Rab GTPases regulate all stages of membrane trafficking, including vesicle budding, cargo sorting, transport, tethering, and fusion1, 2. In the inactive (GDP-bound) conformation, accessory factors facilitate the targeting of Rab GTPases to intracellular compartments3–8. Following nucleotide exchange to the active (GTP-bound) conformation, Rab GTPases interact with functionally diverse effectors including lipid kinases, motor proteins, and tethering complexes. How effectors distinguish between homologous Rab GTPases represents an unresolved problem with respect to the specificity of vesicular trafficking. Using a structural proteomic approach, we have determined the specificity and structural basis underlying the interaction of the multivalent effector Rabenosyn-5 with the Rab family. The results demonstrate that even structurally similar effectors can achieve highly selective recognition of distinct subsets of Rab GTPases exclusively through interactions with the switch and interswitch regions. The observed specificity is determined at a family-wide level by structural diversity in the active conformation, which governs the spatial disposition of critical conserved recognition determinants, and by a small number of both positive and negative sequence determinants that allow further discrimination between Rab GTPases with similar switch conformations.
Rab GTPases like Ras-related monomeric GTPases are well known to regulate intracellular vesicle trafficking by cycling between membrane-bound and cytosolic states. The functions of these proteins are controlled by upstream regulators and downstream effectors. Ypt/Rabs transmit signals to downstream effectors in a GTP-dependent manner. GDP-bound Rab proteins are extracted from their target membrane by cytosolic proteins known as GDP dissociation inhibitors (GDIs), and the Rab GTPase is recruited to the membrane compartment following dissociation from the GDI by GDI displacement factor (GDF). Now, we're going to discuss the role of plant PRA concerted with Rab and GDI proteins by recycling Rab between membrane and cytosol for intracellular trafficking of cargo proteins.
GDF; GDI; PRA1; Rab; vacuolar trafficking; vesicle trafficking
Intracellular membrane traffic defines a complex network of pathways that connects many of the membrane-bound organelles of eukaryotic cells. Although each pathway is governed by its own set of factors, they all contain Rab GTPases that serve as master regulators. In this review, we discuss how Rabs can regulate virtually all steps of membrane traffic from the formation of the transport vesicle at the donor membrane to its fusion at the target membrane. Some of the many regulatory functions performed by Rabs include interacting with diverse effector proteins that select cargo, promoting vesicle movement, and verifying the correct site of fusion. We describe cascade mechanisms that may define directionality in traffic and ensure that different Rabs do not overlap in the pathways that they regulate. Throughout this review we highlight how Rab dysfunction leads to a variety of disease states ranging from infectious diseases to cancer.
Recent studies indicate that lipid droplets isolated from a variety of different cells are rich in proteins known to regulate membrane traffic. Among these proteins are multiple Rab GTPases. Rabs are GTP switches that regulate intracellular membrane traffic through an ability to control membrane-membrane docking as well as vesicle motility. Here we present evidence that the multiple Rabs associated with droplets have a function in regulating membrane traffic. Droplet Rabs are removed by Rab GDP-dissociation inhibitor (RabGDI) in a GDP-dependent reaction, and are recruited to Rab-depleted droplets from cytosol in a GTP-dependent reaction. Rabs also control the recruitment of the early endosome (EE) marker EEA1 from cytosol. We use an in vitro reconstitution assay to show that transferrin receptor positive EEs bind to the droplet in a GTP/Rab-dependent reaction that appears not to lead to membrane fusion. This docking reaction is insensitive to ATPγs but is blocked by ATP. Finally, we show that when GTP bound active or GDP bound inactive Rab5 is targeted to the droplet, the active form recruits EEA1. We conclude that the Rabs associated with droplets may be capable of regulating the transient interaction of specific membrane systems, probably to transport lipids between membrane compartments.
The Ras-superfamily of small G proteins is a family of GTP hydrolases that is regulated by GTP/GDP binding states. One member of the Ras-superfamily, Rab, is involved in the regulation of vesicle trafficking, which is critical to endocytosis, biosynthesis, secretion, cell differentiation and cell growth. The active form of the Rab proteins, which contains GTP, can recruit specific binding partners, such as sorting adaptors, tethering factors, kinases, phosphatases and motor proteins, thereby influencing vesicle formation, transport, and tethering. Many Rab proteins share the same interacting partners and perform unique roles in specific locations. Because functional loss of the Rab pathways has been implicated in a variety of diseases, the Rab GTPase family has been extensively investigated. In this review, we summarize Rab GTPase- mediated membrane trafficking while focusing on the structures of Rab protein and Rab-effector complexes. This review provides detailed information that helps explain how the Rab GTPase family is involved in membrane trafficking.
membrane trafficking; ras-superfamily; small G protein; rab GTPase; protein structure
Cellular sophistication is not exclusive to multicellular organisms, and unicellular eukaryotes can resemble differentiated animal cells in their complex network of membrane-bound structures. These comparisons can be illuminated by genome-wide surveys of key gene families. We report a systematic analysis of Rabs in a complex unicellular Ciliate, including gene prediction and phylogenetic clustering, expression profiling based on public data, and Green Fluorescent Protein (GFP) tagging. Rabs are monomeric GTPases that regulate membrane traffic. Because Rabs act as compartment-specific determinants, the number of Rabs in an organism reflects intracellular complexity. The Tetrahymena Rab family is similar in size to that in humans and includes both expansions in conserved Rab clades as well as many divergent Rabs. Importantly, more than 90% of Rabs are expressed concurrently in growing cells, while only a small subset appears specialized for other conditions. By localizing most Rabs in living cells, we could assign the majority to specific compartments. These results validated most phylogenetic assignments, but also indicated that some sequence-conserved Rabs were co-opted for novel functions. Our survey uncovered a rare example of a nuclear Rab and substantiated the existence of a previously unrecognized core Rab clade in eukaryotes. Strikingly, several functionally conserved pathways or structures were found to be associated entirely with divergent Rabs. These pathways may have permitted rapid evolution of the associated Rabs or may have arisen independently in diverse lineages and then converged. Thus, characterizing entire gene families can provide insight into the evolutionary flexibility of fundamental cellular pathways.
Single-celled organisms appear simple compared to multicellular organisms, but this may not be true at the level of the individual cell. In fact, microscopic observations suggest that protists can possess networks of organelles just as elaborate as those in animal cells. Consistent with this idea, recent analysis has identified large families of genes in protists that are predicted to act as determinants for complex membrane networks. To test these predictions and to probe relationships between cellular structures across a wide swath of evolution, we focused on one gene family in the single-celled organism Tetrahymena. These genes control the traffic between organelles, with each gene controlling a single step in this traffic. We asked three questions about each of 56 genes in the family. First, what is the gene related to in humans? Second, under what conditions is the gene being used in Tetrahymena? Third, what is the role of each gene? The results provide insights into both the dynamics and evolution of membrane traffic, including the finding that some pathways appearing both structurally and functionally similar in protists and animals are likely to have arisen independently in the two lineages.
Rab GTPases regulate vesicular traffic in eukaryotic cells by cycling between the active GTP-bound and inactive GDP-bound states. Their functions are modulated by the diverse selection of effector proteins that bind to specific Rabs in their activated state. We previously described the expression of Rab13 in bone cells. To search for novel Rab13 interaction partners, we screened a newborn rat bone marrow cDNA library for Rab13 effectors with a bacterial two-hybrid system. We found that Rab13 binds to the C-terminus of Endospanin-2, a small transmembrane protein. In addition to Rab13 also Rab8 bound to Endospanin-2, while no binding of Rab7, Rab10, Rab11 or Rab32 was observed. Rab13 and Rab8 also interacted with Endospanin-1, a close homolog of Endospanin-2. Rab13 and Endospanin-2 colocalised in perinuclear vesicular structures in Cos1 cells suggesting direct binding also in vivo. Endospanin-2 is implicated in the regulation of the cell surface growth hormone receptor (GHR), but the inhibition of Rab13 expression did not affect GHR cell surface expression. This suggests that the Rab13–Endospanin-2 interaction may have functions other than GHR regulation. In conclusion, we have identified a novel interaction for Rab13 and Rab8 with Endospanin-2 and Endospanin-1. The role of this interaction in cell physiology, however, remains to be elucidated.
▸ Rab13 and Rab8 both interact with Endospanin-2 and Endospanin-1. ▸ Rab13 and Rab8 binding to endospanins is specific; Rabs 7, 10, 11 and 32 do not bind. ▸ Rab13 binding to Endospanin-2 is nucleotide-dependent. ▸ Rab13 and Endospanin-2 colocalise in perinuclear vesicles and at the cell periphery.
Vesicle trafficking; Rab13; Rab effector; Protein interaction; Endospanin; Osteoclast; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; GHR, growth hormone receptor; GST, glutathione-S-transferase; HA, human influenza hemagglutinin; MBP, maltose binding protein; OB-R, leptin receptor; VPS55, vacuolar protein sorting 55.
During apoptosis, apoptotic cells are recognized and quickly engulfed by phagocytes. The internalized cell corpses are enclosed within membrane-bound vesicles called phagosomes. Cell corpse degradation depends on the phagosomes undergoing a maturation process, but regulation of phagosomal maturation is not well understood. Recently, we identified C. elegans Rab GTPase 14 as a novel regulator of apoptotic cell degradation. Loss of rab-14 function affects several steps of phagosome maturation, causing accumulation of persistent cell corpses. RAB-14 and UNC-108 (Rab GTPase 2) function redundantly to regulate phagosome maturation. Three Rabs, RAB-14, UNC-108/RAB2 and RAB-7, act cooperatively to control phagolysosome formation. RAB-14 and UNC-108 recruit lysosomes, while RAB-7 mediates fusion of lysosomes to phagosomes. Our data thus reveal the sequential action of Rab GTPases in regulating tethering, docking and fusion of lysosomes to phagosomes.
Rab GTPase; RAB-14; apoptotic cell degradation; phagosome maturation; phagolysosome; C. elegans
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).
Crystals of Rab11 in complex with the Rab-binding domain (RBD) of its effector Rab11-FIP2 were grown and synchrotron diffraction data were collected at 2.8 Å resolution.
The small GTPase Rab11 regulates the recycling of endosomes back to the plasma membrane. In its active GTP-bound form, Rab11 binds a novel set of effectors termed the Rab11 family of interacting proteins (Rab11-FIPs) which contain a conserved C-terminal Rab-binding domain (RBD) of unknown structure. Here, a complex of Rab11 with the RBD of Rab11-FIP2 has been purified and crystallized in the trigonal space group P3121, with unit-cell parameters a = 64.99, b = 64.99, c = 112.59 Å. Static light-scattering analyses of the molecular weight of the complex in solution are consistent with two copies of Rab11 and two copies of Rab11-FIP2 in the complex.
GTPases; Rab11; Rab11 family of interacting proteins
The Rab11-Family Interacting Protein (Rab11-FIP) group of effector proteins contain a highly conserved region in their C-termini that bind the GTPase, Rab11. Rab11 belongs to the largest family of small GTPases and is believed to regulate vesicle docking with target membranes and vesicle fusion. The amino acid sequence of the Rab11-FIP proteins predicts coiled-coil formation in the conserved C-terminal domain. In this study on Rab11-FIP2, we found experimental evidence for the coiled-coil and then defined the minimal structured core using limited proteolysis. We also showed that the Rab11-FIP2 coiled-coil domain forms a parallel homodimer in solution using cross-linking and mutagenesis and sedimentation equilibrium experiments. Various constructs representing the C-terminal domain of Rab11-FIP2 were characterized by circular dichroism and their affinity with Rab11 measured using isothermal titration calorimetry. The longest construct was both well-structured and bound Rab11. A construct truncated at the N-terminus was poorly structured, but retained the same affinity for binding to Rab11. Conformational changes were also demonstrated upon complex formation between Rab11 and Rab11-FIP2. A construct truncated at the C-terminus, which was the minimal coiled-coil domain defined by limited proteolysis, did not retain the ability to interact with Rab11 although it was as well-structured as the longer peptide. These data show that coiled-coil formation and Rab11 binding are separable functions of the C-terminal domain of Rab11-FIP2. The dissection of Rab11 binding from the formation of defined structure in a coiled-coil provides a potential mechanism for regulating Rab11-dependent endosomal trafficking.
Rab11-FIP2; Coiled-coil domain; ITC; CD; Limited proteolysis
Small GTPases of the rab family are involved in the regulation of vesicular transport. It is believed that cycling between the GTP- and GDP-bound forms, and accessory factors regulating this cycling are crucial for rab function. However, an essential role for rab nucleotide exchange factors has not yet been demonstrated. In this report we show the requirement of nucleotide exchange factor activity for Ypt1 GTPase mediated protein transport. The Ypt1 protein, a member of the rab family, plays a role in targeting vesicles to the acceptor compartment and is essential for the first two steps of the yeast secretory pathway. We use two YPT1 dominant mutations that contain alterations in a highly conserved GTP-binding domain, N121I and D124N. YPT1-D124N is a novel mutation that encodes a protein with nucleotide specificity modified from guanine to xanthine. This provides a tool for the study of an individual rab GTPase in crude extracts: a xanthosine triphosphate (XTP)-dependent conditional dominant mutation. Both mutations confer growth inhibition and a block in protein secretion when expressed in vivo. The purified mutant proteins do not bind either GDP or GTP. Moreover, they completely inhibit the ability of the exchange factor to stimulate nucleotide exchange for wild type Ypt1 protein, and are potent inhibitors of ER to Golgi transport in vitro at the vesicle targeting step. The inhibitory effects of the Ypt1-D124N mutant protein on both nucleotide exchange activity and protein transport in vitro can be relieved by XTP, indicating that it is the nucleotide-free form of the mutant protein that is inhibitory. These results suggest that the dominant mutant proteins inhibit protein transport by sequestering the exchange factor from the wild type Ypt1 protein, and that this factor has an essential role in vesicular transport.
The pathology causing stages of the human malaria parasite Plasmodium falciparum reside within red blood cells that are devoid of any regulated transport system. The parasite, therefore, is entirely responsible for mediating vesicular transport within itself and in the infected erythrocyte cytoplasm, and it does so in part via its family of 11 Rab GTPases. Putative functions have been ascribed to Plasmodium Rabs due to their homology with Rabs of yeast, particularly with Saccharomyces that has an equivalent number of rab/ypt genes and where analyses of Ypt function is well characterized.
Rabs are important regulators of vesicular traffic due to their capacity to recruit specific effectors. In order to identify P. falciparum Rab (PfRab) effectors, we first built a Ypt-interactome by exploiting genetic and physical binding data available at the Saccharomyces genome database (SGD). We then constructed a PfRab-interactome using putative parasite Rab-effectors identified by homology to Ypt-effectors. We demonstrate its potential by wet-bench testing three predictions; that casein kinase-1 (PfCK1) is a specific Rab5B interacting protein and that the catalytic subunit of cAMP-dependent protein kinase A (PfPKA-C) is a PfRab5A and PfRab7 effector.
The establishment of a shared set of physical Ypt/PfRab-effector proteins sheds light on a core set Plasmodium Rab-interactants shared with yeast. The PfRab-interactome should benefit vesicular trafficking studies in malaria parasites. The recruitment of PfCK1 to PfRab5B+ and PfPKA-C to PfRab5A+ and PfRab7+ vesicles, respectively, suggests that PfRab-recruited kinases potentially play a role in early and late endosome function in malaria parasites.
Interactom; Kinase; Plasmodium; Rab; Yeast; Ypt
Small GTPases of the rab family are crucial elements of the machinery
that controls membrane traffic. In the present study, we examined the
distribution and function of rab11. Rab11 was shown by confocal
immunofluorescence microscopy and EM to colocalize with internalized
transferrin in the pericentriolar recycling compartment of CHO and BHK
cells. Expression of rab11 mutants that are preferentially in the GTP- or
GDP-bound state caused opposite effects on the distribution of
transferrin-containing elements; rab11-GTP expression caused accumulation
of labeled elements in the perinuclear area of the cell, whereas rab11-GDP
caused a dispersion of the transferrin labeling. Functional studies showed
that the early steps of uptake and recycling for transferrin were not
affected by overexpression of rab11 proteins. However, recycling from the
later recycling endosome was inhibited in cells overexpressing the
rab11-GDP mutant. Rab5, which regulates early endocytic trafficking, acted
before rab11 in the transferrin-recycling pathway as expression of rab5-GTP
prevented transport to the rab11- positive recycling endosome. These
results suggest a novel role for rab11 in controlling traffic through the
A systematic screen of the entire human Rab GTPase family for interactions with myosin Va identified 10 novel Rab partners for myosin Va, all of which belong to the endocytic recycling and post-Golgi secretory membrane network. However, Rab10 and Rab11 appear to be the major determinants of its recruitment to intracellular membranes.
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.
Rab GTPases are crucial regulators of organelle biogenesis, maintenance, and transport. Multiple Rabs are expressed in all cells, and each is localized to a distinct set of organelles, but little is known regarding the mechanisms by which Rabs are targeted to their resident organelles. Integral membrane proteins have been postulated to serve as receptors that recruit Rabs from the cytosol in a complex with the Rab chaperone, GDI, to facilitate the dissociation of Rab and GDI, hence facilitating loading of Rabs on membranes. We show here that the yeast (Saccharomyces cerevisiae) Golgi Rab GTPase Ypt1p can be copurified with the integral membrane protein Yip3p from detergent cell extracts. In addition, a member of the highly conserved reticulon protein family, Rtn1p, is also associated with Yip3p in vivo. However, Ypt1p did not copurify with Rtn1p, indicating that Yip3p is a component of at least two different protein complexes. Yip3p and Rtn1p are only partially colocalized in cells, with Yip3p localized predominantly to the Golgi and secondarily to the endoplasmic reticulum, whereas Rtn1p is localized predominantly to the endoplasmic reticulum and secondarily to the Golgi. Surprisingly, the intracellular localization of Rabs was not perturbed in yip3Δ or rtn1Δ mutants, suggesting that these proteins do not play a role in targeting Rabs to intracellular membranes. These data indicate that Yip3p may have multiple functions and that its interaction with Rabs is not critical for their recruitment to organelle membranes.
Dual-specific A-kinase-anchoring protein 2 (D-AKAP2/AKAP10), which interacts at its carboxyl terminus with protein kinase A and PDZ domain proteins, contains two tandem regulator of G-protein signaling (RGS) domains for which the binding partners have remained unknown. We show here that these RGS domains interact with Rab11 and GTP-bound Rab4, the first demonstration of RGS domains binding small GTPases. Rab4 and Rab11 help regulate membrane trafficking through the endocytic recycling pathways by recruiting effector proteins to specific membrane domains. Although D-AKAP2 is primarily cytosolic in HeLa cells, a fraction of the protein localizes to endosomes and can be recruited there to a greater extent by overexpression of Rab4 or Rab11. D-AKAP2 also regulates the morphology of the Rab11-containing compartment, with co-expression causing accumulation of both proteins on enlarged endosomes. Knockdown of D-AKAP2 by RNA interference caused a redistribution of both Rab11 and the constitutively recycling transferrin receptor to the periphery of cells. Knockdown also caused an increase in the rate of transferrin recycling, suggesting that D-AKAP2 promotes accumulation of recycling proteins in the Rab4/Rab11-positive endocytic recycling compartment.
Chlamydia species are obligate intracellular bacteria that replicate within a membrane-bound vacuole, the inclusion, which is trafficked to the peri-Golgi region by processes that are dependent on early chlamydial gene expression. Although neither the host nor the chlamydial proteins that regulate the intracellular trafficking have been clearly defined, several enhanced green fluorescent protein (EGFP)-tagged Rab GTPases, including Rab6, are recruited to Chlamydia trachomatis inclusions. To further characterize the association of Rab6 with C. trachomatis inclusions, we examined the intracellular localization of guanine nucleotide-binding mutants of Rab6 and demonstrated that only active GTP-bound and not inactive GDP-bound EGFP-Rab6 mutants were recruited to the inclusion, suggesting that EGFP-Rab6 interacts with the inclusion via a host Rab6 effector or a chlamydial protein that mimics a Rab6 effector. Using EGFP-tagged fusion proteins, we also demonstrated that the Rab6 effector Bicaudal D1 (BICD1) localized to C. trachomatis inclusions in a biovar-specific manner. In addition, we demonstrated that EGFP-Rab6 and its effector EGFP-BICD1 are recruited to the inclusion in a microtubule- and Golgi apparatus-independent but chlamydial gene expression-dependent mechanism. Finally, in contrast to the Rab6-dependent Golgi apparatus localization of endogenous BICD1, EGFP-BICD1 was recruited to the inclusion by a Rab6-independent mechanism. Collectively, these data demonstrate that neither Rab6 nor BICD1 is trafficked to the inclusion via a Golgi apparatus-localized intermediate, suggesting that each protein is trafficked to the C. trachomatis serovar L2 inclusion by a unique, but as-yet-undefined, mechanism.
This review discusses how kinetic proofreading by Rab GTPases provides a speed-dating mechanism defining the identity of membrane domains in vesicle trafficking.
Rab GTPases are highly conserved components of vesicle trafficking pathways that help to ensure the fusion of a vesicle with a specific target organelle membrane. Specific regulatory pathways promote kinetic proofreading of membrane surfaces by Rab GTPases, and permit accumulation of active Rabs only at the required sites. Emerging evidence indicates that Rab activation and inactivation are under complex feedback control, suggesting that ultrasensitivity and bistability, principles established for other cellular regulatory networks, may also apply to Rab regulation. Such systems can promote the rapid membrane accumulation and removal of Rabs to create time-limited membrane domains with a unique composition, and can explain how Rabs define the identity of vesicle and organelle membranes.
Rab GTPases regulate discrete steps in vesicular transport pathways. Rabs require activation by specific guanine nucleotide exchange factors (GEFs) that stimulate the exchange of GDP for GTP. Rab27a controls motility and regulated exocytosis of secretory granules and related organelles. In melanocytes, Rab27a regulates peripheral transport of mature melanosomes by recruiting melanophilin and myosin Va. Here, we studied the activation of Rab27a in melanocytes. We identify Rab3GEP, previously isolated as a GEF for Rab3a, as the non-redundant Rab27a GEF. Similar to Rab27a-deficient ashen melanocytes, Rab3GEP-depleted cells show both clustering of melanosomes in the perinuclear area and loss of the Rab27a effector Mlph. Consistent with a role as an activator, levels of Rab27a-GTP are decreased in cells lacking Rab3GEP. Recombinant Rab3GEP exhibits guanine nucleotide exchange activity against Rab27a and Rab27b in vitro, in addition to its previously documented activity against Rab3. Our results indicate promiscuity in Rab GEF action and suggest that members of related but functionally distinct Rab subfamilies can be controlled by common activators.
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.
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.
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.
Organelles; G proteins; humans; last eukaryotic common ancestor
Salmonella typhimurium survives and replicates intracellular in a membrane-bound compartment, the Salmonella-containing vacuole (SCV). In HeLa cells, the SCV matures through interactions with the endocytic pathway, but Salmonella avoids fusion with mature lysosomes. The exact mechanism of the inhibition of phagolysosomal fusion is not understood. Rab GTPases control several proteins involved in membrane fusion and vesicular transport. The small GTPase Rab7 regulates the transport of and fusion between late endosomes and lysosomes and associates with the SCV. We show that the Rab7 GTPase cycle is not affected on the SCV. We then manipulated a pathway downstream of the small GTPase Rab7 in HeLa cells infected with Salmonella. Expression of the Rab7 effector RILP induces recruitment of the dynein/dynactin motor complex to the SCV. Subsequently, SCV fuse with lysosomes. As a result, the intracellular replication of Salmonella is inhibited. Activation of dynein-mediated vesicle transport can thus control intracellular survival of Salmonella.
Rab5 regulates endocytic membrane traffic by specifically recruiting cytosolic effector proteins to their site of action on early endosomal membranes. We have characterized a new Rab5 effector complex involved in endosomal fusion events. This complex includes a novel protein, Rabenosyn-5, which, like the previously characterized Rab5 effector early endosome antigen 1 (EEA1), contains an FYVE finger domain and is recruited in a phosphatidylinositol-3-kinase–dependent fashion to early endosomes. Rabenosyn-5 is complexed to the Sec1-like protein hVPS45. hVPS45 does not interact directly with Rab5, therefore Rabenosyn-5 serves as a molecular link between hVPS45 and the Rab5 GTPase. This property suggests that Rabenosyn-5 is a closer mammalian functional homologue of yeast Vac1p than EEA1. Furthermore, although both EEA1 and Rabenosyn-5 are required for early endosomal fusion, only overexpression of Rabenosyn-5 inhibits cathepsin D processing, suggesting that the two proteins play distinct roles in endosomal trafficking. We propose that Rab5-dependent formation of membrane domains enriched in phosphatidylinositol-3-phosphate has evolved as a mechanism for the recruitment of multiple effector proteins to mammalian early endosomes, and that these domains are multifunctional, depending on the differing activities of the effector proteins recruited.
endocytosis; Rab5; hVPS45; EEA1; Rabenosyn-5
Retroviruses take advantage of cellular trafficking machineries to assemble and release new infectious particles. Rab proteins regulate specific steps in intracellular membrane trafficking by recruiting tethering, docking and fusion factors, as well as the actin- and microtubule-based motor proteins that facilitate vesicle traffic. Using virological tests and RNA interference targeting Rab proteins, we demonstrate that the late endosome-associated Rab7A is required for HIV-1 propagation. Analysis of the late steps of the HIV infection cycle shows that Rab7A regulates Env processing, the incorporation of mature Env glycoproteins into viral particles and HIV-1 infectivity. We also show that siRNA-mediated Rab7A depletion induces a BST2/Tetherin phenotype on HIV-1 release. BST2/Tetherin is a restriction factor that impedes HIV-1 release by tethering mature virus particles to the plasma membrane. Our results suggest that Rab7A contributes to the mechanism by which Vpu counteracts the restriction factor BST2/Tetherin and rescues HIV-1 release. Altogether, our results highlight new roles for a major regulator of the late endocytic pathway, Rab7A, in the late stages of the HIV-1 replication cycle.
Human immunodeficiency virus (HIV) propagation requires the assistance of host cell factors at all stages of the infection cycle. HIV exploits components of the cellular membrane sorting machinery for its assembly, budding and release. Rab GTPases are key regulators of membrane-trafficking events, including exocytosis and endocytosis, in eukaryotic cells. Here we show that the late endosome associated Rab7A plays a major role in HIV-1 replication. We find that Rab7A regulates the production of infectious HIV-1 particles at two critical stages. First, Rab7A is required for efficient Env processing and, thus, for the incorporation of mature HIV-1 envelope glycoproteins into virions. Second, Rab7A contributes to the mechanism that counteracts the restriction imposed on HIV-1 release by the cellular restriction factor BST2/Tetherin that physically tethers viral particles to the plasma membrane of infected cells. Altogether these data highlight new roles for a major player of the late endocytic pathway, Rab7A, in the late stages of the HIV-1 replication cycle.
Rab/Ypt GTPases are key regulators of membrane trafficking and together with SNARE proteins mediate selective fusion of vesicles with target compartments. A family of GTPase-activating enzymes (GAPs) specific for Rab/Ypt GTPases has been discovered, but little is known about their function and substrate specificity in vivo. Here we show that the GAP activity of Gyp1p, a yeast member of this family, is specifically required for recycling of the SNARE Snc1p and the membrane dye FM4-64, implying that inactivation of a Rab/Ypt GTPase may be necessary for recycling of membrane material. Interestingly, recycling of GFP-Snc1p in gyp1Δ cells is partially restored by reducing the activity of Ypt1p. Moreover, GFP-Snc1p accumulated intracellularly in wild-type cells expressing a GTP-locked, mutant form of Ypt1p (Ypt1p-Q67L), suggesting that GTP hydrolysis of Ypt1p is essential for recycling. Ypt6p is known to be required for the fusion of recycling vesicles to the late Golgi compartment. Interestingly, the deletions of GYP1 and YPT6 were synthetic lethal, raising the possibility that at least two distinct pathways are involved in recycling of membrane material.