Search

RAB GTPases interact with specific effector molecules in a spatiotemporally regulated manner to induce various downstream reactions. To clarify the overall picture of RAB GTPase functions, it is important to elucidate the cellular locale where RAB and its effectors interact. Here, we applied a bimolecular fluorescence complementation (BiFC) assay to analyze where RAB GTPase interacted with effectors in endosomal trafficking.

Specific recognition of the cargo that molecular motors transport or tether to cytoskeleton tracks allows them to perform precise cellular functions at particular times and positions in cells. However, very little is known about how evolution has favored conservation of functions for some isoforms, while also allowing for the generation of new recognition sites and specialized cellular functions. Here we present several crystal structures of the myosin Va or the myosin Vb globular tail domain (GTD) that gives insights into how the motor is linked to the recycling membrane compartments via Rab11 or to the melanosome membrane via recognition of the melanophilin adaptor that binds to Rab27a. The structures illustrate how the Rab11-binding site has been conserved during evolution and how divergence at another site of the GTD allows more specific interactions such as the specific recognition of melanophilin by the myosin Va isoform. With atomic structural insights, these structures also show how either the partner or the GTD structural plasticity upon association is critical for selective recruitment of the motor.

The small GTPase Rab7 is a key regulator of endosomal maturation in eukaryotic cells. Mutations in rab7 are thought to cause the dominant neuropathy Charcot-Marie-Tooth 2B (CMT2B) by a gain-of-function mechanism. Here we show that loss of rab7, but not overexpression of rab7 CMT2B mutants, causes adult-onset neurodegeneration in a Drosophila model. All CMT2B mutant proteins retain 10-50% function based on quantitative imaging, electrophysiology, and rescue experiments in sensory and motor neurons in vivo. Consequently, expression of CMT2B mutants at levels between 0.5 and 10-fold their endogenous levels fully rescues the neuropathy-like phenotypes of the rab7 mutant. Live imaging reveals that CMT2B proteins are inefficiently recruited to endosomes, but do not impair endosomal maturation. These findings are not consistent with a gain-of-function mechanism. Instead, they indicate a dosage-dependent sensitivity of neurons to rab7-dependent degradation. Our results suggest a therapeutic approach opposite to the currently proposed reduction of mutant protein function. DOI: http://dx.doi.org/10.7554/eLife.01064.001.

Rab small GTPases are the master regulators of intracellular trafficking in eukaryotes. They mediate spatial and temporal recruitment of effector proteins to distinct cellular compartments through GTP-induced changes in their conformation. Despite numerous structural studies, the molecular basis for Rab/effector specificity and subsequent biological activity remains poorly understood. Rab25, also known as Rab11c, which is epithelial-specific, has been heavily implicated in ovarian cancer development and independently appears to act as a tumour suppressor in the context of a distinct subset of carcinomas. Here, we show that Rab25 associates with FIP2 and can recruit this effector protein to endosomal membranes. We report the crystal structure of Rab25 in complex with the C-terminal region of FIP2, which consists of a central dimeric FIP2 coiled-coil that mediates a heterotetrameric Rab25-(FIP2)2-Rab25 complex. Thermodynamic analyses show that, despite a relatively conserved interface, FIP2 binds to Rab25 with an approximate 3-fold weaker affinity than to Rab11a. Reduced affinity is mainly associated with lower enthalpic gains for Rab25:FIP2 complex formation, and can be attributed to subtle differences in the conformations of switch 1 and switch 2. These cellular, structural and thermodynamic studies provide insight into the Rab11/Rab25 subfamily of small GTPases that regulate endosomal trafficking pathways in eukaryotes., (© 2013.)

Small G-proteins of the Ras superfamily control the temporal and spatial coordination of intracellular signaling networks by acting as molecular on/off switches. Guanine nucleotide exchange factors (GEFs) regulate the activation of these G-proteins through catalytic replacement of GDP by GTP. During nucleotide exchange, three distinct substrate·enzyme complexes occur: a ternary complex with GDP at the start of the reaction (G-protein·GEF·GDP), an intermediary nucleotide-free binary complex (G-protein·GEF), and a ternary GTP complex after productive G-protein activation (G-protein·GEF·GTP). Here, we show structural snapshots of the full nucleotide exchange reaction sequence together with the G-protein substrates and products using Rabin8/GRAB (GEF) and Rab8 (G-protein) as a model system. Together with a thorough enzymatic characterization, our data provide a detailed view into the mechanism of Rabin8/GRAB-mediated nucleotide exchange.

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., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Clathrin-independent endocytosis (CIE) mediates the internalization of many plasma membrane (PM) proteins involved in homeostasis, immune response, and signaling. CIE cargo molecules are internalized independent of clathrin, and dynamin, and modulated by the small G protein Arf6. After internalization the CIE cargo proteins either follow a default pathway of trafficking to lysosomes for degradation or follow a pathway where they are routed directly to the recycling endosomes for return to the PM. The selective endosomal sorting of molecules like CD44, CD98, and CD147, which are involved in cell-cell and cell-extracellular interactions, indicates that sorting mechanisms dictate the post-endocytic fate of CIE cargo proteins. In a recent study, we identified sorting signals that specify the endosomal trafficking of CIE cargo proteins and uncover a role for Hook1 as an endosomal cargo adaptor that routes CIE cargo to the recycling endosomes. Furthermore, we found that Hook1, microtubules, and Rab22a work in coordination to directly recycle the cargo and facilitate cell spreading. Here, we discuss our current view on the endosomal sorting of CIE cargo proteins and their molecular regulators.

GTPase activating proteins (GAPs) from pathogenic bacteria and eukaryotic host organisms deactivate Rab GTPases by supplying catalytic arginine and glutamine fingers in trans and utilizing the cis-glutamine in the DXXGQ motif of the GTPase for binding rather than catalysis. Here, we report the transition state mimetic structure of the Legionella pneumophila GAP LepB in complex with Rab1 and describe a comprehensive structure-based mutational analysis of potential catalytic and recognition determinants. The results demonstrate that LepB does not simply mimic other GAPs but instead deploys an expected arginine finger in conjunction with a novel glutamic acid finger, which forms a salt bridge with an indispensible switch II arginine that effectively locks the cis-glutamine in the DXXGQ motif of Rab1 in a catalytically competent though unprecedented transition state configuration. Surprisingly, a heretofore universal transition state interaction with the cis-glutamine is supplanted by an elaborate polar network involving critical P-loop and switch I serines. LepB further employs an unusual tandem domain architecture to clamp a switch I tyrosine in an open conformation that facilitates access of the arginine finger to the hydrolytic site. Intriguingly, the critical P-loop serine corresponds to an oncogenic substitution in Ras and replaces a conserved glycine essential for the canonical transition state stereochemistry. In addition to expanding GTP hydrolytic paradigms, these observations reveal the unconventional dual finger and non-canonical catalytic network mechanisms of Rab GAPs as necessary alternative solutions to a major impediment imposed by substitution of the conserved P-loop glycine.

Arf and Rab proteins, members of small GTPases superfamily, localize to specific subcellular compartments and regulate intracellular trafficking. To carry out their cellular functions, Arfs/Rabs interact with numerous and structurally diverse effector proteins. Over the years, a number of Arf/Rab:effector complexes have been crystallized and their structures reveal shared binding modes including α-helical packing, β-β complementation, and heterotetrameric assemblies. We review available structural information and provide a framework for in-depth analysis of complexes. The unifying features that we identify are organized into a classification scheme for different modes of Arf/Rab:effector interactions, which includes "all-α-helical," "mixed α-helical," "β-β zipping," and "bivalent" modes of binding. Additionally, we highlight structural determinants that are the basis of effector specificity. We conclude by expanding on functional implications that are emerging from available structural information under our proposed classification scheme., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: The 60(+) members of the mammalian Rab protein family group into subfamilies postulated to share common functionality. The Rab VI subfamily contains 5 Rab proteins, Rab6a/a', Rab6b, Rab6c and Rab41. High-level knockdown of Rab6a/a' has little effect on the tightly organized Golgi ribbon in HeLa cells as seen by fluorescence microscopy. In striking contrast, we found Rab41 was strongly required for normal Golgi ribbon organization., Methods/results: Treatment of HeLa cells with Rab41 siRNAs scattered the Golgi ribbon into clustered, punctate Golgi elements. Overexpression of GDP-locked Rab41, but not wild type or GTP-locked Rab41, produced a similar Golgi phenotype. By electron microscopy, Rab41 depletion produced short, isolated Golgi stacks. Golgi-associated vesicles accumulated. At low expression levels, wild type and GTP-locked Rab41 showed little concentration in the Golgi region, but puncta were observed and most were in ruffled regions at the cell periphery. There was 25% co-localization of GTP-locked Rab41 with the ER marker, Sec61p. GDP-locked Rab41, as expected, displayed an entirely diffuse cytoplasmic distribution. Depletion of Rab41 or overexpression of GDP-locked Rab41 partially inhibited ER-to-Golgi transport of VSV-G protein. However, Rab41 knockdown had little, if any, effect on endosome-to-Golgi transport of SLTB. Additionally, after a 2-day delay, treatment with Rab41 siRNA inhibited cell growth, while overexpression of GDP-locked Rab41, but not wild type or GTP-locked Rab41, produced a rapid, progressive cell loss. In double knockdown experiments with Rab6, the Golgi ribbon was fragmented, a result consistent with Rab41 and Rab6 acting in parallel., Conclusion: We provide the first evidence for distinctive Rab41 effects on Golgi organization, ER-to-Golgi trafficking and cell growth. When combined with the evidence that Rab6a/a' and Rab6b have diverse roles in Golgi function, while Rab6c regulates mitotic function, our data indicate that Rab VI subfamily members, although related by homology and structure, share limited functional conservation.

A fundamental question in cell biology is how cells determine membrane compartment identity and the directionality with which cargoes pass through the secretory and endocytic pathways. The discovery of so-called 'Rab cascades' provides a satisfying molecular mechanism that helps to resolve this paradox. One Rab GTPase has the ability to template the localization of the subsequent acting Rab GTPase along a given transport pathway. Thus, in addition to determining compartment identity and functionality, Rab GTPases are likely able to order the events of membrane trafficking. This review will highlight recent advances in our understanding of Rabs and Rab cascades., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Disruption of vacuolar biogenesis in the pathogenic yeast Candida albicans causes profound defects in polarized hyphal growth. However, the precise vacuolar pathways involved in yeast-hypha differentiation have not been determined. Previously we focused on Vps21p, a Rab GTPase involved in directing vacuolar trafficking through the late endosomal prevacuolar compartment (PVC). Herein, we identify two additional Vps21p-related GTPases, Ypt52p and Ypt53p, that colocalize with Vps21p and can suppress the hyphal defects of the vps21Δ/Δ mutant. Phenotypic analysis of gene deletion strains revealed that loss of both VPS21 and YPT52 causes synthetic defects in endocytic trafficking to the vacuole, as well as delivery of the virulence-associated vacuolar membrane protein Mlt1p from the Golgi compartment. Transcription of all three GTPase-encoding genes is increased under hyphal growth conditions, and overexpression of the transcription factor Ume6p is sufficient to increase the transcription of these genes. While only the vps21Δ/Δ single mutant has hyphal growth defects, these were greatly exacerbated in a vps21Δ/Δ ypt52Δ/Δ double mutant. On the basis of relative expression levels and phenotypic analysis of gene deletion strains, Vps21p is the most important of the three GTPases, followed by Ypt52p, while Ypt53p has an only marginal impact on C. albicans physiology. Finally, disruption of a nonendosomal AP-3-dependent vacuolar trafficking pathway in the vps21Δ/Δ ypt52Δ/Δ mutant, further exacerbated the stress and hyphal growth defects. These findings underscore the importance of membrane trafficking through the PVC in sustaining the invasive hyphal growth form of C. albicans.

Warburg Micro syndrome and Martsolf syndrome (MS) are heterogeneous autosomal-recessive developmental disorders characterized by brain, eye, and endocrine abnormalities. Causative biallelic germline mutations have been identified in RAB3GAP1, RAB3GAP2, or RAB18, each of which encode proteins involved in membrane trafficking. This report provides an up to date overview of all known disease variants identified in 29 previously published families and 52 new families. One-hundred and forty-four Micro and nine Martsolf families were investigated, identifying mutations in RAB3GAP1 in 41% of cases, mutations in RAB3GAP2 in 7% of cases, and mutations in RAB18 in 5% of cases. These are listed in Leiden Open source Variation Databases, which was created by us for all three genes. Genotype-phenotype correlations for these genes have now established that the clinical phenotypes in Micro syndrome and MS represent a phenotypic continuum related to the nature and severity of the mutations present in the disease genes, with more deleterious mutations causing Micro syndrome and milder mutations causing MS. RAB18 has not yet been linked to the RAB3 pathways, but mutations in all three genes cause an indistinguishable phenotype, making it likely that there is some overlap. There is considerable genetic heterogeneity for these disorders and further gene identification will help delineate these pathways., (© 2013 Wiley Periodicals, Inc.)

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.

The cytoplasm of a eukaryotic cell consists of a wide variety of membrane bound cell organelles and continuous flow of proteins amongst these organelles is a major challenge and must be stringently maintained in order to continue the correct biochemical functioning inside a cell. The transportation of various proteins amongst these organelles is facilitated by a vast Tubulo-vesicular network mediated by carrier proteins. The Rabs belong to small G proteins super family involved in the regulation and vesicle transport in between the organelles by shuttling between the active GTP and inactive GDP bound states. In this paper we put forth the homology modeling and docking studies of Rab6A proteins (Mus musculus, Gallus gallus and Caenorhabditis elegans) with GTP, GMP-PNP and GDP molecules and a comparative study between these proteins is done to identify key residues out of which serine of the phosphate binding loop (P - loop) and aspartic acid showed prominent interactions with the GTP, GDP and GMP-PNP nucleotides and cogitate that aspartic acid might also help in the stabilization of the switch I region of the Rab proteins besides serine.

The Rab GTPase Ypt11 is a Myo2-binding protein implicated in mother-to-bud transport of the cortical endoplasmic reticulum (ER), late Golgi, and mitochondria during yeast division. However, its reported subcellular localization does not reflect all of these functions. Here we show that Ypt11 is normally a low-abundance protein whose ER localization is only detected when the protein is highly overexpressed. Although it has been suggested that ER-localized Ypt11 and ER-mitochondrial contact sites might mediate passive transport of mitochondria into the bud, we found that mitochondrial, but not ER, association is essential for Ypt11 function in mitochondrial inheritance. Our studies also reveal that Ypt11 function is regulated at multiple levels. In addition to membrane targeting and GTPase domain-dependent effector interactions, the abundance of active Ypt11 forms is controlled by phosphorylation status and degradation. We present a model that synthesizes these new features of Ypt11 function and regulation in mitochondrial inheritance.

Membrane fusion along the endocytic pathway occurs in a sequence of tethering, docking, and fusion. At endosomes and vacuoles, the CORVET (class C core vacuole/endosome tethering) and HOPS (homotypic fusion and vacuole protein sorting) tethering complexes require their organelle-specific Rabs for localization and function. Until now, despite the absence of experimental evidence, it has been assumed that CORVET is a membrane-tethering factor. To test this theory and understand the mechanistic analogies with the HOPS complex, we set up an in vitro system, and establish CORVET as a bona-fide tether for Vps21-positive endosome/vacuole membranes. Purified CORVET binds to SNAREs and Rab5/Vps21-GTP. We then demonstrate that purified CORVET can specifically tether Vps21-positive membranes. Tethering via CORVET is dose-dependent, stimulated by the GEF Vps9, and inhibited by Msb3, the Vps21-GAP. Moreover, CORVET supports fusion of isolated membranes containing Vps21. In agreement with its role as a tether, overexpressed CORVET drives Vps21, but not the HOPS-specific Ypt7 into contact sites between vacuoles, which likely represent vacuole-associated endosomes. We therefore conclude that CORVET is a tethering complex that promotes fusion of Rab5-positive membranes and thus facilitates receptor down-regulation and recycling at the late endosome.

Rab small GTPases regulate vesicle transport in eukaryotes by interacting with various effectors. Guanine nucleotide-exchange factor (GEF) catalyzes the transition from inactive GDP-bound Rab to active GTP-bound Rab. The existence of several GDP-bound intermediates containing the Arabidopsis thaliana Rab5 homologue ARA7 and the GEF VPS9a prior to the formation of a nucleotide-free binary complex has been proposed [Uejima et al. (2010), J. Biol. Chem. 285, 36689-36697]. During this process, VPS9a directly interacts with the β-phosphate of GDP and the P-loop lysine of ARA7 via a catalytically important aspartate finger, which promotes the release of GDP from ARA7. However, it is unclear how VPS9a removes Mg2+ from ARA7 before forming the GDP-bound ternary complex. Here, the structure of the ARA7-GDP-Ca2+-VPS9a complex is reported, in which the aspartate finger directly coordinates the divalent metal ion. Ca2+ is bound to the canonical Mg2+-binding site, coordinated by the β-phosphate of GDP and the P-loop serine of ARA7. Unexpectedly, Ca2+ is further coordinated by the aspartate finger and the main chain of VPS9a. This structure may represent the earliest intermediate step in the GEF-catalyzed nucleotide-exchange reaction of ARA7 before the metal-free GDP-bound intermediates are created.

White spot disease is a devastating disease of shrimp Penaeus monodon in which the shrimp receptor protein PmRab7 interacts with viral envelop protein VP28 to form PmRab7-VP28 complex, which causes initiation of the disease. The molecular mechanism implicated in the disease, the dynamic behavior of proteins as well as interaction between both the biological counterparts that crafts a micro-environment feasible for entry of virus into the shrimp is still unknown. In the present study, we applied molecular modeling (MM), molecular dynamics (MD) and docking to compute surface mapping of infective amino acid residues between interacting proteins. Our result showed that α-helix of PmRab7 (encompassing Ser74, Ile143, Thr184, Arg53, Asn144, Thr184, Arg53, Arg79) interacts with β-sheets of VP28 (containing Ser74, Ile143, Thr184, Arg53, Asn144, Thr184, Arg53, Arg79) and Arg69-Ser74, Val75-Ile143, Leu73-Ile143, Arg79-Asn144, Ala198-Ala182 bonds contributed in the formation of PmRab7-VP28 complex. Further studies on the amino acid residues and bonds may open new possibilities for preventing PmRab7-VP28 complex formation, thus reducing chances of WSD. The quantitative predictions provide a scope for experimental testing in future as well as endow with a straightforward evidence to comprehend cellular mechanisms underlying the disease.

The biogenesis of peroxisomes is mediated by peroxins (PEXs). PEX7 is a cytosolic receptor that imports peroxisomal targeting signal type 2 (PTS2)-containing proteins. Although PEX7 is important for protein transport, the mechanisms that mediate its function are unknown. In this study, we performed proteomic analysis to identify PEX7-binding proteins using transgenic Arabidopsis expressing green fluorescent protein (GFP)-tagged PEX7. Our analysis identified RabE1c, a small GTPase, as a PEX7 binding partner. In vivo analysis revealed that GTP-bound RabE1c binds to PEX7 and that a subset of RabE1c localizes to peroxisomes and interacts with PEX7 on the peroxisome membrane. Unlike endogenous PEX7, which is predominantly localized to the cytosol, GFP-PEX7 accumulates abnormally on the peroxisomal membrane and induces degradation of endogenous PEX7, concomitant with a reduction in import of PTS2-containing proteins and decreased peroxisomal β-oxidation activity. Thus, GFP-PEX7 on the peroxisomal membrane exerts a dominant negative effect. Mutation of RabE1c restored endogenous PEX7 protein expression and import of PTS2-containing proteins as well as peroxisomal β-oxidation activity. Treatment with proteasome inhibitors also restored endogenous PEX7 protein levels in GFP-PEX7-expressing seedlings. Based on these findings, we conclude that RabE1c binds PEX7 and facilitates PEX7 degradation in the presence of immobile GFP-PEX7 accumulated at the membrane.

Eukaryotic cells critically depend on the correct regulation of intracellular vesicular trafficking to transport biological material. The Rab subfamily of small guanosine triphosphatases controls these processes by acting as a molecular on/off switch. To fulfill their function, active Rab proteins need to localize to intracellular membranes via posttranslationally attached geranylgeranyl lipids. Each member of the manifold Rab family localizes specifically to a distinct membrane, but it is unclear how this specific membrane recruitment is achieved. Here, we demonstrate that Rab-activating guanosine diphosphate/guanosine triphosphate exchange factors (GEFs) display the minimal targeting machinery for recruiting Rabs from the cytosol to the correct membrane using the Rab-GEF pairs Rab5A-Rabex-5, Rab1A-DrrA, and Rab8-Rabin8 as model systems. Specific mistargeting of Rabex-5/DrrA/Rabin8 to mitochondria led to catalytic recruitment of Rab5A/Rab1A/Rab8A in a time-dependent manner that required the catalytic activity of the GEF. Therefore, RabGEFs are major determinants for specific Rab membrane targeting.

Legionella pneumophila is an intracellularly surviving pathogen that releases about 270 different proteins into the host cell during infection. A set of secreted proteins takes control of the vesicular trafficking regulator Rab1. Legionella LepB inactivates Rab1 by acting as a GTPase-activating protein (GAP). We present the crystal structure of the Rab1b:LepB complex together with a thorough biochemical analysis and show that the GAP domain of LepB consists of an unusual fold. LepB acts by an atypical RabGAP mechanism that is reminiscent of classical GAPs and therefore sets the protein apart from mammalian TBC-like GAPs. Surprisingly, LepB can function as a GAP for Rab3, Rab8, Rab13 and Rab35, too, suggesting that it has a broader cellular role than previously thought.

The graph theory was combined with fluctuation dynamics to investigate the structural communication in four small G proteins, Arf1, H-Ras, RhoA, and Sec4. The topology of small GTPases is such that it requires the presence of the nucleotide to acquire a persistent structural network. The majority of communication paths involves the nucleotide and does not exist in the unbound forms. The latter are almost devoid of high-frequency paths. Thus, small Ras GTPases acquire the ability to transfer signals in the presence of nucleotide, suggesting that it modifies the intrinsic dynamics of the protein through the establishment of regions of hyperlinked nodes with high occurrence of correlated motions. The analysis of communication paths in the inactive (S(GDP)) and active (S(GTP)) states of the four G proteins strengthened the separation of the Ras-like domain into two dynamically distinct lobes, i.e. lobes 1 and 2, representing, respectively, the N-terminal and C-terminal halves of the domain. In the framework of this separation, interfunctional states and interfamily differences could be inferred. The structure network undergoes a reshaping depending on the bound nucleotide. Nucleotide-dependent divergences in structural communication reach the maximum in Arf1 and the minimum in RhoA. In Arf1, the nucleotide-dependent paths essentially express a communication between the G box 4 (G4) and distal portions of lobe 1. In the S(GDP) state, the G4 communicates with the N-term, while, in the S(GTP) state, the G4 communicates with the switch II. Clear differences could be also found between Arf1 and the other three G proteins. In Arf1, the nucleotide tends to communicate with distal portions of lobe 1, whereas in H-Ras, RhoA, and Sec4 it tends to communicate with a cluster of aromatic/hydrophobic amino acids in lobe 2. These differences may be linked, at least in part, to the divergent membrane anchoring modes that would involve the N-term for the Arf family and the C-term for the Rab/Ras/Rho families.

Rabs comprise the largest member of the Ras superfamily of small GTPases with over 60 proteins in mammals and 11 proteins in yeast. Like all small GTPases, Rabs oscillate between an inactive GDP-bound conformation and an active GTP-bound state that is tethered to lipid membranes via a C-terminal prenylation site on conserved cysteine residues. In their active state, Rabs regulate various aspects of membrane trafficking, including vesicle formation, transport, docking, and fusion. The critical element of biological activity is the recruitment of cytosolic effector proteins to specific endomembranes by active Rabs. The importance of Rabs in cellular processes is apparent from their links to genetic disorders, immunodeficiency, cancer, and pathogen invasion. During the last decade, numerous structures of complexes have shed light on the molecular basis for Rab/effector specificity and their topological organization on subcellular membranes. Here, I review the known structures of Rab/effector complexes and their modes of oligomerization. This is followed by a brief discussion on the thermodynamics of effector recruitment, which has not been documented sufficiently in previous reviews. A summary of diseases associated with Rab/effector trafficking pathways concludes this chapter., (Copyright © 2013 Elsevier Inc. All rights reserved.)

One of the main reasons of the high diversity and complexity of the human proteome compared to the human genome is the extensive work performed by the posttranslational machinery to incorporate numerous different functionalities on proteins. The covalent attachment of chemical moieties in proteins after translation is known as posttranslational modification (PTM) and has a crucial role in controlling protein localization and activity. Relevant modifications include phosphorylation, carboxymethylation, glycosylation, acetylation, or lipidation. Despite their essential role on protein function, the synthesis of fully posttranslationally modified proteins has been challenging. However, important advances on chemical biology have enabled the synthesis of fully posttranslationally modified peptides and proteins. As a result of this, peptides bearing, i.e., phosphorylated amino acids, C-terminal methylations, lipid modifications, or nonnatural tags have become accessible. These peptides, as well as the corresponding proteins obtained using ligation techniques, have been invaluable tools in biochemical and biophysical studies. As an example of these advances, this chapter describes the methods developed for the synthesis of lipidated peptides from the Ras and Rab families.

Proteins and lipids are known to be transported to targeted cytosolic compartments in vesicles. A similar system in chloroplasts is suggested to transfer lipids from the inner envelope to the thylakoids. However, little is known about both possible cargo proteins and the proteins required to build a functional vesicle transport system in chloroplasts. A few components have been suggested, but only one (CPSAR1) has a verified location in chloroplast vesicles. This protein is localized in the donor membrane (envelope) and vesicles, but not in the target membrane (thylakoids) suggesting it plays a similar role to a cytosolic homologue, Sar1, in the secretory pathway. Thus, we hypothesized that there may be more similarities, in addition to lipid transport, between the vesicle transport systems in the cytosol and chloroplast, i.e. similar vesicle transport components, possible cargo proteins and receptors. Therefore, using a bioinformatics approach we searched for putative chloroplast components in the model plant Arabidopsis thaliana, corresponding mainly to components of the cytosolic vesicle transport system that may act in coordination with previously proposed COPII chloroplast homologues. We found several additional possible components, supporting the notion of a fully functional vesicle transport system in chloroplasts. Moreover, we found motifs in thylakoid-located proteins similar to those of COPII vesicle cargo proteins, supporting the hypothesis that chloroplast vesicles may transport thylakoid proteins from the envelope to the thylakoid membrane. Several putative cargo proteins are involved in photosynthesis, thus we propose the existence of a novel thylakoid protein pathway that is important for construction and maintenance of the photosynthetic machinery.

During epithelial junctional development, both vesicle transport and reorganization of the actin cytoskeleton must be spatiotemporally regulated. Coordination of these cellular functions is especially important, but the precise mechanism remains elusive. Previously, we identified junctional Rab13-binding protein (JRAB)/molecules interacting with CasL-like 2 (MICAL-L2) as an effector of the Rab13 small G protein, and we found that the Rab13-JRAB system may be involved in the formation of cell-cell adhesions via transport of adhesion molecules. Here, we showed that JRAB interacts with two actin-binding proteins, actinin-1 and -4, and filamentous actin via different domains and regulates actin cross-linking and stabilization through these interactions. During epithelial junctional development, JRAB is prominently enriched in the actin bundle at the free border; subsequently, JRAB undergoes a Rab13-dependent conformational change that is required for maturation of cell-cell adhesion sites. These results suggest that Rab13 and JRAB regulate reorganization of the actin cytoskeleton throughout epithelial junctional development from establishment to maturation of cell-cell adhesion.

The molecular mechanisms underlying the transport from the Golgi to the cell surface of G protein-coupled receptors remain poorly elucidated. Here we determined the role of Rab26, a Ras-like small GTPase involved in vesicle-mediated secretion, in the cell surface export of α(2)-adrenergic receptors. We found that transient expression of Rab26 mutants and siRNA-mediated depletion of Rab26 significantly attenuated the cell surface numbers of α(2A)-AR and α(2B)-AR, as well as ERK1/2 activation by α(2B)-AR. Furthermore, the receptors were extensively arrested in the Golgi by Rab26 mutants and siRNA. Moreover, Rab26 directly and activation-dependently interacted with α(2B)-AR, specifically the third intracellular loop. These data demonstrate that the small GTPase Rab26 regulates the Golgi to cell surface traffic of α(2)-adrenergic receptors, likely through a physical interaction. These data also provide the first evidence implicating an important function of Rab26 in coordinating plasma membrane protein transport.

Rab proteins constitute the largest family within the Ras superfamily of small GTPases (>60 in mammals) and are essential regulators of transport between intracellular organelles. Key to this activity is their targeting to specific compartments within the cell. However, although great strides have been made over the last 25 years in assigning functions to individual Rabs and identifying their downstream effectors, the mechanism(s) regulating their targeting to specific subcellular membranes remains less well understood. In the present paper, we review the evidence supporting the proposed mechanisms of Rab targeting and highlight insights into this process provided by studies of Rab27a.

The fundamental separation of Golgi function between subcompartments termed cisternae is conserved across all eukaryotes. Likewise, Rab proteins, small GTPases of the Ras superfamily, are putative common coordinators of Golgi organization and protein transport. However, despite sequence conservation, e.g., Rab6 and Ypt6 are conserved proteins between humans and yeast, the fundamental organization of the organelle can vary profoundly. In the yeast Saccharomyces cerevisiae, the Golgi cisternae are physically separated from one another, while in mammalian cells, the cisternae are stacked one upon the other. Moreover, in mammalian cells, many Golgi stacks are typically linked together to generate a ribbon structure. Do evolutionarily conserved Rab proteins regulate secretory membrane trafficking and diverse Golgi organization in a common manner? In mammalian cells, some Golgi-associated Rab proteins function in coordination of protein transport and maintenance of Golgi organization. These include Rab6, Rab33B, Rab1, Rab2, Rab18, and Rab43. In yeast, these include Ypt1, Ypt32, and Ypt6. Here, based on evidence from both yeast and mammalian cells, we speculate on the essential role of Rab proteins in Golgi organization and protein transport.

Despite over two decades of research, the mechanism of Rab targeting to specific intracellular membranes is still not completely understood. Present evidence suggests that the original hypothesis that the message for targeting resides solely in the hypervariable C-terminus is incorrect, and a second mechanism involving a GDF [GDI (guanine-nucleotide-dissociation inhibitor) displacement factor] to disrupt stable Rab-GDI complexes has only been shown to apply in one case, despite the need for targeting over 60 human Rab proteins. Evidence for the involvement of Rab-effector interactions has only been presented for a few cases or in a very specific context. There is mounting evidence that GEFs (guanine-nucleotide-exchange factors) are essential for membrane targeting, although contributions from additional factors are likely to be of importance, at least in specific cases.

Clathrin coat accessory proteins play key roles in transport mediated by clathrin-coated vesicles. Yeast Irc6p and the related mammalian p34 are putative clathrin accessory proteins that interact with clathrin adaptor complexes. We present evidence that Irc6p functions in clathrin-mediated traffic between the trans-Golgi network and endosomes, linking clathrin adaptor complex AP-1 and the Rab GTPase Ypt31p. The crystal structure of the Irc6p N-terminal domain revealed a G-protein fold most related to small G proteins of the Rab and Arf families. However, Irc6p lacks G-protein signature motifs and high-affinity GTP binding. Also, mutant Irc6p lacking candidate GTP-binding residues retained function. Mammalian p34 rescued growth defects in irc6 cells, indicating functional conservation, and modeling predicted a similar N-terminal fold in p34. Irc6p and p34 also contain functionally conserved C-terminal regions. Irc6p/p34-related proteins with the same two-part architecture are encoded in genomes of species as diverse as plants and humans. Together these results define Irc6p/p34 as a novel type of conserved clathrin accessory protein and founding members of a new G protein-like family.

Rab6A, a member of the Ras superfamily of small G proteins, is involved in the regulation of vesicle trafficking, which is critical for endocytosis, cell differentiation and cell growth. Rab6A can exist in two isoforms termed Rab6A and Rab6A'. The substitution of Gln72 by Leu (Q72L) in the Rab6A family blocks GTP-hydrolysis activity, and this mutation usually causes the Rab6A protein to be in a constitutively active form. In this study, in order to understand the functional uniqueness of Rab6A' and the molecular mechanism of the control of activity by GTP and GDP from the crystal structure, a Rab6A'(Q72L) mutant form was overexpressed in Escherichia coli with an engineered N-terminal His tag. Rab6A'(Q72L) was then purified to homogeneity and crystallized at 293 K. X-ray diffraction data were collected to a resolution of 1.9 Å from a crystal belonging to space group P22(1)2(1) with unit-cell parameters a = 36.84, b = 96.78, c = 109.99 Å. The asymmetric unit was estimated to contain two molecules.

Caenorhabditis elegans RAB-10 and mammalian Rab10 are key regulators of endocytic recycling, especially in the basolateral recycling pathways of polarized epithelial cells. To understand better how RAB-10 contributes to recycling endosome function, we sought to identify RAB-10 effectors. One RAB-10-binding partner that we identified, CNT-1, is the only C. elegans homolog of the mammalian Arf6 GTPase-activating proteins ACAP1 and ACAP2. Arf6 is known to regulate endosome-to-plasma membrane transport, in part through activation of type I phophatidylinositol-4-phosphate 5 kinase. Here we show that CNT-1 binds to RAB-10 through its C-terminal ankyrin repeats and colocalizes with RAB-10 and ARF-6 on recycling endosomes in vivo. Furthermore, we find that RAB-10 is required for the recruitment of CNT-1 to endosomal membranes in the intestinal epithelium. Consistent with negative regulation of ARF-6 by RAB-10 and CNT-1, we found overaccumulation of endosomal phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] in cnt-1 and rab-10 mutants and reduced endosomal PI(4,5)P2 levels in arf-6 mutants. These mutants produced similar effects on endosomal recruitment of the PI(4,5)P2-dependent membrane-bending proteins RME-1/Ehd and SDPN-1/Syndapin/Pacsin and resulted in endosomal trapping of specific recycling cargo. Our studies identify a RAB-10-to-ARF-6 regulatory loop required to regulate endosomal PI(4,5)P2, a key phosphoinositide in membrane traffic.

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.

NESCA, a newly discovered signaling adapter protein in the NGF-pathway, contains a RUN domain at its N-terminus. Here we report the crystal structure of the NESCA RUN domain determined at 2.0-Å resolution. The overall fold of the NESCA RUN domain comprises nine helices, resembling the RUN domain of RPIPx and the RUN1 domain of Rab6IP1. However, compared to the other RUN domains, the RUN domain of NESCA has significantly different surface electrostatic distributions at the putative GTPase-interacting interface. We demonstrate that the RUN domain of NESCA can bind H-Ras, a downstream signaling molecule of TrkA, with high affinity. Moreover, NESCA RUN can directly interact with TrkA. These results provide new insights into how NESCA participates in the NGF-TrkA signaling pathway.

The Ras small G protein-superfamily is a family of GTP hydrolases whose activity is regulated by GTP/GDP binding states. Rab6A, a member of the Ras superfamily, is involved in the regulation of vesicle trafficking, which is critical for endocytosis, biosynthesis, secretion, cell differentiation and cell growth. Rab6A exists in two isoforms, termed RabA and Rab6A'. Substitution of Gln72 to Leu72 (Q72L) at Rab6 family blocks GTP hydrolysis activity and this mutation usually causes the Rab6 protein to be constitutively in an active form. Here, we report the crystal structure of the human Rab6A'(Q72L) mutant form at 1.9Å resolution. Unexpectedly, we found that Rab6A'(Q72L) possesses GDP/Mg(2+) in the GTP binding pockets, which is formed by a flexible switch I and switch II. Large conformational changes were also detected in the switch I and switch II regions. Our structure revealed that the non-hydrolysable, constitutively active form of Rab6A' can accommodate GDP/Mg(2+) in the open conformation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The retromer complex, composed of sorting nexin subunits and a Vps26/Vps29/Vps35 trimer, mediates sorting of retrograde cargo from the endosome to the trans-Golgi network. The retromer trimer subcomplex is an effector of Rab7 (Ypt7 in yeast). Whereas endosome targeting of human retromer has been shown to require Rab7-GTP, targeting of yeast retromer to the endosome is independent of Ypt7-GTP and requires the Vps5 and Vps17 retromer sorting nexin subunits. An evolutionarily conserved amino acid segment within Vps35 is required for Ypt7/Rab7 recognition in vivo by both yeast and human retromer, establishing that Rab recognition is a conserved feature of this subunit. Recognition of Ypt7 by retromer is required for its function in retrograde sorting, and in yeast cells lacking the guanine nucleotide exchange factor for Ypt7, retrograde cargo accumulates in endosomes that are decorated with retromer, revealing an additional role for Rab recognition at the cargo export stage of the retromer functional cycle. In addition, yeast retromer trimer antagonizes Ypt7-regulated organelle tethering and fusion of endosomes/vacuoles via recognition of Ypt7. Thus retromer has dual roles in retrograde cargo export and in controlling the fusion dynamics of the late endovacuolar system.

Membrane trafficking relies on dynamic changes in membrane identities that are determined by the regulation of distinct RAB GTPases and phosphoinositides. RABs and phosphoinositides both act to spatiotemporally recruit effectors of membrane remodelling, including sequential RAB and phosphoinositide activities. New ideas on coordinated regulation of specific RABs and phosphoinositides, achieved by direct physical and functional interactions between their regulatory enzymes, are emerging as a central mechanism to ensure precision and fidelity of membrane trafficking.

Autophagy is a bulk degradation process characterized by the formation of double membrane vesicles called autophagosomes. The exact molecular mechanism of autophagosome formation and the origin of the autophagosomal membrane remain unclear. We screened 38 human Tre-2/Bub2/Cdc16 domain-containing Rab guanosine triphosphatase-activating proteins (GAPs) and identified 11 negative regulators of starvation-induced autophagy. One of these putative RabGAPs, TBC1D14, colocalizes and interacts with the autophagy kinase ULK1. Overexpressed TBC1D14 tubulates ULK1-positive recycling endosomes (REs), impairing their function and inhibiting autophagosome formation. TBC1D14 binds activated Rab11 but is not a GAP for Rab11, and loss of Rab11 prevents TBC1D14-induced tubulation of REs. Furthermore, Rab11 is required for autophagosome formation. ULK1 and Atg9 are found on Rab11- and transferrin (Tfn) receptor (TfnR)-positive recycling endosomes. Amino acid starvation causes TBC1D14 to relocalize from REs to the Golgi complex, whereas TfnR and Tfn localize to forming autophagosomes, which are ULK1 and LC3 positive. Thus, TBC1D14- and Rab11-dependent vesicular transport from REs contributes to and regulates starvation-induced autophagy.

Intracellular vesicular trafficking is regulated by approximately 60 members of the Rab subfamily of small Ras-like GDP/GTP binding proteins. Rab proteins cycle between inactive and active states as well as between cytosolic and membrane bound forms. Membrane extraction/delivery and cytosolic distribution of Rabs is mediated by interaction with the protein GDP dissociation inhibitor (GDI) that binds to prenylated inactive (GDP-bound) Rab proteins. Because the Rab:GDP:GDI complex is of high affinity, the question arises of how GDI can be displaced efficiently from Rab protein in order to allow the necessary recruitment of the Rab to its specific target membrane. While there is strong evidence that DrrA, as a bacterially encoded GDP/GTP exchange factor, contributes to this event, we show here that posttranslational modifications of Rabs can also modulate the affinity for GDI and thus cause effective displacement of GDI from Rab:GDI complexes. These activities have been found associated with the phosphocholination and adenylylation activities of the enzymes AnkX and DrrA/SidM, respectively, from the pathogenic bacterium Legionella pneumophila. Both modifications occur after spontaneous dissociation of Rab:GDI complexes within their natural equilibrium. Therefore, the effective GDI displacement that is observed is caused by inhibition of reformation of Rab:GDI complexes. Interestingly, in contrast to adenylylation by DrrA, AnkX can covalently modify inactive Rabs with high catalytic efficiency even when GDP is bound to the GTPase and hence can inhibit binding of GDI to Rab:GDP complexes. We therefore speculate that human cells could employ similar mechanisms in the absence of infection to effectively displace Rabs from GDI.

A previous report described lipid mixing of reconstituted proteoliposomes made using lipid mixtures that mimic the composition of yeast vacuoles. This lipid mixing required SNARE {SNAP [soluble NSF (N-ethylmaleimide-sensitive factor)-attachment protein] receptor} proteins, Sec18p and Sec17p (yeast NSF and α-SNAP) and the HOPS (homotypic fusion and protein sorting)-Class C Vps (vacuole protein sorting) complex, but not the vacuolar Rab GTPase Ypt7p. The present study investigates the activity of Ypt7p in proteoliposome lipid mixing. Ypt7p is required for the lipid mixing of proteoliposomes lacking cardiolipin [1,3-bis-(sn-3'-phosphatidyl)-sn-glycerol]. Omission of other lipids with negatively charged and/or small head groups does not cause Ypt7p dependence for lipid mixing. Yeast vacuoles made from strains disrupted for CRD1 (cardiolipin synthase) fuse to the same extent as vacuoles from strains with functional CRD1. Disruption of CRD1 does not alter dependence on Rab GTPases for vacuole fusion. It has been proposed that the recruitment of the HOPS complex to membranes is the main function of Ypt7p. However, Ypt7p is still required for lipid mixing even when the concentration of HOPS complex in lipid-mixing reactions is adjusted such that cardiolipin-free proteoliposomes with or without Ypt7p bind to equal amounts of HOPS. Ypt7p therefore must stimulate membrane fusion by a mechanism that is in addition to recruitment of HOPS to the membrane. This is the first demonstration of such a stimulatory activity--that is, beyond bulk effector recruitment--for a Rab GTPase.

Mutations of the inositol-5-phosphatase OCRL1 cause Lowe syndrome. Lowe syndrome is an inherited disease characterized by renal dysfunction and impaired development of the eye and the nervous system. OCRL1 is a Rab effector protein that can bind to a large number of different Rab proteins. We have recently determined the X-ray structure of the Rab-binding domain of OCRL1 in complex with Rab8. Furthermore, we have characterized point mutations that abolish binding to Rab proteins and cause Lowe syndrome. Here we shortly review our recent biophysical and structural work and discuss possible functional implications of our finding that Rab8 binds with the highest affinity to OCRL1 among the Rab proteins tested. This could direct further work on OCRL1 leading to a better understanding of the complex disease mechanism of Lowe syndrome.

Background: The closely related GTPases Rab11 and Rab25 promote cell migration by regulating vesicular transport and recycling of surface receptors. Rab25 carries a constitutively activating mutation in its GTPase domain. Increased expression of Rab25 has been associated with the aggressiveness of migrating tumor cells. Here, we aimed to elucidate potential differences in the role of those two GTPases in vesicle trafficking during cell migration., Methods: We expressed Rab11 and Rab25 wildtype and mutant constructs in HeLa and MDA-MB231 cells and measured their effect on cell morphology, vesicle dynamics and migration behaviour. In prostate cancer samples we analyzed the expression of both GTPases., Results: Cells grown on fibronectin displayed a more stretched morphology when Rab11 was inactivated, whereas inactivation of Rab25 led to reduced stretching. Overexpression of both Rab11 and Rab25 accelerated cell migration. Analysis of vesicular movement revealed higher transport efficiency in the inner cell compartment for Rab11 positive vesicles and in proximity to the membrane for Rab25 positive vesicles. Interestingly, we found Rab25 to be highly expressed in prostate cancer tissue., Conclusion: Taken together, our data suggest that Rab11 is mainly responsible for basal long-distance transport from the rear end to the front of the migrating cell, whereas Rab25 acts predominantly in the small-scale fast recycling within the tips of the cell. Our results further support the idea of Rab25 as a promoter of tumor development., (Copyright © 2012 S. Karger AG, Basel.)

It is known that cancer progresses by vertical gene transfer, but this paradigm ignores that DNA circulates in higher organisms and that it is biologically active upon its uptake by recipient cells. Here we confirm previous observations on the ability of cell-free DNA to induce in vitro cell transformation and tumorigenesis by treating NIH3T3 recipient murine cells with serum of colon cancer patients and supernatant of SW480 human cancer cells. Cell transformation and tumorigenesis of recipient cells did not occur if serum and supernatants were depleted of DNA. It is also demonstrated that horizontal cancer progression mediated by circulating DNA occurs via its uptake by recipient cells in an in vivo model where immunocompetent rats subjected to colon carcinogenesis with 1,2-dimethylhydrazine had increased rate of colonic tumors when injected in the dorsum with human SW480 colon carcinoma cells as a source of circulating oncogenic DNA, which could be offset by treating these animals with DNAse I and proteases. Though the contribution of biologically active molecules other than DNA for this phenomenon to occur cannot be ruled out, our results support the fact that cancer cells emit into the circulation biologically active DNA to foster tumor progression. Further exploration of the horizontal tumor progression phenomenon mediated by circulating DNA is clearly needed to determine whether its manipulation could have a role in cancer therapy.

The past decade has seen a significant expansion in our understanding of membrane traffic in Tetrahymena thermophila, facilitated by the development of new experimental tools and by the availability of the macronuclear genome sequence. Here we review studies on multiple pathways of uptake and secretion, as well as work on metabolism of membrane lipids. We discuss evidence for conservation versus innovation in the mechanisms used in ciliates compared with those in other eukaryotic lineages, and raise the possibility that existing gene expression databases can be exploited to analyze specific pathways of membrane traffic in these cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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).

Eukaryotic cells have developed a diverse repertoire of Rab GTPases to regulate vesicle trafficking pathways. Together with their effector proteins, Rabs mediate various aspects of vesicle formation, tethering, docking and fusion, but details of the biological roles elicited by effectors are largely unknown. Human Rab6 is involved in the trafficking of vesicles at the level of Golgi via interactions with numerous effector proteins. We have previously determined the crystal structure of Rab6 in complex with DENND5, alternatively called Rab6IP1, which comprises two RUN domains (RUN1 and RUN2) separated by a PLAT domain. The structure of Rab6/RUN1-PLAT (Rab6/R1P) revealed the molecular basis for Golgi recruitment of DENND5 via the RUN1 domain, but the functional role of the RUN2 domain has not been well characterized. Here we show that a soluble DENND5 construct encompassing the RUN2 domain binds to the N-terminal region of sorting nexin 1 by surface plasmon resonance analyses.

Rab GTPases play a key role in the regulation of membrane trafficking. Post-translational geranylgeranylation is critical for their biological activity and is conferred by Rab geranylgeranyl transferease (RabGGTase), together with an accessory factor, Rab escort protein (REP). Mechanistic studies of Rab prenylation and identification of RabGGTase inhibitors require sensitive reporters of Rab prenylation. In the present work, a combination of protein engineering and expressed protein ligation was used to construct a library of semisynthetic Rab7 fluorescent conjugates. In order to avoid synthesis of a large number of fluorescently labeled peptides, we developed a strategy that combined thiol-reactive dye-labeling of cysteine with in vitro protein ligation. Application of this strategy required optimization of labeling and ligation conditions to promote thiol labeling and disfavor intramolecular cyclization. Using this approach, we constructed 46 fluorescent sensors with different spectral properties that reported on the interaction of Rab7 with RabGGTase, REP-1, and the overall prenylation reaction. Two constructs, Rab7Δ3CCK(NBD) and Rab7Δ2SCCC-dans, displayed 2.5- and 1.5-fold increase in fluorescence, respectively, upon prenylation. Moreover, dansyl-, NBD (4-nitro-benzofurazan)-, I-BA-, and I-SO-labeled Rab7 conjugates exhibited two- to tenfold change in fluorescence upon binding to REP or RabGGTase. These fluorescent sensors allowed us to monitor Rab prenylation in real time and to investigate the assembly of Rab-REP binary and Rab-REP-RabGGTase ternary complexes., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)