa group’ ) .', 'The second is Entamoeba histolytica [30] , a Unikont from the phylum of Amoebozoa that is thus marginally closer to the sequences that dominate our reference database , but has a heavily expanded and diverse Rab repertoire which makes it challenging to assign Rab subfamilies .', 'The third organism , Monosiga brevicollis from the class of Choanoflagellates , was chosen as a representative of a phylum ( Choanozoa ) for which no information on the Rab family is available yet .', 'In this third case , we compare the automated predictions against a manual analysis we performed in this study ( Figure 2E ) , and which we will discuss below .', 'The first aspect we assessed is the ability of the Rabifier to distinguish Rabs from other GTPases ( summarised in Figure 2A ) .', 'We present the Rabifier with the set of GTPases from the above organisms and count how often we miss a Rab ( false negative—FN ) , and how often we incorrectly classify a non-Rab as a Rab ( false positive—FP ) .', 'For T . brucei , we correctly classified 101 out of 102 GTPases as being a Rab or not , 292 out of 295 in E . histolytica and finally all 125 GTPases in M . brevicollis .', 'Altogether , we have no FP and 4 FN , which means that for this particular set of genomes we make correct decisions about whether a protein is a Rab in 99 . 2% of the cases with no differences amongst the organisms .', 'In order to understand the sources of the misannotations at family level , we inspected the false negatives individually .', 'The Rabifier disagrees with the manual curation of [32] in T . brucei for TbRabX3 , a RabL2-like protein , that is counted as a false negative .', 'We explicitly added RabL2 sequences to our negative data set as we do not consider these proteins as members of the Rab family ( see Materials and Methods ) .', 'The remaining disagreements between the Rabifier and the manual annotations are three false negative proteins in E . histolytica in which we cannot find any detectable RabF motif , and one protein which has no similarity to any member of our reference dataset of small GTPases .', 'We conclude that these proteins are likely misclassified in [30] , and hence that the above failures of the Rabifier to identify Rabs are artificially introduced by our validation procedure .', 'Secondly , we established the accuracy by which a given Rab sequence is assigned to the right subfamily ( summarised in Figure 2A ) .', 'Concretely , for those sequences which were correctly identified as Rabs , we checked whether the proposed subfamily agreed either with the public annotation or our own one for M . brevicollis .', 'We distinguished between two operating modes of the Rabifier: a normal one which does not consider the confidence levels the Rabifier attributes to its classifications , and a high-confidence mode which accepts only the high-confidence annotations above a certain confidence threshold , whereas those below are classified as belonging to the undetermined subfamily RabX .', 'Ignoring the information provided by the classification confidence , we correctly called 16 out of 17 Rabs for T . brucei , 59 out of 91 in E . histolytica and 20 out of 25 for M . brevicollis , leading to an overall fraction of 71 . 4% correct decisions ( 79 . 7% on average per organism ) .', 'However , if one defines a threshold below which a classification is systematically considered as belonging to the undefined subfamily RabX , the accuracy can be substantially improved .', 'To illustrate this , Figure 2B displays the distribution of scores associated to correct and wrong calls , which shows that wrong calls clearly have lower confidence scores on average .', 'In order to test for all possible thresholds exploiting this difference , we performed a ROC curve analysis presented in Figure 2C .', 'This machine learning technique allows to summarise and quantify the classification performance for all thresholds ( Area Under the Curve ( AUC ) [39] , here 0 . 94 ) , and enables to objectively choose a threshold providing an optimal TP/FP-tradeoff .', "Here , we opted for 0 . 4 , which we propose as a default choice for the interpretation of the Rabifier's results .", 'Yet , the use of this threshold is not fixed as it may vary depending on the dataset , and can be freely modified by users of the Rabifier .', 'The consequences of applying a cutoff on the classification accuracy are quantified by the inlay in Figure 2C: only trusting calls with confidence higher or equal to 0 . 4 greatly reduces the amount of misclassified Rabs from non-human subfamilies and improves the overall accuracy to 90% ( 92 . 01% on average per organism ) .', 'In summary , we conclude that our workflow is able to correctly discern Rabs from other GTPases .', 'Furthermore , calls both at family and subfamily level have an associated confidence score which correctly captures uncertainty in the decision .', 'Relying on the information provided by the confidence level , the Rabifier suggests correct subfamilies around 90% of the time even in difficult and phylogenetically isolated cases .', 'After having established the correctness of our procedure , we wished to assess the improvement it represents over possible alternative large-scale approaches in an objective manner .', 'This excludes benchmarking against methods for example based on phylogenetic trees , as reasoning over them is difficult to automate and not feasible for thousands of sequences .', 'We chose to compare the Rabifier to the Conserved Domain Database at the NCBI [40] , the only resource we are aware of that specifically scores for RabF motifs .', 'To this end , we implemented an alternative decision scheme which given a protein retrieves the protein name and CDD domain annotation of its BH in the NCBI protein database .', 'Note that if the protein is in the NCBI database , the BH retrieves the protein itself .', 'As for the choice of genome , the Rabifier has to be benchmarked against an organism whose Rab family has not been manually curated , as our alternative procedure would simply retrieve that annotation .', 'Moreover , an organism from a taxon which is both close to Metazoa and for which no information on the Rab family exists best ensures an unbiased measurement .', 'These requirements are met by the Choanoflagellate M . brevicollis , which we analysed ourselves and is thus an ideal candidate for a direct comparison .', 'The results of this experiment are detailed in Figure 2D ( see also Table S1 ) .', 'As above , we distinguished between the ability to discern Rabs from other GTPases and to actually propose the correct subfamily for a given Rab .', 'First , while the Rabifier achieved 100% accuracy in separating Rabs from other GTPases in M . brevicollis , the alternative strategy—although not introducing false positives—misses 8 of 25 Rabs leading to an overall drop in sensitivity .', 'On top of these 8 sequences , the Rabifier correctly suggests subfamilies for 4 further proteins wrongly classified by the alternative strategy , leading to an overall difference of 12 sequences correctly classified only by the Rabifier .', 'Thus , our annotation pipeline represents a significant improvement over currently available large scale approaches , both in terms of sensitive identification of Rabs and especially with regards to the difficult automatic classification of Rabs into subfamilies .', 'In order to make our pipeline useful to the cell biology community interested in Rabs , we provide access to the Rabifier in form of a web tool ( Figure 3A ) .', 'Via the graphical interface users can submit up to five protein sequences at a time , and the classifications generated by our workflow are returned together with their associated degree of confidence .', 'We envisage users who want to quickly generate hypotheses about one or a few candidate proteins .', 'Users wishing to classify more sequences are encouraged to contact us .', 'We emphasise that the Rabifier works without need for phylogenetic information about the input , hence any set of protein sequences can be submitted .', 'In addition , we generated a database of nearly 8 , 000 classified Rab sequences in 247 eukaryotic genomes , which we make publicly available at www . RabDB . org ( Figure 3A ) together with basic browsing and visualisation tools .', 'Our database is built on top of the Superfamily database [41] ( September 2009 release ) , which allows us to follow its release cycle and include predictions for all newly sequenced genomes contained therein .', 'Figure 3B details the phylogenetic distribution of genomes in RabDB and the number of Rabs we predict in each of those eukaryotic branches .', 'The correctness of the content in www . RabDB . org is not manually confirmed systematically .', 'However , we constantly inspect and manually curate the generated predictions and update our internal reference database accordingly .', 'Furthermore , we provide users the possibility to notify us of a potential mis-annotation found in the database such that we can correct the classification of the Rab in question .', 'These measures further enhance the expected quality of future releases of www . RabDB . org .', 'As can be noticed from Figure 3B , the Rabifier detected a large number of Rabs not belonging to any subfamily represented in our reference set , i . e . most subfamilies which have been described before .', 'By definition these sequences show no similarity to any functionally characterised Rab , hence a bioinformatic annotation is not possible .', 'However , in order to structure the space of new sequences and provide a starting point to study this yet unexplored diversity , we clustered these Rabs with respect to their sequence identity and propose several hypothetical Rab subfamilies ( see Material and Methods for details ) .', 'The result of this procedure is shown in Figure 4 , which details the amount of hypothetical subfamilies according to the breadth of their occurrence ( see Figure S7 for an overview of the amount of Rabs falling into each of these classes ) .', 'We integrated these new subfamilies both in our database , where they can be browsed with help of the visualisation tools we provide , and in the online version of the Rabifier .', 'Note that in addition to these new hypothetical subfamilies we still find hundreds of Rabs that we cannot group with others .', 'Those may result from erroneous gene models in less well curated genomes , represent cases where our simple clustering procedure failed , or indeed be bona fide singletons .', 'A detailed phylogenetic analysis may be required to resolve these cases which is out of the scope of this study .', 'A dataset of 8 , 000 Rabs allows us to take a global view of the Rab sequence space , and to address previously inaccessible questions .', 'Here , we investigate the patterns of Rab repertoire expansion in the eukaryotic tree ( Figure 5 ) .', 'Expansion of certain protein families has been found to correlate with organismal complexity [42] .', 'The anecdotal evidence of Rab profiles in different organisms suggests at least three possible scenarios: a conserved core of Rabs present in all organisms; tinkering with a core of subfamilies by taxon- or species-specific expansions of existing subfamilies; a major variation of the Rab machinery with taxon- or species-specific Rab repertoires .', 'We asked whether any such scenario is apparent for the Rab family across the eukaryotic tree , or if different ones predominate in different branches .', 'We observe a tremendous heterogeneity in the sizes of Rab repertoires , ranging from 5 to several hundreds of Rabs in Encephalitozoon cuniculi and Trichomonas vaginalis respectively .', 'Genomic analyses have shown a general trend for more and larger families in bigger genomes [43] , [44] .', 'In the case of Rabs , linear regression over all taxa reveals that genome size explains roughly 60% of the observed variance in numbers of Rabs in an organism ( Figure S2 ) .', 'However , due to the current bias in fully sequenced genomes towards Ophistokonts ( compare Figure 3B ) , it is unclear whether these numbers will remain as such in the future .', 'We find that closely related organisms tend to have similar Rab repertoires in size , but at the level of phyla we encounter marked differences indicating taxon-specific adaptations .', 'For example , although Ciliophora and Apicomplexa belong to the same superphylum ( Alveolata ) , these sister phyla show very different repertoires , highly expanded in the first case , and streamlined in the second .', 'The smaller Rab repertoires in Apicomplexan genomes , mostly dominated by intracellular parasites , may be due to secondary gene loss , similar to that reported in bacterial intracellular parasites and endosymbionts [45] and in the obligate intracellular parasitic Microsporidia [45] .', 'Another example of reduction of Rab repertoires is observed in the fungal branch , as we reported previously [26] and now confirm based on an extended set of 103 genomes .', 'It is noteworthy that Fungi are Unikonts , a taxon which comprises Metazoa and Amoebozoa , i . e . branches that appeared to have suffered independent expansions of their Rab repertoires [24] , [30] .', 'We observe large expansions in Diplomonadida/Trichomonadida , Ciliophora and Amoebozoa .', 'Much of these expansions are accounted for by species-specific subfamilies ( see Figure 4 ) .', 'This demonstrates that there is frequent invention of new Rabs , perhaps in a taxon-specific manner—a hypothesis that will have to await broader sampling of the genomes space to be tested in most taxa .', 'On the other hand , inspection of Figure 5 reveals that for those Rabs that can be classified , different subfamilies expanded in each branch of the tree .', "For example , Rab7 forms the largest subfamily in Diplomonadida/Trichomonadida and Amoebozoa , whereas Ciliophora's most expanded subfamily is Rab2 .", 'This suggests that these are independent expansions , which has already been observed for example within the Rab5 subfamily [26] , [46] .', 'Note that we repeated these analyses for different confidence cutoffs and observed no significant consequences on the broad picture .', 'In summary , the global evolution of Rab repertoires is highly dynamic with frequent taxon-specific subfamily expansions , gain of new Rabs and losses .', 'Hence , we observe a scenario where a core set of Rabs tends to be universally conserved , and can coexist in different taxa with subfamily expansions and/or taxon- or species-specific Rabs .', 'It is clear that no unique path to cellular complexity and specialisation exists , implying that any conclusion about the evolution of Rabs in a given taxon is not necessarily true for other eukaryotic taxa .', 'The systematic identification and classification of Rab repertoires in multiple branches of the eukaryotic tree of life allows the establishment of a phylogenetic profile for each Rab subfamily .', 'As Metazoa and Fungi are the most extensively sampled and best annotated groups , we profiled human subfamilies ( Figure 6 ) and determined their likely time of origin ( Figure 7 ) .', 'For a detailed analysis of fungal Rabs see [26] .', 'We further established the direction of duplication , i . e . from which Rab subfamily another emerged by duplication and subsequent divergence , by crossing their likely time of origin with a phylogenetic tree of the human Rab family .', 'We reasoned that for two closely related Rabs , the one that is present in more taxa is likely the ancestral one .', 'Since all Rabs are by definition paralogs and especially the deeper evolutionary relationships are unclear , we restricted the inference of direction of duplication to well supported branches .', 'Here , we define well supported branches as those with bootstrap support higher than 58% in a tree of human Rabs , which is chosen to include the branch between Rab5 and Rab22 as their association is commonly accepted [47]–[51] .', 'As further support , we note that all branches selected according to this criterion are also present in the tree of mouse Rabs we present below , however , in general 58% is not a strong branch support and should not be used indiscriminately on trees of other Rabs .', 'Based on a 58% cutoff , one obtains directed duplication scenarios for a number of subfamilies as summarised in Figure 7 .', 'We term subfamilies with a clear origin as ‘derived’ .', 'This analysis suggests new candidates for ancestral Rabs .', 'Previously Rab1 , 2 , 4 , 5 , 6 , 7 , 8 and Rab11 [17] , Rab18 [31] , [52] , Rab21 [30] , [53] as well as Rab23 and 28 [32] could be mapped to more than one major branch of the eukaryotic tree , making them likely candidates to be present in the LECA .', 'Our results support these assignments and reveal a new set of proteins that can be found in two or more basal eukaryotic taxa , namely Rab14 , 32 and RabL4 .', 'Applying the same parsimony argument as previous studies suggests that these Rabs were part of the ancestral set of Rab in the LECA .', 'Are these putative ancestral Rabs an artefact due to incorrect assignments or convergent evolution ?', 'We validated the automated subfamily classification by phylogenetic trees , and could not disprove their annotation ( Figures S4 A–C ) .', 'The possibility of convergent evolution is however harder to rule out .', 'Regardless , an organism with 15 Rabs is not surprising and comparable with some unicellular eukaryotes [32] , [33] , and free living fungi frequently have less [26] .', 'It is remarkable that with every new analysis the LECA appears to become increasingly more complex [54] .', 'On functional grounds , mapping these Rabs to the LECA is plausible .', 'RabL4 , also known as IFT27 , plays a role in ciliogenesis as part of the Intra Flagella Transport ( IFT ) machinery [55] .', 'Flagella are believed to be ancestral characters , present in the LECA [56] , [57] .', 'Rab32 regulates transport to the pigmented/secretory granules [58] , an animal-specific function , but it has also been claimed to have a mitochondria-related function [59] , [60] .', 'The known function of Rab14 in phagosome maturation and a recycling step at the TGN [61] , [62] is less clearly ancestral , but it may lend support for a phagotrophic LECA as previously proposed [63] .', 'In summary , our results support the claim that the LECA had a highly complex endomembrane system , and that secondary Rab losses have been dominant in the evolution of the major eukaryotic taxa [17] .', 'The emergence of multicellularity is one of the major transitions in evolution [64] , which happened independently multiple times ( see [65] for a recent review ) .', 'There are several critical features necessary for the evolution of multicellular organisms , for example mechanisms for cell adhesion , cell polarity and inter-cellular communication .', 'Little is known about how protein trafficking has evolved during this transition .', 'We take advantage of our extensive annotation of the Rab family to derive the Rab complement prior to and after the emergence of multicellularity in Metazoa .', 'Monosiga brevicollis belongs to the Choanozoa , the closest unicellular relatives of Metazoa .', 'The genome of this organism was only recently sequenced [66] , and in the context of the validation of the Rabifier we conducted a detailed analysis of its Rab family .', 'The phylogenetic tree in Figure 2E reveals a relatively large Rab family with nearly no subfamily expansions ( see also Figure 5 ) , i . e . mostly with a single member per subfamily ( only Rab32 has two members ) .', 'This is also observed in simpler animals like D . melanogaster and C . elegans [52] , suggesting that larger subfamilies observed in mammals represent taxon-specific duplications .', 'Secondly , we observe several organism-specific Rabs , which we labeled MbRabX .', 'Consistent with results from the last section , the “invention” of new Rabs is a recurrent feature in multiple branches of the tree of life ( e . g . [28] , [30] , [32] , [52] ) .', 'We observed the emergence of three novel sub-families , Rab9 , 22 , 29 , none playing ‘animal-specific’ roles .', 'The function of Rab29 is unknown , but Rab9 and Rab22 both appear to be involved in late endocytic traffic [49] , [50] , [67] , [68] .', 'Surprisingly , the genome of M . brevicollis codes for prote
['Introduction', 'Results/Discussion', 'Materials and Methods']
['Rab proteins are small GTPases that act as essential regulators of vesicular trafficking .', '44 subfamilies are known in humans , performing specific sets of functions at distinct subcellular localisations and tissues .', 'Rab function is conserved even amongst distant orthologs .', 'Hence , the annotation of Rabs yields functional predictions about the cell biology of trafficking .', 'So far , annotating Rabs has been a laborious manual task not feasible for current and future genomic output of deep sequencing technologies .', 'We developed , validated and benchmarked the Rabifier , an automated bioinformatic pipeline for the identification and classification of Rabs , which achieves up to 90% classification accuracy .', 'We cataloged roughly 8 . 000 Rabs from 247 genomes covering the entire eukaryotic tree .', 'The full Rab database and a web tool implementing the pipeline are publicly available at www . RabDB . org .', 'For the first time , we describe and analyse the evolution of Rabs in a dataset covering the whole eukaryotic phylogeny .', 'We found a highly dynamic family undergoing frequent taxon-specific expansions and losses .', 'We dated the origin of human subfamilies using phylogenetic profiling , which enlarged the Rab repertoire of the Last Eukaryotic Common Ancestor with Rab14 , 32 and RabL4 .', 'Furthermore , a detailed analysis of the Choanoflagellate Monosiga brevicollis Rab family pinpointed the changes that accompanied the emergence of Metazoan multicellularity , mainly an important expansion and specialisation of the secretory pathway .', 'Lastly , we experimentally establish tissue specificity in expression of mouse Rabs and show that neo-functionalisation best explains the emergence of new human Rab subfamilies .', 'With the Rabifier and RabDB , we provide tools that easily allows non-bioinformaticians to integrate thousands of Rabs in their analyses .', 'RabDB is designed to enable the cell biology community to keep pace with the increasing number of fully-sequenced genomes and change the scale at which we perform comparative analysis in cell biology .']
['Intracellular compartmentalisation via membrane-delimited organelles is a fundamental feature of the eukaryotic cell .', 'Understanding its origins and specialisation into functionally distinct compartments is a major challenge in evolutionary cell biology .', 'We focus on the Rab enzymes , critical organisers of the trafficking pathways that link the endomembrane system .', 'Rabs form a large family of evolutionarily related proteins , regulating distinct steps in vesicle transport .', 'They mark pathways and organelles due to their specific subcellular and tissue localisation .', 'We propose a solution to the problem of identifying and annotating Rabs in hundreds of sequenced genomes .', 'We developed an accurate bioinformatics pipeline that is able to take into account pre-existing and often inconsistent , manual annotations .', 'We made it available to the community in form of a web tool , as well as a database containing thousands of Rabs assigned to sub-families , which yields clear functional predictions .', 'Thousands of Rabs allow for a new level of analysis .', 'We illustrate this by characterising for the first time the global evolutionary dynamics of the Rab family .', 'We dated the emergence of subfamilies and suggest that the Rab family expands by duplicates acquiring new functions .']
['computer applications', 'genome expression analysis', 'cellular structures', 'subcellular organelles', 'genome evolution', 'evolutionary biology', 'molecular genetics', 'information technology', 'sequence analysis', 'comparative genomics', 'biology', 'databases', 'computer science', 'web-based applications', 'cell biology', 'gene identification and analysis', 'genetics', 'gene duplication', 'genomics', 'molecular cell biology', 'genomic evolution', 'computational biology', 'genetics and genomics']