Untangling the evolution of Rab G proteins: implications of a comprehensive genomic analysis

Background

Membrane-bound organelles are a defining feature of eukaryotic cells, and play a central role in most of their fundamental processes. The Rab G proteins are the single largest family of proteins that participate in the traffic between organelles, with 66 Rabs encoded in the human genome. Rabs direct the organelle-specific recruitment of vesicle tethering factors, motor proteins, and regulators of membrane traffic. Each organelle or vesicle class is typically associated with one or more Rab, with the Rabs present in a particular cell reflecting that cell's complement of organelles and trafficking routes.

Results

Through iterative use of hidden Markov models and tree building, we classified Rabs across the eukaryotic kingdom to provide the most comprehensive view of Rab evolution obtained to date. A strikingly large repertoire of at least 20 Rabs appears to have been present in the last eukaryotic common ancestor (LECA), consistent with the 'complexity early' view of eukaryotic evolution. We were able to place these Rabs into six supergroups, giving a deep view into eukaryotic prehistory.

Conclusions

Tracing the fate of the LECA Rabs revealed extensive losses with many extant eukaryotes having fewer Rabs, and none having the full complement. We found that other Rabs have expanded and diversified, including a large expansion at the dawn of metazoans, which could be followed to provide an account of the evolutionary history of all human Rabs. Some Rab changes could be correlated with differences in cellular organization, and the relative lack of variation in other families of membrane-traffic proteins suggests that it is the changes in Rabs that primarily underlies the variation in organelles between species and cell types.     

The C. elegans Rab Family: Identification,Classification and Toolkit Construction

Rab monomeric GTPases regulate specific aspects of vesicle transport in eukaryotes including coat recruitment, uncoating, fission, motility, target selection and fusion. Moreover, individual Rab proteins function at specific sites within the cell, for example the ER, golgi and early endosome. Importantly, the localization and function of individual Rab subfamily members are often conserved underscoring the significant contributions that model organisms such as Caenorhabditis elegans can make towards a better understanding of human disease caused by Rab and vesicle trafficking malfunction. With this in mind, a bioinformatics approach was first taken to identify and classify the complete C. elegans Rab family placing individual Rabs into specific subfamilies based on molecular phylogenetics. For genes that were difficult to classify by sequence similarity alone, we did a comparative analysis of intron position among specific subfamilies from yeast to humans. This two-pronged approach allowed the classification of 30 out of 31 C. elegans Rab proteins identified here including Rab31/Rab50, a likely member of the last eukaryotic common ancestor (LECA). Second, a molecular toolset was created to facilitate research on biological processes that involve Rab proteins. Specifically, we used Gateway-compatible C. elegans ORFeome clones as starting material to create 44 full-length, sequence-verified, dominant-negative (DN) and constitutive active (CA) rab open reading frames (ORFs). Development of this toolset provided independent research projects for students enrolled in a research-based molecular techniques course at California State University, East Bay (CSUEB).     

A Highlights from MBoC Selection: Identification and characterization of multiple novel Rab–myosin Va interactions

Myosin Va is a widely expressed actin-based motor protein that binds members of the Rab GTPase family (3A, 8A, 10, 11A, 27A) and is implicated in many intracellular trafficking processes. To our knowledge, myosin Va has not been tested in a systematic screen for interactions with the entire Rab GTPase family. To that end, we report a yeast two-hybrid screen of all human Rabs for myosin Va-binding ability and reveal 10 novel interactions (3B, 3C, 3D, 6A, 6A′, 6B, 11B, 14, 25, 39B), which include interactions with three new Rab subfamilies (Rab6, Rab14, Rab39B). Of interest, myosin Va interacts with only a subset of the Rabs associated with the endocytic recycling and post-Golgi secretory systems. We demonstrate that myosin Va has three distinct Rab-binding domains on disparate regions of the motor (central stalk, an alternatively spliced exon, and the globular tail). Although the total pool of myosin Va is shared by several Rabs, Rab10 and Rab11 appear to be the major determinants of its recruitment to intracellular membranes. We also present evidence that myosin Va is necessary for maintaining a peripheral distribution of Rab11- and Rab14-positive endosomes.     

Specific Rab GTPase-activating proteins define the Shiga toxin and epidermal growth factor uptake pathways

Rab family guanosine triphosphatases (GTPases) together with their regulators define specific pathways of membrane traffic within eukaryotic cells. In this study, we have investigated which Rab GTPase-activating proteins (GAPs) can interfere with the trafficking of Shiga toxin from the cell surface to the Golgi apparatus and studied transport of the epidermal growth factor (EGF) from the cell surface to endosomes. This screen identifies 6 (EVI5, RN-tre/USP6NL, TBC1D10A-C, and TBC1D17) of 39 predicted human Rab GAPs as specific regulators of Shiga toxin but not EGF uptake. We show that Rab43 is the target of RN-tre and is required for Shiga toxin uptake. In contrast, RabGAP-5, a Rab5 GAP, was unique among the GAPs tested and reduced the uptake of EGF but not Shiga toxin. These results suggest that Shiga toxin trafficking to the Golgi is a multistep process controlled by several Rab GAPs and their target Rabs and that this process is discrete from ligand-induced EGF receptor trafficking.     

Rab GTPases and microtubule motors

Rab proteins are a family of small GTPases which, since their initial identification in the late 1980s, have emerged as master regulators of all stages of intracellular trafficking processes in eukaryotic cells. Rabs cycle between distinct conformations that are dependent on their guanine-nucleotide-bound status. When active (GTP-bound), Rabs are distributed to the cytosolic face of specific membranous compartments where they recruit downstream effector proteins. Rab-effector complexes then execute precise intracellular trafficking steps, which, in many cases, include vesicle motility. Microtubule-based kinesin and cytoplasmic dynein motor complexes are prominent among the classes of known Rab effector proteins. Additionally, many Rabs associate with microtubule-based motors via effectors that act as adaptor molecules that can simultaneously associate with the GTP-bound Rab and specific motor complexes. Thus, through association with motor complexes, Rab proteins can allow for membrane association and directional movement of various vesicular cargos along the microtubule cytoskeleton. In this mini-review, we highlight the expanding repertoire of Rab/microtubule motor protein interactions, and, in doing so, present an outline of the multiplicity of transport processes which result from such interactions.     

Rab GTPase Mediated Procollagen Trafficking in Ascorbic Acid Stimulated Osteoblasts

Despite advances in investigating functional aspects of osteoblast (OB) differentiation, especially studies on how bone proteins are deposited and mineralized, there has been little research on the intracellular trafficking of bone proteins during OB differentiation. Collagen synthesis and secretion is the major function of OBs and is markedly up-regulated upon ascorbic acid (AA) stimulation, significantly more so than in fibroblast cells. Understanding the mechanism by which collagen is mobilized in specialized OB cells is important for both basic cell biology and diseases involving defects in bone protein secretion and deposition. Protein trafficking along the exocytic and endocytic pathways is aided by many molecules, with Rab GTPases being master regulators of vesicle targeting. In this study, we used microarray analysis to identify the Rab GTPases that are up-regulated during a 5-day AA differentiation of OBs, namely Rab1, Rab3d, and Rab27b. Further, we investigated the role of identified Rabs in regulating the trafficking of collagen from the site of synthesis in the ER to the Golgi and ultimately to the plasma membrane utilizing Rab dominant negative (DN) expression. We also observed that experimental halting of biosynthetic trafficking by these mutant Rabs initiated proteasome-mediated degradation of procollagen and ceased global protein translation. Acute expression of Rab1 and Rab3d DN constructs partially alleviated this negative feedback mechanism and resulted in impaired ER to Golgi trafficking of procollagen. Similar expression of Rab27b DN constructs resulted in dispersed collagen vesicles which may represent failed secretory vesicles sequestered in the cytosol. A significant and strong reduction in extracellular collagen levels was also observed implicating the functional importance of Rab1, Rab3d and Rab27b in these major collagen-producing cells.     

Rab proteins: The key regulators of intracellular vesicle transport

Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes.     

Structural determinants of Rab and Rab Escort Protein interaction: Rab family motifs define a conserved binding surface

Rab proteins are a large family of monomeric GTPases with 60 members identified in the human genome. Rab GTPases require an isoprenyl modification to their C-terminus for membrane association and function in the regulation of vesicular trafficking pathways. This reaction is catalysed by Rab geranylgeranyl transferase, which recognises as protein substrate any given Rab in a 1:1 complex with Rab Escort Protein (REP). REP is therefore able to bind many distinct Rab proteins but the molecular basis for this activity is still unclear. We recently identified conserved motifs in Rabs termed RabF motifs, which we proposed to mediate a conserved mode of interaction between Rabs and REPs. Here, we tested this hypothesis. We first used REP1 as a bait in the yeast two-hybrid system and isolated strictly full-length Rabs, suggesting that REP recognises multiple regions within and properly folded Rabs. We introduced point mutations in Rab3a as a model Rab and assessed the ability of the mutants to interact with REP using the yeast two-hybrid system and an in vitro prenylation assay. We identified several residues that affect REP:Rab binding in the RabF1, RabF3, and RabF4 regions (which include parts of the switch I and II regions), but not other RabF regions. These results support the hypothesis that Rabs bind REP via conserved RabF motifs and provide a molecular explanation for the preferential recognition of the GDP-bound conformation of Rab by REP.     

Decoding the regulation of mast cell exocytosis by networks of rab GTPases

Exocytosis is a key event in mast cell functions. By this process, mast cells release inflammatory mediators, contained in secretory granules (SGs), which play important roles in immunity and wound healing but also provoke allergic and inflammatory responses. The mechanisms underlying mast cell exocytosis remained poorly understood. An essential step toward deciphering the mechanisms behind exocytosis is the identification of the cellular components that regulate this process. Because Rab GTPases regulate specific trafficking pathways, we screened 44 Rabs for their functional impacts on exocytosis triggered by the FcεRI or combination of Ca(2+) ionophore and phorbol ester. Because exocytosis involves the continuous reorganization of the actin cytoskeleton, we also repeated our screen in the presence of cytochalasin D that inhibits actin polymerization. In this paper, we report on the identification of 30 Rabs as regulators of mast cell exocytosis, the involvement of 26 of which has heretofore not been recognized. Unexpectedly, these Rabs regulated exocytosis in a stimulus-dependent fashion, unless the actin skeleton was disrupted. Functional clustering of the identified Rabs suggested their classification as Rabs involved in SGs biogenesis or Rabs that control late steps of exocytosis. The latter could be further divided into Rabs that localize to the SGs and Rabs that regulate transport from the endocytic recycling compartment. Taken together, these findings unveil the Rab networks that control mast cell exocytosis and provide novel insights into their mechanisms of action.     

Large scale screening for novel rab effectors reveals unexpected broad Rab binding specificity

Small GTPase Rab is generally thought to control intracellular membrane trafficking through interaction with specific effector molecules. Because of the large number of Rab isoforms in mammals, however, the effectors of most of the mammalian Rabs have never been identified, and the Rab binding specificity of the Rab effectors previously reported has never been thoroughly investigated. In this study we systematically screened for novel Rab effectors by a yeast two-hybrid assay with 28 different mouse or human Rabs (Rab1-30) as bait and identified 27 Rab-binding proteins, including 19 novel ones. We further investigated their Rab binding specificity by a yeast two-hybrid assay with a panel of 60 different GTP-locked mouse or human Rabs. Unexpectedly most (17 of 27) of the Rab-binding proteins we identified exhibited broad Rab binding specificity and bound multiple Rab isoforms. As an example, inositol-polyphosphate 5-phosphatase OCRL (oculocerebrorenal syndrome of Lowe) bound the greatest number of Rabs (i.e. 16 distinct Rabs). Others, however, specifically recognized only a single Rab isoform or only two closely related Rab isoforms. The interaction of eight of the novel Rab-binding proteins identified (e.g. INPP5E and Cog4) with a specific Rab isoform was confirmed by co-immunoprecipitation assay and/or colocalization analysis in mammalian cell cultures, and the novel Rab2B-binding domain of Golgi-associated Rab2B interactor (GARI) and GARI-like proteins was identified by deletion and homology search analyses. The findings suggest that most Rab effectors (or Rab-binding proteins) regulate intracellular membrane trafficking through interaction with several Rab isoforms rather than through a single Rab isoform.     

A Ypt/Rab GTPase module makes a PAS

Organization of membrane micro-domains by Ypt/Rab GTPases is key for all membrane trafficking events in eukaryotic cells. Since autophagy is a membrane trafficking process, it was expected that these GTPases would play a role in autophagy as well. While evidence about participation of Ypt/Rabs in autophagy is beginning to emerge, the mechanisms by which they act in this process are still not clear. Moreover, it is still questionable if and how Ypt/Rabs coordinate autophagy with other cellular trafficking processes. Yeast Ypt1 and its mammalian homolog Rab1 are required for both endoplasmic reticulum (ER)-to-Golgi transport and autophagy, suggesting that they coordinate these two processes. In our recent paper, we identify Atg11, a bona fide phagophore assembly site (PAS) component, as a downstream effector of Ypt1. Moreover, we show that three components of a GTPase module—the Ypt1 activator, Trs85-containing TRAPP complex, Ypt1, and the Atg11 effector—interact on the PAS and are required for PAS formation during selective autophagy. We propose that Ypt/Rabs coordinate the secretory and the autophagic pathways by recruiting process-specific effectors.     

Conservation and function of Rab small GTPases in Entamoeba: Annotation of E. invadens Rab and its use for the understanding of Entamoeba biology

Entamoeba invadens is a reptilian enteric protozoan parasite closely related to the human pathogen Entamoeba histolytica and a good model organism of encystation. To understand the molecular mechanism of vesicular trafficking involved in the encystation of Entamoeba, we examined the conservation of Rab small GTPases between the two species. E. invadens has over 100 Rab genes, similar to E. histolytica. Most of the Rab subfamilies are conserved between the two species, while a number of species-specific Rabs are also present. We annotated all E. invadens Rabs according to the previous nomenclature [Saito-Nakano, Y., Loftus, B.J., Hall, N., Nozaki, T., 2005. The diversity of Rab GTPases in Entamoeba histolytica. Experimental Parasitology 110, 244-252]. Comparative genomic analysis suggested that the fundamental vesicular traffic machinery is well conserved, while there are species-specific protein transport mechanisms. We also reviewed the function of Rabs in Entamoeba, and proposed the use of the annotation of E. invadens Rab genes to understand the ubiquitous importance of Rab-mediated membrane trafficking during important biological processes including differentiation in Entamoeba.     

Large-scale profiling of Rab GTPase trafficking networks: the membrome

Rab GTPases and SNARE fusion proteins direct cargo trafficking through the exocytic and endocytic pathways of eukaryotic cells. We have used steady state mRNA expression profiling and computational hierarchical clustering methods to generate a global overview of the distribution of Rabs, SNAREs, and coat machinery components, as well as their respective adaptors, effectors, and regulators in 79 human and 61 mouse nonredundant tissues. We now show that this systems biology approach can be used to define building blocks for membrane trafficking based on Rab-centric protein activity hubs. These Rab-regulated hubs provide a framework for an integrated coding system, the membrome network, which regulates the dynamics of the specialized membrane architecture of differentiated cells. The distribution of Rab-regulated hubs illustrates a number of facets that guides the overall organization of subcellular compartments of cells and tissues through the activity of dynamic protein interaction networks. An interactive website for exploring datasets comprising components of the Rab-regulated hubs that define the membrome of different cell and organ systems in both human and mouse is available at http://www.membrome.org/.     

Role of endosomal Rab GTPases in cytokinesis

Completion of cytokinesis requires Rab 11-dependent membrane trafficking events to deliver new membrane to the furrow and for abscission. Many Rabs have overlapping endosomal distributions, hence, we examined whether these Rabs also function in cytokinesis. Analysis of the distribution of Rabs 4, 5, 7, 8, 9, 11, 21, and 22 revealed that only Rab 11 was enriched within the furrow of cells in telophase or present within the midbody. By contrast, Rabs 4, 5, 7, 8, and 9 were mainly localised within a peri-nuclear compartment facing away from the furrow. Using RNA interference and dominant negative Rab mutants, we evaluated the role of these Rabs in furrowing and abscission. Consistent with previous work, we find that Rab 11 is intimately involved in abscission. However, we further found that depletion of Rab 4 slowed but did not prevent abscission. Depletion of any other Rab species had little effect on furrowing or abscission. These data suggest that the membrane trafficking events required for completion of cytokinesis are largely controlled by Rab 11 and not other endosomal Rab proteins, and further suggest that the relocation of Rab 11-specific cargo is an integral facet of abscission. Arf6 knockdown was without effect on cytokinesis, but when both Rab 11 and Arf6 were knocked-down, we found the furrow rapidly regressed and the cells were unable to form a stable midbody. We suggest that Rab 11 and Arf6 function synergistically in the switch from furrowing to abscission, as well as in the terminal stage of abscission.     

Evolution of the Rab family of small GTP-binding proteins.

Rab proteins are small GTP-binding proteins that form the largest family within the Ras superfamily. Rab proteins regulate vesicular trafficking pathways, behaving as membrane-associated molecular switches. Here, we have identified the complete Rab families in the Caenorhabditis elegans (29 members), Drosophila melanogaster (29), Homo sapiens (60) and Arabidopsis thaliana (57), and we defined criteria for annotation of this protein family in each organism. We studied sequence conservation patterns and observed that the RabF motifs and the RabSF regions previously described in mammalian Rabs are conserved across species. This is consistent with conserved recognition mechanisms by general regulators and specific effectors. We used phylogenetic analysis and other approaches to reconstruct the multiplication of the Rab family and observed that this family shows a strict phylogeny of function as opposed to a phylogeny of species. Furthermore, we observed that Rabs co-segregating in phylogenetic trees show a pattern of similar cellular localisation and/or function. Therefore, animal and fungi Rab proteins can be grouped in "Rab functional groups" according to their segregating patterns in phylogenetic trees. These functional groups reflect similarity of sequence, localisation and/or function, and may also represent shared ancestry. Rab functional groups can help the understanding of the functional evolution of the Rab family in particular and vesicular transport in general, and may be used to predict general functions for novel Rab sequences.     

Rab35: GEFs,GAPs and Effectors

Rabs are the largest family of small GTPases and are master regulators of membrane trafficking. Following activation by guanine‐nucleotide exchange factors (GEFs), each Rab binds a specific set of effector proteins that mediate the various downstream functions of that Rab. Then, with the help of GTPase‐activating proteins, the Rab converts GTP to GDP, terminating its function. There are over 60 Rabs in humans and only a subset has been analyzed in any detail. Recently, Rab35 has emerged as a key regulator of cargo recycling at endosomes, with an additional role in regulation of the actin cytoskeleton. Here, we will focus on the regulation of Rab35 activity by the connecdenn/DENND1 family of GEFs and the TBC1D10/EPI64 family of GTPase‐activating proteins. We will describe how analysis of these proteins, as well as a plethora of Rab35 effectors has provided insights into Rab35 function. Finally, we will describe how Rab35 provides a novel link between the Rab and Arf family of GTPases with implications for tumor formation and invasiveness .      

VipD of Legionella pneumophila Targets Activated Rab5 and Rab22 to Interfere with Endosomal Trafficking in Macrophages

Upon phagocytosis, Legionella pneumophila translocates numerous effector proteins into host cells to perturb cellular metabolism and immunity, ultimately establishing intracellular survival and growth. VipD of L. pneumophila belongs to a family of bacterial effectors that contain the N-terminal lipase domain and the C-terminal domain with an unknown function. We report the crystal structure of VipD and show that its C-terminal domain robustly interferes with endosomal trafficking through tight and selective interactions with Rab5 and Rab22. This domain, which is not significantly similar to any known protein structure, potently interacts with the GTP-bound active form of the two Rabs by recognizing a hydrophobic triad conserved in Rabs. These interactions prevent Rab5 and Rab22 from binding to downstream effectors Rabaptin-5, Rabenosyn-5 and EEA1, consequently blocking endosomal trafficking and subsequent lysosomal degradation of endocytic materials in macrophage cells. Together, this work reveals endosomal trafficking as a target of L. pneumophila and delineates the underlying molecular mechanism.     

The diversity of Rab GTPases in Entamoeba histolytica

Rab proteins are ubiquitous small GTP-binding proteins that form a highly conserved family and regulate vesicular trafficking. Recent completion of the genome of the enteric protozoan parasite Entamoeba histolytica enabled us to identify an extremely large number (>90) of putative Rab genes. Multiple alignment and phylogenic analysis of amebic, human, and yeast Rab showed that only 22 amebic Rab proteins including EhRab1, EhRab2, EhRab5, EhRab7, EhRab8, EhRab11, and EhRab21 showed significant similarity to Rab from other organisms. The 69 remaining amebic Rab proteins showed only moderate similarity (<40% identity) to Rab proteins from other organisms. Approximately one-third of Rab proteins including Rab7, Rab11, and RabC form 15 subfamilies, which contain up to nine isoforms. Approximately 70% of amebic Rab genes contain single or multiple introns, and this proportion is significantly higher than that of common genes in this organism. Twenty-five Rabs possess an atypical carboxyl terminus such as CXXX, XCXX, XXCX, XXXC, and no cysteine. We propose annotation of amebic Rab genes and discuss biological significance of this extraordinary diversity of EhRab proteins in this organism.     

Family-wide characterization of the DENN domain Rab GDP-GTP exchange factors

A key requirement for Rab function in membrane trafficking is site-specific activation by GDP-GTP exchange factors (GEFs), but the majority of the 63 human Rabs have no known GEF. We have performed a systematic characterization of the 17 human DENN domain proteins and demonstrated that they are specific GEFs for 10 Rabs. DENND1A/1B localize to clathrin patches at the plasma membrane and activate Rab35 in an endocytic pathway trafficking Shiga toxin to the trans-Golgi network. DENND2 GEFs target to actin filaments and control Rab9-dependent trafficking of mannose-6-phosphate receptor to lysosomes. DENND4 GEFs target to a tubular membrane compartment adjacent to the Golgi, where they activate Rab10, which suggests a function in basolateral polarized sorting in epithelial cells that compliments the non-DENN GEF Sec2 acting on Rab8 in apical sorting. DENND1C, DENND3, DENND5A/5B, MTMR5/13, and MADD activate Rab13, Rab12, Rab39, Rab28, and Rab27A/27B, respectively. Together, these findings provide a basis for future studies on Rab regulation and function.     

Dual arginine recognition of LRRK2 phosphorylated Rab GTPases

Parkinson’s-disease-associated LRRK2 is a multidomain Ser/Thr kinase that phosphorylates a subset of Rab GTPases to control their effector functions. Rab GTPases are the prime regulators of membrane trafficking in eukaryotic cells. Rabs exert their biological effects by recruitment of effector proteins to subcellular compartments via their Rab-binding domain (RBD). Effectors are modular and typically contain additional domains that regulate various aspects of vesicle formation, trafficking, fusion, and organelle dynamics. The RBD of effectors is typically an α-helical coiled coil that recognizes the GTP conformation of the switch 1 and switch 2 motifs of Rabs. LRRK2 phosphorylates Rab8a at T72 (pT72) of its switch 2 α-helix. This post-translational modification enables recruitment of RILPL2, an effector that regulates ciliogenesis in model cell lines. A newly identified RBD motif of RILPL2, termed the X-cap, has been shown to recognize the phosphate via direct interactions between an arginine residue (R132) and pT72 of Rab8a. Here, we show that a second distal arginine (R130) is also essential for phospho-Rab binding by RILPL2. Through structural, biophysical, and cellular studies, we find that R130 stabilizes the primary R132:pT72 salt bridge through favorable enthalpic contributions to the binding affinity. These findings may have implications for the mechanism by which LRRK2 activation leads to assembly of phospho-Rab complexes and subsequent control of their membrane trafficking functions in cells.