Abstract
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.
Highlights
Intracellular compartmentalisation is found in all cellular lifeforms, yet eukaryotes have evolved extensive membranous compartments unique to this domain of life
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]
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
Summary
Intracellular compartmentalisation is found in all cellular lifeforms, yet eukaryotes have evolved extensive membranous compartments unique to this domain of life. 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 It is unclear how these data map across organisms, and how general the mechanisms characterised in single species are. 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). They are small GTPases that work as molecular switches to regulate vesicle
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