Rabs grab motors: defining the connections between Rab GTPases and motor proteins (original) (raw)
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Rabs and their effectors: Achieving specificity in membrane traffic
Proceedings of the National Academy of Sciences, 2006
Rab proteins constitute the largest branch of the Ras GTPase superfamily. Rabs use the guanine nucleotide-dependent switch mechanism common to the superfamily to regulate each of the four major steps in membrane traffic: vesicle budding, vesicle delivery, vesicle tethering, and fusion of the vesicle membrane with that of the target compartment. These different tasks are carried out by a diverse collection of effector molecules that bind to specific Rabs in their GTP-bound state. Recent advances have not only greatly extended the number of known Rab effectors, but have also begun to define the mechanisms underlying their distinct functions. By binding to the guanine nucleotide exchange proteins that activate the Rabs certain effectors act to establish positive feedback loops that help to define and maintain tightly localized domains of activated Rab proteins, which then serve to recruit other effector molecules. Additionally, Rab cascades and Rab conversions appear to confer directio...
Rab proteins: The key regulators of intracellular vesicle transport
Experimental Cell Research, 2014
Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes.
Cellular functions of Rab GTPases at a glance
Journal of cell science, 2015
Rab GTPases control intracellular membrane traffic by recruiting specific effector proteins to restricted membranes in a GTP-dependent manner. In this Cell Science at a Glance and the accompanying poster, we highlight the regulation of Rab GTPases by proteins that control their membrane association and activation state, and provide an overview of the cellular processes that are regulated by Rab GTPases and their effectors, including protein sorting, vesicle motility and vesicle tethering. We also discuss the physiological importance of Rab GTPases and provide examples of diseases caused by their dysfunctions.
Traffic control: Rab GTPases and the regulation of interorganellar transport
News in Physiological Sciences, 2001
V iewed through a microscope, the architecture of a eukaryotic cell suggests the internal partitions of a complex building. Unlike inert walls, however, cellular membranes are highly dynamic structures. For example, the entire surface area of the plasma membrane turns over every hour in a typical cell. This turnover permits changes in the protein and lipid composition of cellular membranes and mediates transfer of soluble macromolecules among intracellular organelles. Multicellular organisms make extensive use of traffic to and from the plasma membrane to regulate systems physiology. For example, hormones are secreted by exocytosis in the afferent limb of endocrine signaling, glucose and water transporters are reversibly inserted into the plasma membrane in response to endocrine signaling, and adhesion molecules are translocated to the cell surface to mediate intercellular attachment. How eukaryotic cells maintain their structure in the face of this heavy traffic is a classic problem of cellular biology that is rapidly yielding its secrets; the identities of numerous components of the molecular machines that mediate vesicular transport are known, high resolution structures of several components are available, and the molecular physiology of these machines is now coming into focus. Rab GTPases are ubiquitous components of vesicle trafficking machines, with different Rab proteins regulating traffic between different intracellular compartments (Fig. 1). Here we review the current understanding of the structure and function of Rab proteins in relation to other components of trafficking machines.
Rab7b at the intersection of intracellular trafficking and cell migration
Communicative & Integrative Biology, 2015
Rab proteins are small GTPases essential for controlling and coordinating intracellular traffic. The small GTPase Rab7b regulates the retrograde transport from late endosomes toward the Trans-Golgi Network (TGN), and is important for the proper trafficking of several receptors such as Toll-like receptors (TLRs) and sorting receptors. We recently identified the actin motor protein myosin II as a new interaction partner for Rab7b, and found that Rab7b transport is dependent on myosin II. Interestingly, we also discovered that Rab7b influences the phosphorylation state of myosin II by controlling the activation status of the small GTPase RhoA. Consequently, Rab7b is important for the remodeling of actin filaments in processes such as stress fiber formation, cell adhesion, polarization and cell migration. Our finding that Rab7b can control actomyosin reorganization reveals yet another important role for Rab proteins, in addition to their already established role as master regulators of intracellular transport. Here we discuss our findings and speculate how they can explain the importance of Rab7b in dendritic cells (DCs).
Toward a Comprehensive Map of the Effectors of Rab GTPases
Developmental Cell, 2014
The Rab GTPases recruit peripheral membrane proteins to intracellular organelles. These Rab effectors typically mediate the motility of organelles and vesicles and contribute to the specificity of membrane traffic. However, for many Rabs, few, if any, effectors have been identified; hence, their role remains unclear. To identify Rab effectors, we used a comprehensive set of Drosophila Rabs for affinity chromatography followed by mass spectrometry to identify the proteins bound to each Rab. For many Rabs, this revealed specific interactions with Drosophila orthologs of known effectors. In addition, we found numerous Rab-specific interactions with known components of membrane traffic as well as with diverse proteins not previously linked to organelles or having no known function. We confirm over 25 interactions for Rab2,
Structural Mechanisms for Regulation of Membrane Traffic by Rab GTPases
Traffic, 2009
In all eukaryotic organisms, Rab GTPases function as critical regulators of membrane traffic, organelle biogenesis and maturation, and related cellular processes. The numerous Rab proteins have distinctive yet overlapping sub-cellular distributions throughout the endomembrane system. Intensive investigation has clarified the underlying molecular and structural mechanisms for several ubiquitous Rab proteins that control membrane traffic between tubular-vesicular organelles in the exocytic, endocytic and recycling pathways. In this review, we focus on structural insights that inform our current understanding of the organization of the Rab family as well as the mechanisms for membrane targeting and activation, interaction with effectors, deactivation, and specificity determination.
A rab protein is required for the assembly of SNARE complexes in the docking of transport vesicles
Cell, 1994
Rab proteins are generally required for transport vesicle docking. We have exploited yeast secretion mutants to demonstrate that a rab protein is required for V-SNARES and t-SNARES to assemble. The absence of the rab protein in the docking complex suggests that, in a broad sense, rab proteins participate in a reaction catalyzing SNARE complex assembly. In so doing, rab proteins could help impart an additional layer of specificity to vesicle docking. This mechanism likely involves the Secl homolog Slyl, which we identified in isolated docking complexes.
Rab proteins and endocytic trafficking: potential targets for therapeutic intervention
Advanced Drug Delivery Reviews, 2003
Rab GTPases serve as master regulators of vesicular membrane transport on both the exo-and endocytic pathways. In their active forms, rab proteins serve in cargo selection and as scaffolds for the sequential assembly of effectors requisite for vesicle budding, cytoskeletal transport, and target membrane fusion. Rab protein function is in turn tightly regulated at the level of protein expression, localization, membrane association, and activation. Alterations in the rab GTPases and associated regulatory proteins or effectors have increasingly been implicated in causing human disease. Some diseases such as those resulting in bleeding and pigmentation disorders (Griscelli syndrome), mental retardation, neuropathy (Charcot -Marie -Tooth), kidney disease (tuberous sclerosis), and blindness (choroideremia) arise from direct loss of function mutations of rab GTPases or associated regulatory molecules. In contrast, in a number of cancers (prostate, liver, breast) as well as vascular, lung, and thyroid diseases, the overexpression of select rab GTPases have been tightly correlated with disease pathogenesis. Unique therapeutic opportunities lie ahead in developing strategies that target rab proteins and modulate the endocytic pathway. D
Targeting of Rab GTPases to Cellular Membranes
Biochemical Society Transactions, 2005
Rab proteins are members of the superfamily of Ras-like small GTPases and are involved in several cellular processes relating to membrane trafficking and organelle mobility throughout the cell. Like other small GTPases, Rab proteins are initially synthesized as soluble proteins and for membrane attachment they require the addition of lipid moiety(ies) to specific residues of their polypeptide chain. Despite their welldocumented roles in regulating cellular trafficking, Rab proteins own trafficking is still poorly understood. We still need to elucidate the molecular mechanisms of their recruitment to cellular membranes and the structural determinants for their specific cellular localization. Recent results indicate that Rab cellular targeting might be Rab-dependent, and this paper briefly reviews our current knowledge of this process.
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European journal of human genetics : EJHG, 2013
Spinal muscular atrophy (SMA) is a monogenic disorder that is subdivided into four different types and caused by survival motor neuron gene 1 (SMN1) deletion. Discordant cases of SMA suggest that there exist additional severity modifying factors, apart from the SMN2 gene copy number. Here we performed the first genome-wide methylation profiling of SMA patients and healthy individuals to study the association of DNA methylation status with the severity of the SMA phenotype. We identified strong significant differences in methylation level between SMA patients and healthy controls in CpG sites close to the genes CHML, ARHGAP22, CYTSB, CDK2AP1 and SLC23A2. Interestingly, the CHML and ARHGAP22 genes are associated with the activity of Rab and Rho GTPases, which are important regulators of vesicle formation, actin dynamics, axonogenesis, processes that could be critical for SMA development. We suggest that epigenetic modifications may influence the severity of SMA and that these novel genetic positions could prove to be valuable biomarkers for the understanding of SMA pathogenesis.
Organellar Proteomics: Analysis of Pancreatic Zymogen Granule Membranes
Molecular & Cellular Proteomics, 2005
1 The abbreviations used are: ZG, zymogen granule; iTRAQ, isobaric tag for relative and absolute quantification; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TM-HMM, Transmembrane Hidden Markov Model; VAMP, vesicle-associated membrane protein; 2D, two-dimensional; GPI, glycosylphosphatidylinositol; GE, gel electrophoresis; GTP␣S, guanosine 5Ј-O-(1-thiotriphosphate).
PLoS Neglected Tropical Diseases, 2010
Background: Giardia passes through two stages during its life cycle, the trophozoite and the cyst. Cyst formation involves the synthesis of cyst wall proteins (CWPs) and the transport of CWPs into encystation-specific vesicles (ESVs). Active vesicular trafficking is essential for encystation, but the molecular machinery driving vesicular trafficking remains unknown. The Rab proteins are involved in the targeting of vesicles to several intracellular compartments through their association with cytoskeletal motor proteins.
Photoreceptor cGMP Phosphodiesterase Subunit (PDE ) Functions as a Prenyl-binding Protein
Journal of Biological Chemistry, 2004
Bovine PDE␦ was originally copurified with rod cGMP phosphodiesterase (PDE) and shown to interact with prenylated, carboxymethylated C-terminal Cys residues. Other studies showed that PDE␦ can interact with several small GTPases including Rab13, Ras, Rap, and Rho6, all of which are prenylated, as well as the Nterminal portion of retinitis pigmentosa GTPase regulator and Arl2/Arl3, which are not prenylated. We show by immunocytochemistry with a PDE␦-specific antibody that PDE␦ is present in rods and cones. We find by yeast two-hybrid screening with a PDE␦ bait that it can interact with farnesylated rhodopsin kinase (GRK1) and that prenylation is essential for this interaction. In vitro binding assays indicate that both recombinant farnesylated GRK1 and geranylgeranylated GRK7 co-precipitate with a glutathione S-transferase-PDE␦ fusion protein. Using fluorescence resonance energy transfer techniques exploiting the intrinsic tryptophan fluorescence of PDE␦ and dansylated prenyl cysteines as fluorescent ligands, we show that PDE␦ specifically binds geranylgeranyl and farnesyl moieties with a K d of 19.06 and 0.70 M, respectively. Our experiments establish that PDE␦ functions as a prenyl-binding protein interacting with multiple prenylated proteins.
PLoS Pathogens, 2009
The final step during cell division is the separation of daughter cells, a process that requires the coordinated delivery and assembly of new membrane to the cleavage furrow. While most eukaryotic cells replicate by binary fission, replication of apicomplexan parasites involves the assembly of daughters (merozoites/tachyzoites) within the mother cell, using the socalled Inner Membrane Complex (IMC) as a scaffold. After de novo synthesis of the IMC and biogenesis or segregation of new organelles, daughters bud out of the mother cell to invade new host cells. Here, we demonstrate that the final step in parasite cell division involves delivery of new plasma membrane to the daughter cells, in a process requiring functional Rab11A. Importantly, Rab11A can be found in association with Myosin-Tail-Interacting-Protein (MTIP), also known as Myosin Light Chain 1 (MLC1), a member of a 4-protein motor complex called the glideosome that is known to be crucial for parasite invasion of host cells. Ablation of Rab11A function results in daughter parasites having an incompletely formed IMC that leads to a block at a late stage of cell division. A similar defect is observed upon inducible expression of a myosin A tail-only mutant. We propose a model where Rab11A-mediated vesicular traffic driven by an MTIP-Myosin motor is necessary for IMC maturation and to deliver new plasma membrane to daughter cells in order to complete cell division.
2016
The innovation of the eukaryote cytoskeleton enabled phagocytosis, intracellular transport and cytokinesis, and is responsible for diverse eukaryotic morphologies. Still, the relationship between phenotypic innovations in the cytoskeleton and their underlying genotype is poorly understood. To explore the genetic mechanism of morphological evolution of the eukaryotic cytoskeleton we provide the first single cell transcriptomes from uncultivable, free-living unicellular eukaryotes: the radiolarian speciesLithomelissa setosaandSticholonche zanclea. Analysis of the genetic components of the cytoskeleton and mapping of the evolution of these to a revised phylogeny of Rhizaria reveals lineage-specific gene duplications and neo-functionalization of α and β tubulin in Retaria, actin in Retaria and Endomyxa, and Arp2/3 complex genes in Chlorarachniophyta. We show how genetic innovations have shaped cytoskeletal structures in Rhizaria, and how single cell transcriptomics can be applied for re...
Melanosomes on the move: a model to understand organelle dynamics
Biochemical Society Transactions, 2011
Advances in live-cell microscopy have revealed the extraordinarily dynamic nature of intracellular organelles. Moreover, movement appears to be critical in establishing and maintaining intracellular organization and organellar and cellular function. Motility is regulated by the activity of organelle-associated motor proteins, kinesins, dyneins and myosins, which move cargo along polar MT (microtubule) and actin tracks. However, in most instances, the motors that move specific organelles remain mysterious. Over recent years, pigment granules, or melanosomes, within pigment cells have provided an excellent model for understanding the molecular mechanisms by which motor proteins associate with and move intracellular organelles. In the present paper, we discuss recent discoveries that shed light on the mechanisms of melanosome transport and highlight future prospects for the use of pigment cells in unravelling general molecular mechanisms of intracellular transport.
Molecular mechanisms of membrane polarity in renal epithelial cells
Reviews of Physiology, Biochemistry and Pharmacology
Exciting discoveries in the last decade have cast light onto the fundamental mechanisms that underlie polarized trafficking in epithelial cells. It is now clear that epithelial cell membrane asymmetry is achieved by a combination of intracellular sorting operations, vectorial delivery mechanisms and plasmalemma-specific fusion and retention processes. Several well-defined signals that specify polarized segregation, sorting, or retention processes have, now, been described in a number of proteins. The intracellular machineries that decode and act on these signals are beginning to be described. In addition, the nature of the molecules that associate with intracellular trafficking vesicles to coordinate polarized delivery, tethering, docking, and fusion are also becoming understood. Combined with direct visualization of polarized sorting processes with new technologies in live-cell fluorescent microscopy, new and surprising insights into these once-elusive trafficking processes are emerging. Here we provide a review of these recent advances within an historically relevant context. Abbreviations AEE: Apical Early Endosomes • AP: Adaptins or clathrin-adaptor complexes • ARF: ADP-ribosylation factor, Ras-like small G-proteins involved in vesicular trafficking • BFA: Brefeldin A, a fungal metabolite that inhibits ARF-dependent attachment of coat proteins • BEE: Basolateral Early Endosomes • CASK: Calcium/calmodulin-dependent protein kinase, MAGUK protein, also known as Lin-2 • CRE: Common Recycling Endosome • DRM: Detergent Resistant Membrane, a biochemical hallmark of a raft • ECM: Extracellular Matrix • EEA1: Early Endosome Antigen 1, a Rab effector and a marker for the early endosome • FYVE: Zinc-binding domain named after the proteins that it is found in; targets proteins to membrane lipids via interaction with phosphatidylinositol-3-phosphate, "PI3P finger protein" • GFP:
Retrograde traffic in the biosynthetic-secretory route
Histochemistry and Cell Biology, 2008
In the biosynthetic-secretory route from the rough endoplasmic reticulum, across the pre-Golgi intermediate compartments, the Golgi apparatus stacks, trans Golgi network, and post-Golgi organelles, anterograde transport is accompanied and counterbalanced by retrograde traYc of both membranes and contents. In the physiologic dynamics of cells, retrograde Xow is necessary for retrieval of molecules that escaped from their compartments of function, for keeping the compartments' balances, and maintenance of the functional integrities of organelles and compartments along the secretory route, for repeated use of molecules, and molecule repair. Internalized molecules may be transported in retrograde direction along certain sections of the secretory route, and compartments and machineries of the secretory pathway may be misused by toxins. An important example is the toxin of Shigella dysenteriae, which has been shown to travel from the cell surface across endosomes, and the Golgi apparatus en route to the endoplasmic reticulum, and the cytosol, where it exerts its deleterious eVects. Most importantly in medical research, knowledge about the retrograde cellular pathways is increasingly being utilized for the development of strategies for targeted delivery of drugs to the interior of cells. Multiple details about the molecular transport machineries involved in retrograde traYc are known; a high number of the molecular constituents have been characterized, and the complicated Wne structural architectures of the compartments involved become more and more visible. However, multiple contradictions exist, and already established traYc models again are in question by contradictory results obtained with diverse cell systems, and/or diVerent techniques. Additional problems arise by the fact that the conditions used in the experimental protocols frequently do not reXect the physiologic situations of the cells. Regular and pathologic situations often are intermingled, and experimental treatments by themselves change cell organizations. This review addresses physiologic and pathologic situations, tries to correlate results obtained by diVerent cell biologic techniques, and asks questions, which may be the basis and starting point for further investigations.
Involvement of Myosin Vb in Glutamate Receptor Trafficking
Journal of Biological Chemistry, 2005
Myosin V motors mediate cargo transport; however, the identity of neuronal molecules transported by these proteins remains unknown. Here we show that myosin Vb is expressed in several neuronal populations and associates with the ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type glutamate receptor subunit GluR1. In developing hippocampal neurons, expression of the tail domain of myosin Vb, but not myosin Va, enhanced GluR1 accumulation in the soma and reduced its surface expression. These changes were accompanied by reduced GluR1 clustering and diminished frequency of excitatory but not inhibitory synaptic currents. Similar effects were observed upon expression of full-length myosin Vb lacking a C-terminal region required for binding to the small GTPase Rab11. In contrast, mutant myosin Vb did not change the localization of several other neurotransmitter receptors, including the glutamate receptor subunit NR1. These results reveal a novel mechanism for the transport of a specific glutamate receptor subunit in neurons mediated by a member of the myosin V family. Proper sorting and transport of excitatory neurotransmitter receptors and associated proteins is essential for neuronal activity and plasticity. Recent studies have identified several proteins that regulate clustering of neurotransmitter receptors at the synapse (1). However, it remains unknown what proteins mediate sorting and delivery of receptors from the soma to postsynaptic sites. Molecular motors that regulate cargo trafficking on both actin filaments and microtubules have been implicated in initial transport and delivery to specific subcellular sites (2, 3). In particular, class V of unconventional myosins is actin-based motors thought to regulate trafficking of organelles and associated proteins in neuronal cells . Three known members of the myosin V family have been detected in brain extracts. The most studied member, myosin Va, is widely expressed in the brain (5). Dilute mice, which possess mutation in the myosin Va gene, suffer from impaired melanosome transport and severe seizures and die within 2-3 weeks after birth (6). These observations suggest that alteration in the transport of important yet unknown cargos contributed to the observed defects in neuronal function. In neu-
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Coordinated recruitment of Spir actin nucleators and myosin V motors to Rab11 vesicle membranes
eLife, 2016
There is growing evidence for a coupling of actin assembly and myosin motor activity in cells. However, mechanisms for recruitment of actin nucleators and motors on specific membrane compartments remain unclear. Here we report how Spir actin nucleators and myosin V motors coordinate their specific membrane recruitment. The myosin V globular tail domain (MyoV-GTD) interacts directly with an evolutionarily conserved Spir sequence motif. We determined crystal structures of MyoVa-GTD bound either to the Spir-2 motif or to Rab11 and show that a Spir-2:MyoVa:Rab11 complex can form. The ternary complex architecture explains how Rab11 vesicles support coordinated F-actin nucleation and myosin force generation for vesicle transport and tethering. New insights are also provided into how myosin activation can be coupled with the generation of actin tracks. Since MyoV binds several Rab GTPases, synchronized nucleator and motor targeting could provide a common mechanism to control force generati...
Journal of Biological Chemistry
Rab proteins occur in the cytosol bound to Rab-GDP dissociation inhibitor (GDI). We demonstrate here that cytosolic complexes of Rab9 bound to GDI represent a functional pool of Rab9 protein that can be utilized for transport from late endosomes to the trans Golgi network in vitro. Immunodepletion of GDI and Rab proteins bound to GDI led to the loss of cytosol activity; readdition of pure Rab9-GDI complexes fully restored cytosol activity. Delipidated serum albumin could solubilize prenylated Rab9 protein, but unlike Rab9-GDI complexes, Rab9-serum albumin complexes led to indiscriminate membrane association of Rab9 protein. Rab9 delivered to membranes by serum albumin was functional, but GDI increased the efficiency of Rab9 utilization, presumably because it suppressed Rab9 protein mistargeting. Finally, GDI inhibited transport of proteins from late endosomes to the trans Golgi network, likely because of its capacity to inhibit the membrane recruitment of cytosolic Rab9. These experiments show that GDI contributes to the selectivity of Rab9 membrane recruitment and presents functional Rab9 to the endosome-trans Golgi network transport machinery.
Current Biology, 2001
Many intracellular compartments, including MHC Results and discussion RILP requires GTP-Rab7 for clustering of late endosomes class II-containing lysosomes [1], melanosomes and lysosomes around the MTOC [2], and phagosomes [3], move along microtubules Rab7 specifically associates with late endosomal/lysoin a bidirectional manner and in a stop-and-go somal compartments [8-11] and might thus regulate motor fashion due to the alternating activities of a plusprotein recruitment to these compartments. In order to end directed kinesin motor and a minus-end identify Rab7 binding proteins involved in this process, directed dynein-dynactin motor [4]. It is largely an Epstein-Barr virus-transformed human B lymphocyte unclear how motor proteins are targeted cDNA library was screened by yeast two-hybrid assay. A specifically to different compartments. Rab GTPases protein was isolated that specifically interacted with acrecruit and/or activate several proteins involved in tive, GTP bound Rab7Q67L but not with inactive, GDP membrane fusion and vesicular transport [5, 6]. They bound Rab7T23N. The same protein (called RILP) was associate with specific compartments after isolated by Cantalupo et al. [7]. When overexpressed by activation, which makes Rab GTPases ideal nuclear microinjection of cDNA in Mel JuSo cells expresscandidates for controlling motor protein binding ing MHC class II-GFP, RILP induced a collapse of class to specific membranes. We and others [7] have II-containing lysosomal compartments (see Supplemenidentified a protein, called RILP (for Rab7tary material available with this article online). This effect interacting lysosomal protein), that interacts with could be inhibited by coexpression of dominant-negative active Rab7 on late endosomes and lysosomes. Rab7T22N (see Supplementary material). Expression of Here we show that RILP prevents further cycling of the C-terminal half of RILP (denoted ⌬N) resulted in a
Biophysical Analysis of the Interaction of Rab6a GTPase with Its Effector Domains
Journal of Biological Chemistry, 2008
Rab GTPases are key regulators of intracellular vesicular transport that control vesicle budding, cargo sorting, transport, tethering, and fusion. In the inactive (GDP-bound) conformation, Rab GTPases are targeted to intracellular compartments where they are converted into the active GTP-bound form and recruit effector domain containing proteins. Rab6a has been implicated in dynein-mediated vesicle movement at the Golgi apparatus and shown to interact with a plethora of effector proteins. In this study, we identify minimal Rab6a binding domains of three Rab6a effector proteins: PIST, BicaudalD2, and p150 glued. All three domains are >15-kDa fragments predicted to form coiled-coil structures that display no sequence homology to each other. Complex formation with BicaudalD2 and p150 has a moderate inhibitory effect on the intrinsic GTPase activity of Rab6a, while interaction with PIST has no influence on the hydrolysis rate. The effectors bind activated Rab6a with comparable affinities with K d values ranging from high nanomolar to low micromolar. Transient kinetic analysis revealed that effectors bind to Rab6a in an apparent single-step reaction characterized by relatively rapid on-and off-rates. We propose that the high off-rates of Rab6⅐effector complexes enable GTPase-activating protein-mediated net dissociation, which would not be possible if the off-rate were significantly slower. Vesicle-mediated transport between intracellular membrane-bound compartments of eukaryotic cells is essential for biosynthesis, secretion, endocytosis, cell differentiation, and growth. Transport involves sequestering of the cargo molecules into vesicles, vesicular budding, transport processes, and finally docking and fusion of vesicles at the target membrane (1). GTPases of the Rab family play a central role in the regulation of these processes by recruiting specific partner (effector) proteins (2). Like all other GTPases involved in regulatory processes, Rab proteins interconvert between active, GTP-bound forms that are capable of effector interactions, and inactive,
Regulation of Receptor Trafficking by Rab GTPases
2003
Human cells contain more than 60 Ras-like, Rab GTPases that are localized to the surfaces of distinct membrane-bound compartments. Rabs are master regulators of membrane traffic events: they can catalyze the formation of membrane microdomains, the collection of cargo into transport vesicles, the motility of vesicles along cytoskeletal tracks, the docking of vesicles with their targets, and the subsequent membrane fusion process. Rab protein function has broad implication for our understanding of human disease. For example, mutations in Rab27 lead to Griscelli syndrome due to defects in melanosome transport in melanocytes and loss of cytotoxic killing activity in T cells. Other genetic diseases are caused by loss of function of multiple Rab proteins due to mutations in common Rab regulators. My laboratory studies the Rab9 GTPase that is essential for transport of mannose 6-phosphate receptors between late endosomes and the Golgi complex. Rab9 forms a distinct domain in late endosome ...
The Rab family of proteins: 25 years on
Biochemical Society Transactions, 2012
Intracellular membrane trafficking requires the complex interplay of several classes of trafficking proteins. Rab proteins, the largest subfamily of the Ras superfamily of small G-proteins, are central regulators of all aspects of intracellular trafficking processes including vesicle budding and uncoating, motility, tethering and fusion. In the present paper, we discuss the discovery, evolution and characterization of the Rab GTPase family. We examine their basic functional roles, their important structural features and the regulatory proteins which mediate Rab function. We speculate on outstanding issues in the field, such as the mechanisms of Rab membrane association and the co-ordinated interplay between distinct Rab proteins. Finally, we summarize the data implicating Rab proteins in an ever increasing number of diseases.
The Journal of Cell Biology, 1996
Rab8 is a small Ras-like GTPase that regulates polarized membrane transport to the basolateral membrane in epithelial cells and to the dendrites in neurons. It has recently been demonstrated that fibroblasts sort newly synthesized proteins into two different pathways for delivery to the cell surface that are equivalent to the apical and the basolateral post-Golgi routes in epithelial cells (Yoshimori, T., P. Keller, M.G. Roth, and K. Simons. 1996. J. Cell Biol. 133:247-256). To determine the role of Rab8 in fibroblasts, we used both transient expression systems and stable cell lines expressing mutant or wild-type (wt) Rab8. A dramatic change in cell morphology occurred in BHK cells expressing both the wt Rab8 and the activated form of the GTPase, the Rab8Q67L mutant. These cells formed processes as a result of a reorganization of both their actin filaments and microtubules. Newly synthesized vesicular stomatitis virus G glycoprotein, a basolateral marker protein in MDCK cells, was preferentially delivered into these cell outgrowths. Based on these findings, we propose that Rab8 provides a link between the machinery responsible for the formation of cell protrusions and polarized biosynthetic membrane traffic.
Role of Rab Proteins in Epithelial Membrane Traffic
International Review of Cytology, 2003
Small GTPase rab proteins play an important role in various aspects of membrane traffic, including cargo selection, vesicle budding, vesicle motility, tethering, docking, and fusion. Recent data suggest also that rabs, and their divalent effector proteins, organize organelle subdomains and as such may define functional organelle identity. Most rabs are ubiquitously expressed. However, some rabs are preferentially expressed in epithelial cells where they appear intimately associated with the epithelial-specific transcytotic pathway and/or tight junctions. This review discusses the role of rabs in epithelial membrane transport.
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