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The small GTPase Rab5 is the key regulator of early endosomal fusion. It is post-translationally modified by covalent attachment of two geranylgeranyl (GG) chains to adjacent cysteine residues of the C-terminal hypervariable region (HVR). The GDP dissociation inhibitor (GDI) recognizes membrane-associated Rab5(GDP) and serves to release it into the cytoplasm where it is kept in a soluble state. A detailed new structural and dynamic model for human Rab5(GDP) recognition and binding with human GDI at the early endosome membrane and in its dissociated state is presented. In the cytoplasm, the GDI protein accommodates the GG chains in a transient hydrophobic binding pocket. In solution, two different binding modes of the isoprenoid chains inserted into the hydrophobic pocket of the Rab5(GDP):GDI complex can be identified. This equilibrium between the two states helps to stabilize the protein-protein complex in solution. Interprotein contacts between the Rab5 switch regions and characteristic patches of GDI residues from the Rab binding platform (RBP) and the C-terminus coordinating region (CCR) reveal insight on the formation of such a stable complex. GDI binding to membrane-anchored Rab5(GDP) is initially mediated by the solvent accessible switch regions of the Rab-specific RBP. Formation of the membrane-associated Rab5(GDP):GDI complex induces a GDI reorientation to establish additional interactions with the Rab5 HVR. These results allow to devise a detailed structural model for the process of extraction of GG-Rab5(GDP) by GDI from the membrane and the dissociation from targeting factors and effector proteins prior to GDI binding.

Rab guanosine nucleotide dissociation inhibitor (Rab-GDI) proteins are important Rab GTPase effectors, which control the cycling of small GTPases between membrane and cytosol byinteracting with the prenylated GDP bound small GTPases. However, little is known about the function of Rab-GDIs in fruit plants. In this study, we present the cloning and characterisation of a putative MiRab-GDI (GenBank accession no. KF768565 ) from Mangifera indica . The gene encoded a 444 amino acid protein with a molecular weight of 49.75 kDa and a theoretical isoelectric point of 5.48. The deduced amino acid sequence exhibited high homology with Nicotiana tabacum (92% similarity) and contained five structurally and functionally defined sequence conserved residues (SCRs). SDS–PAGE and Western blot analysis indicated that recombinant MiRab-GDI was successfully expressed in E. coli and the molecular weight of the solute protein was the same as the predicted value. Real-time RT–PCR analysis demonstrated that MiRab-GDI was ubiquitously expressed in various plant tissues and organs at different levels. The expression of MiRab-GDI was down-regulated in the early stages of fruit development and up-regulated during later stages of fruit ripening. In addition, it could be induced by various treatments such as cold, NaCl, drought, abscisic acid (ABA), salicylic acid (SA), and hydrogenperoxide (H 2 O 2 ). These results provide insights into the role of the MiRab-GDI gene in fruit ripening and abiotic stresses in the mango plants.

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.

Rab guanosine triphosphatases (GTPases) regulate vesicle trafficking in eukaryotic cells by reversibly associating with lipid membranes. Inactive Rab GTPases are maintained in the cytosol by binding to GDP-dissociation inhibitor (GDI). It is believed that specialized proteins are required to displace GDI from Rab GTPases before Rab activation by guanosine diphosphate–guanosine 5′-triphosphate (GDP-GTP) exchange factors (GEFs). Here, we found that SidM from Legionella pneumophila could act as both GEF and GDI-displacement factor (GDF) for Rab1. Rab1 released from GDI was inserted into liposomal membranes and was used as a substrate for SidM-mediated nucleotide exchange. During host cell infection, recruitment of Rab1 to Legionella -containing vacuoles depended on the GDF activity of SidM. Thus, GDF and GEF activity can be promoted by a single protein, and GDF activity can coordinate Rab1 recruitment from the GDI-bound pool.

The Ras superfamily of small GTP-binding proteins (also known as small GTPases) comprises more than 80 highly conserved proteins of the Ras, Rho, and Rab subfamilies that are involved in multiple intracellular signalling pathways. These proteins are able to function as molecular switches in the transduction of signals from membrane receptors by cycling between an inactive, GDP-bound state and an active, GTP-bound state, which can then interact with a number of different effector molecules (Fig. 1). The activity of small GTPases is regulated by three classes of regulatory proteins: guanine nucleotide exchange factors (GEFs), which catalyse the exchange of GDP for GTP, thereby activating the small GTPase; GTPase-activating proteins (GAPs), which enhance the intrinsic ability of small GTPases to hydrolyse GTP, resulting in reversion to the inactive GDP-bound state; and guanine nucleotide dissociation inhibitors (GDIs), which preferentially bind to the GDP-bound GTPases in the cytoplasm, thereby inhibiting the release of GDP and maintaining the GTPase in the inactive state [1]. GDIs have not been identified for all small GTPases, but play an important role in the control of the Rho family GTPases.

The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in cells. Here we have investigated the role of Mg(2+) cofactor in the guanine nucleotide binding and hydrolysis processes of the Rho family members, Cdc42, Rac1, and RhoA. Differing from Ras and Rab proteins, which require Mg(2+) for GDP and GTP binding, the Rho GTPases bind the nucleotides in the presence or absence of Mg(2+) similarly, with dissociation constants in the submicromolar concentration. The presence of Mg(2+), however, resulted in a marked decrease in the intrinsic dissociation rates of the nucleotides. The catalytic activity of the guanine nucleotide exchange factors (GEFs) appeared to be negatively regulated by free Mg(2+), and GEF binding to Rho GTPase resulted in a 10-fold decrease in affinity for Mg(2+), suggesting that one role of GEF is to displace bound Mg(2+) from the Rho proteins. The GDP dissociation rates of the GTPases could be further stimulated by GEF upon removal of bound Mg(2+), indicating that the GEF-catalyzed nucleotide exchange involves a Mg(2+)-independent as well as a Mg(2+)-dependent mechanism. Although Mg(2+) is not absolutely required for GTP hydrolysis by the Rho GTPases, the divalent ion apparently participates in the GTPase reaction, since the intrinsic GTP hydrolysis rates were enhanced 4-10-fold upon binding to Mg(2+), and k(cat) values of the Rho GTPase-activating protein (RhoGAP)-catalyzed reactions were significantly increased when Mg(2+) was present. Furthermore, the p50RhoGAP specificity for Cdc42 was lost in the absence of Mg(2+) cofactor. These studies directly demonstrate a role of Mg(2+) in regulating the kinetics of nucleotide binding and hydrolysis and in the GEF- and GAP-catalyzed reactions of Rho family GTPases. The results suggest that GEF facilitates nucleotide exchange by destabilizing both bound nucleotide and Mg(2+), whereas RhoGAP utilizes the Mg(2+) cofactor to achieve high catalytic efficiency and specificity.

Guanine nucleotide dissociation inhibitor (GDI) is a 55-kDa protein that functions in vesicular membrane transport to recycle Rab GTPases. We have now determined the crystal structure of bovine alpha-GDI at ultra-high resolution (1.04 A). Refinement at this resolution highlighted a region with high mobility of its main-chain residues. This corresponded to a surface loop in the primarily alpha-helical domain II at the base of alpha-GDI containing the previously uncharacterized sequence-conserved region (SCR) 3A. Site-directed mutagenesis showed that this mobile loop plays a crucial role in binding of GDI to membranes and extraction of membrane-bound Rab. This domain, referred to as the mobile effector loop, in combination with Rab-binding residues found in the multi-sheet domain I at the apex of alpha-GDI may provide flexibility for recycling of diverse Rab GTPases. We propose that conserved residues in domains I and II synergize to form the functional face of GDI, and that domain II mediates a critical step in Rab recycling during vesicle fusion.

Prenylated Rab GTPases occur in the cytosol in their GDP-bound conformations bound to a cytosolic protein termed GDP-dissociation inhibitor (GDI). Rab-GDI complexes represent a pool of active, recycling Rab proteins that can deliver Rabs to specific and distinct membrane-bound compartments. Rab delivery to cellular membranes involves release of GDI, and the membrane-associated Rab protein then exchanges its bound GDP for GTP. We report here the identification of a novel, membrane-associated protein factor that can release prenylated Rab proteins from GDI. This GDI-displacement factor (GDF) is not a guanine nucleotide exchange factor because it did not influence the intrinsic rates of nucleotide exchange by Rabs 5, 7 or 9. Rather, GDF caused the release of each of these endosomal Rabs from GDI, permitting them to exchange nucleotide at their intrinsic rates. GDF displayed the greatest catalytic rate enhancement on Rab9-GDI complexes. However, catalytic rate enhancement paralleled the potency of GDI in blocking nucleotide exchange: GDI was shown to be most potent in blocking nucleotide exchange by Rab9. The failure of GDF to act on Rab1-GDI complexes suggests that it may be specific for endosomal Rab proteins. This novel, membrane-associated activity may be part of the machinery used to localize Rabs to their correct intracellular compartments.

Membrane trafficking is regulated by small Ras-like GDP/GTP binding proteins of the Rab subfamily (Rab GTPases) that cycle between membranes and cytosol depending on their nucleotide state. The GDP dissociation inhibitor (GDI) solubilizes prenylated Rab GTPases from and shuttles them between membranes in the form of a soluble cytosolic complex. We use attenuated total reflection–Fourier transform infrared spectroscopy to directly observe extraction of Rab GTPases from model membranes by GDI. In their native form, most Rab GTPases are doubly geranylgeranylated at the C terminus to achieve localization to the membrane. We find that monogeranylgeranylated Rab35 and Rab1b reversibly bind to a negatively charged model membrane. Correct folding and GTPase activity of the membrane-bound protein can be evaluated. The dissociation kinetics depends on the C-terminal sequence and charge of the GTPases. The attenuated total reflection experiments show that GDI genuinely accelerates the intrinsic Rab membrane dissociation. The extraction process is characterized and occurs in a nucleotide-dependent manner. Furthermore, we find that phosphocholination of Rab35, which is catalyzed by the Legionella pneumophila protein AnkX, interferes with the ability of GDI to extract Rab35 from the membrane. The attenuated total reflection–Fourier transform infrared spectroscopy approach enables label-free investigation of the interaction between GDI and Rab GTPases in a membrane environment. Thereby, GDI is revealed to actively extract monogeranylgeranylated membrane-bound Rab GTPases and, thus, is not merely a solubilization factor.

Rab proteins comprise a family of small GTPases that serve a regulatory role in membrane traffic. These proteins are in part cytosolic and in part associated with the membranes of specific exocytic and endocytic organelles. Smg p25A/rab3A GDI, a cytosolic protein which inhibits the dissociation of GDP from smg p25A/rab3A, Sec4p, and rab11, has also been found to prevent association of rab3A with the membrane. In this study, we have used Madin-Darby canine kidney cells permeabilized with the bacterial toxin streptolysin O to test the general activity of rab3A GDI in modulating the membrane association of various small GTP-binding proteins. Rab3A GDP dissociation inhibitor (GDI) removed from the membrane all rab proteins we have tested and inhibited the membrane binding of in vitro translated rab proteins. However, rab3A GDI had a limited effect on the membrane association of a mutant rab5 protein which contained a farnesylated cysteine motif. Finally, we found that, although rab3A GDI resides primarily in the cytosol, it is also associated with compartments of the exocytic and endocytic pathways. Since rab3A GDI can modulate the membrane association of various rab proteins, we propose to rename it rab GDI.

BACKGROUND: Small GTPases of the Rab family can cycle between a GTP- and a GDP-bound state and also between membrane and cytosol. The latter cycle is mediated by the Guanine Nucleotide Dissociation Inhibitor GDI, which can selectively extract GDP-bound Rab proteins from donor membranes, and then reload them on target membranes. In previous studies, we found that capture of the small GTPase Rab5, a key regulator of endocytic membrane traffic, by GDI is stimulated by oxidative stress via p38MAPK, resulting in increased fluid phase endocytosis. METHODOLOGY/PRINCIPAL FINDINGS: When purifying the GDI stimulating activity we found that that it copurified with a high MW protein complex, which included p38MAPK. Here we report the identification and characterization of another component of this complex as the thioredoxin-like protein TXNL1. Our observations indicate that TXNL1 play a selective role in the regulation of fluid phase endocytosis, by controlling GDI capacity to capture Rab5. CONCLUSIONS/SIGNIFICANCE: Oxidants, which are known to cause cellular damage, can also trigger signaling pathways, in particular via members of the thioredoxin family. We propose that TXNL1 acts as an effector of oxidants or a redox sensor by converting redox changes into changes of GDI capacity to capture Rab5, which in turn modulates fluid phase endocytosis.

Rab is a family of small Ras-like GTPases regulating intracellular vesicle transport. We have previously reported that prenylated Rab acceptor or PRA1 interacts with Rab GTPases and vesicle-associated membrane protein (VAMP2). Structural prediction programs suggest that PRA1, with its two extensive hydrophobic domains, is likely to be an integral membrane protein. However, subcellular fractionation and immunocytochemical analyses indicated that PRA1 is localized both in the cytosol and tightly associated with the membrane compartment. The membrane-bound form can be partially extracted with physiological buffer and urea, suggesting that PRA1 is an extrinsic membrane protein. Deletion of the carboxyl-terminal domain resulted in a protein that behaved as an integral membrane protein, indicating that this domain plays an essential role in maintaining PRA1 in a soluble state. PRA1 can also bind weakly to GDP dissociation inhibitor (GDI), a protein involved in the solubilization of membrane-bound Rab GTPases. Addition of PRA1 inhibited the extraction of membrane-bound Rab3A by GDI, suggesting that membrane localization of Rab GTPases is dependent on the opposing action of PRA1 and GDI. The binding of Rab and VAMP2 to PRA1 is mutually exclusive such that Rab3A can displace VAMP2 in a preformed VAMP2-PRA1 complex.

Guanine nucleotide dissociation inhibitor (GDI) regulates the recycling of Rab GTPases involved in vesicle targeting and fusion. We have analyzed the requirement for conserved amino acid residues in the binding of Rab1A and the function of GDI in transport of cargo between the endoplasmic reticulum (ER) and the Golgi apparatus. Using a new approach to monitor GDI-Rab interactions based on the change in fluorescence associated with the release of methylanthraniloyl guanosine di(tri)phosphate-GDP (mGDP) from Rab, we show that residues previously implicated in the binding of the synapse-specific Rab3A, including Gln-236, Arg-240, and Thr-248, are essential for the binding of Rab1A. Mutation of each of these residues has potent effects on the ability of GDI to remove Rab1A from membranes and inhibit ER to Golgi transport in vitro. Given the sequence divergence between Rab1A and 3A (35% identity), these residues are proposed to play a general role in GDI function in the cell. In contrast, several other residues found within or flanking the Rab-binding region were found to have differential effects in the recognition and recycling of Rab1A and 3A, and therefore direct selective interaction of GDI with individual Rab proteins. Intriguingly, mutation of one residue, Arg-70, led to a reduction of Rab1A binding, failed to extract Rab1A from membranes in vitro, yet bound membranes tightly and potently inhibited ER to Golgi transport. These results provide evidence that novel membrane-associated factor(s) mediate Rab-independent GDI interaction with membranes.

Small GTP binding proteins of the rab/YPT family are essential regulators of vectorial transport in the eukaryotic cell. Members of the rab/YPT1 family are found on the cytoplasmic surface of distinct intracellular membrane compartments. Membrane attachment is facilitated by a C-terminal geranylgeranyl moiety. In this report we investigated posttranslational modification and membrane binding of the rab6 protein, a member of the rab/YPT family located on the Golgi apparatus. A set of point mutations, which simulate the GDP or GTP bound conformation, was introduced into the rab6 cDNA. The mutated cDNAs were expressed in insect cells and the ability of the protein products to undergo geranylgeranyl modification and membrane association was assessed by Triton X-114 partition and cell fractionation. We report here that the modification of rab6 in insect cells depends on protein conformation. Only the GDP bound form, but not the GTP bound form is isoprenylated and subsequently membrane bound.

TRAPPI is a multiprotein tethering complex involved in mediating the initial interaction of endoplasmic reticulum (ER)-derived vesicles at the cis-Golgi, that serves as a guanine nucleotide exchange factor (GEF) for the Rab GTPase, Ypt1p. Activation of Ypt1p by TRAPPI is required for membrane fusion, and thus the mechanism of nucleotide exchange by TRAPPI is a crucial step of regulation in vesicular transport. A minimal complex of five proteins (Bet5p, Trs23p, Trs31p, and 2 copies of Bet3p) is required for GEF activity, suggesting a complex interaction between TRAPPI and Ypt1p.The large majority of GEFs accelerate nucleotide exchange from G-proteins by inserting a glutamate finger wedge into the nucleotide binding pocket. We recently solved the crystal structure of nucleotide-free Ypt1p in complex with TRAPPI, which revealed the absence of the canonical glutamate finger and suggested a novel mechanism of GEF activity by TRAPPI.In this study, we measured the rate and equilibrium constants for nucleotide and TRAPPI binding to Ypt1p. Nucleotide binding is accelerated ∼30-fold and dissociation more than three orders of magnitude, suggesting that the nucleotide binding site is more accessible in the presence of TRAPPI. Analysis of various mutant TRAPPI complexes, a Ypt1 mutant defective in nucleotide binding, and the Mg2+-dependence of exchange do not support a glutamate finger mechanism, and favor nucleotide exchange through a novel mechanism in which TRAPPI accelerates nucleotide dissociation by opening the nucleotide binding site through interactions with switch I and II. Thermodynamic linkage analysis indicates that TRAPPI binds nucleotide-free Ypt1 with greater affinity than Ypt1 with bound nucleotide. However, this increased stability cannot account for the >1000-fold increase in the rate of nucleotide exchange, indicating that GEF activity of TRAPPI is under kinetic control.