Characterization of the ternary complex between Rab7, REP-1 and Rab geranylgeranyl transferase.

Geranylgeranylation is a post-translational modification of Rab GTPases that enables them to associate reversibly with intracellular membranes. Geranylgeranylation of Rab proteins is critical for their activity in controlling intracellular membrane transport. According to the currently accepted model for their action, newly synthesized Rab proteins are recruited by Rab escort protein (REP) and are presented to the Rab geranylgeranyl transferase (RabGGTase) which covalentely modifies the Rab protein with two geranylgeranyl moieties. After prenylation, the Rab protein remains in complex with REP and is delivered to the target membrane by the latter. In this work, we show that RabGGTase can form a stable complex with Rab7-REP in the absence of its lipid substrate geranylgeranyl pyrophosphate. In order to characterize this interaction, we developed three fluorescence assays reporting on the interaction of RabGGTase with the Rab7-REP complex. For this interaction we determined a Kd value of about 120 nM. Association of RabGGTase with the Rab7-REP complex occurs with a rate constant of approximately 108 M-1 x s-1. We demonstrate that the state of the nucleotide bound to Rab7 does not influence the affinity of RabGGTase for the Rab7-REP-1 complex. Finally, we address the issue of substrate specificity of RabGGTase. Titration experiments demonstrate that, in contrast with farnesyl transferase, RabGGTase does not recognize a defined C-terminal sequence motif. Experiments using Rab7 mutants in which the last 16 amino acids were either mutated or truncated revealed that the distal part of the C-terminus makes only a limited contribution to the binding affinity between RabGGTase and the Rab7-REP-1 complex. This demonstrates the functional dissimilarity between RabGGTase and geranylgeranyl transferase I and farnesyl transferase, which interact specifically with the C-terminus of their substrates. Based on these experiments, we propose that RabGGTase recognizes the overall structure arising from the association of Rab and REP and then 'scans' the flexible C-terminus to position the proximal cysteines into the active site.     

Rab GTPases, intracellular traffic and disease.

Membrane and protein traffic in the secretory and endocytic pathways is mediated by vesicular transport. Recent studies of certain key regulators of vesicular transport, the Rab GTPases, have linked Rab dysfunction to human disease. Mutations in Rab27a result in Griscelli syndrome, caused by defects in melanosome transport in melanocytes and loss of cytotoxic killing activity in Tcells. Other genetic diseases are caused by partial dysfunction of multiple Rab proteins resulting from mutations in general regulators of Rab activity; Rab escort protein-1 (choroideremia), Rab geranylgeranyl transferase (Hermansky-Pudlak syndrome) and Rab GDP dissociation inhibitor-alpha (X-linked mental retardation). In infectious diseases caused by intracellular microorganisms, the function of endocytic Rabs is altered either as part of host defences or as part of survival strategy of the pathogen. The human genome is predicted to contain 60 RAB genes, suggesting that future work could reveal further links between Rab dysfunction and disease.     

RabGEFs are a major determinant for specific Rab membrane targeting

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.     

Rab geranylgeranyl transferase. A multisubunit enzyme that prenylates GTP-binding proteins terminating in Cys-X-Cys or Cys-Cys.

Rab proteins are membrane-bound prenylated GTP-binding proteins required for the targeted movement of membrane vesicles from one organelle to another. In the current paper we have characterized and purified an enzyme that attaches geranylgeranyl residues to Rab proteins that bear the COOH-terminal sequence Cys-X-Cys (such as Rab3A) and Cys-Cys (such as Rab1A). This enzyme is designated Rab geranylgeranyl transferase (Rab GG transferase). At high salt concentrations, Rab GG transferase from rat brain cytosol separates into two components, designated A and B, both of which are required for activity. We purified Component B to apparent homogeneity and found that it contains two peptides of 60 and 38 kDa. The purified Rab GG transferase did not attach geranylgeranyl to p21H-ras-CVLL, which is prenylated by a GG transferase of the CAAX type that resembles the CAAX farnesyltransferase. Rab GG transferase was strongly inhibited by Zn2+, a cation that is absolutely required by farnesyltransferase. The Rab GG transferase was also inhibited by NaCl concentrations in excess of 100 mM. Together with previous data, the current findings indicate that mammalian cells possess at least three protein prenyltransferases (CAAX farnesyltransferase, CAAX GG transferase, and Rab GG transferase) that are specific for different classes of low molecular weight GTP-binding proteins and other proteins.     

In vitro assembly,purification, and crystallization of the rab geranylgeranyl transferase:substrate complex

Posttranslational modification with the geranygeranyl moiety is essential for the ability of Rab GTPases to control processes of membrane docking and fusion. This modification is conferred by Rab geranylgeranyltransferase (RabGGTase), which catalyzes the transfer of two 20-carbon geranylgeranyl groups from geranylgeranyl pyrophosphate onto C-terminal cysteine residues of Rab proteins. The enzyme consists of a catalytic alpha/beta heterodimer and an accessory protein termed Rab escort protein (REP-1) that delivers the newly prenylated Rab proteins to their target membrane. In order to understand the structural basis of Rab prenylation, we have investigated in vitro assembly and crystallization of the RabGGTase:REP-1:Rab complex. In order to ensure maximal stability of the ternary complex, we generated its monoprenylated form, which corresponds to a reaction intermediate and displays the highest affinity between the components. This was achieved by expressing the individual components in baculovirus and Escherichia coli systems with subsequent purification followed by in vitro monoprenylation of Rab7 with immobilized recombinant RabGGTase. Purified monoprenylated REP-1:Rab7 was complexed with recombinant RabGGTase and crystallized in hanging drops. The crystals obtained initially diffract to 8 A on an in-house X-ray source.     

A novel statin-mediated “prenylation block-and-release” assay provides insight into the membrane targeting mechanisms of small GTPases

Ras super-family small GTPases regulate diverse cellular processes such as vesicular transport and signal transduction. Critical to these activities is the ability of these proteins to target to specific intracellular membranes. To allow association with membranes Ras-related GTPases are post-translationally modified by covalent attachment of prenyl groups to conserved cysteine residues at or near their C-terminus. Here we used the HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase (HMGCR) inhibitor mevastatin to develop a ‘prenylation block-and-release’ assay that allows membrane targeting of prenylated proteins to be visualized in living cells. Using this assay we investigated the cytosol to membrane targeting of several small GTPases to compartments of the secretory and endocytic pathways. We found that all Rabs tested were targeted directly to the membrane on which they reside at steady-state and not via an intermediate location as reported for Ras and Rho proteins. However, we observed that the kinetics of cytosol to membrane targeting differed for each Rab tested. Comparison of the mevastatin sensitivity and kinetics of membrane targeting of Rab23, Rab23 prenylation motif mutants and H-Ras revealed that these parameters are strongly dependent upon the prenyl transferase with Rab geranylgeranyl transferase substrates exhibiting higher sensitivity and requiring greater time to recover from mevastatin inhibition than farnesyl transferase substrates. We propose that this assay is a useful tool to investigate the kinetics, biological functions and the mechanisms of membrane targeting of prenylated proteins.     

Rab蛋白家族在神经类疾病中的作用

细胞内膜囊泡运输是一个复杂的通路网络,Rab GTPases是膜囊泡运输的主要调节剂,通常被认为是细胞内吞和分泌系统中各种细胞器和囊泡的特异性标记和识别物。与Rab蛋白相关的轴突运输、内体运输发生障碍是造成神经退行性疾病的重要原因之一。本文主要介绍了Rab蛋白在多种神经退行性疾病病理机制中的作用机理与调控机制,同时讨论了线粒体和胶质细胞功能异常与Rab蛋白之间的关联。深入探究Rab蛋白的作用机制对人类神经性疾病的早期诊断和治疗具有潜在的指导意义。     

Semi-synthetic Rab proteins as tools for studying intermolecular interactions

Rab GTPases play a key role in the regulation of membrane traffic. Posttranslational geranylgeranylation is critical for their biological activity and is conferred by a Rab geranylgeranyl transferase (RabGGTase). To study the interactions between Rab proteins and RabGGTase, we used in vitro ligation methodology to generate a fluorescent semi-synthetic Rab7 protein. The obtained protein was functionally active and was used to demonstrate a micromolar affinity interaction of Rab7 with the RabGGTase in the absence of Rab escort protein (REP). This finding is consistent with an earlier proposed model according to which RabGGTase possesses two independent weak binding sites for REP and Rab proteins.     

Structure of doubly prenylated Ypt1:GDI complex and the mechanism of GDI-mediated Rab recycling

In eukaryotic cells Rab/Ypt GTPases represent a family of key membrane traffic controllers that associate with their targeted membranes via C-terminally conjugated geranylgeranyl groups. GDP dissociation inhibitor (GDI) is a general and essential regulator of Rab recycling that extracts prenylated Rab proteins from membranes at the end of their cycle of activity and facilitates their delivery to the donor membranes. Here, we present the structure of a complex between GDI and a doubly prenylated Rab protein. We show that one geranylgeranyl residue is deeply buried in a hydrophobic pocket formed by domain II of GDI, whereas the other lipid is more exposed to solvent and is skewed across several atoms of the first moiety. Based on structural information and biophysical measurements, we propose mechanistic and thermodynamic models for GDI and Rab escort protein-mediated interaction of RabGTPase with intracellular membranes.     

Specific Prenylation of Tomato Rab Proteins by Geranylgeranyl Type-II Transferase Requires a Conserved Cysteine-Cysteine Motif

Posttranslational isoprenylation of some small GTP-binding proteins is required for their biological activity. Rab geranylgeranyl transferase (Rab GGTase) uses geranylgeranyl pyrophosphate to modify Rab proteins, its only known substrates. Geranylgeranylation of Rabs is believed to promote their association with target membranes and interaction with other proteins. Plants, like other eukaryotes, contain Rab-like proteins that are associated with intracellular membranes. However, to our knowledge, the geranylgeranylation of Rab proteins has not yet been characterized from any plant source. This report presents an activity assay that allows the characterization of prenylation of Rab-like proteins in vitro, by protein extracts prepared from plants. Tomato Rab1 proteins and mammalian Rab1a were modified by geranylgeranyl pyrophosphate but not by farnesyl pyrophosphate. This modification required a conserved cysteine-cysteine motif. A mutant form lacking the cysteine-cysteine motif could not be modified, but inhibited the geranylgeranylation of its wild-type homolog. The tomato Rab proteins were modified in vitro by protein extract prepared from yeast, but failed to become modified when the protein extract was prepared from a yeast strain containing a mutant allele for the [alpha] subunit of yeast Rab GGTase (bet4 ts). These results demonstrate that plant cells, like other eukaryotes, contain Rab GGTase-like activity.     

Allosteric regulation of substrate binding and product release in geranylgeranyltransferase type II

GTPases of the Rab family are key components of vesicular transport in eukaryotic cells. Posttranslational attachment of geranylgeranyl moieties is essential for Rab function. Geranylgeranyltransferase type II (GGTase-II) catalyzes the modification of Rab proteins once they are in complex with their escort protein (REP). Upon completion of prenylation, REP and modified Rab leave the enzyme, enabling a new round of catalysis. We have studied the mechanism underlying substrate binding and product release in the geranylgeranylation of Rab proteins. Binding of the Rab7:REP-1 complex to GGTase-II was found to be strongly modulated by geranylgeranyl pyrophosphate (GGpp). The affinity of GGTase-II for the Rab7:REP-1 complex increases from ca. 120 nM to ca. 2 nM in the presence of GGpp. To study the effect of GGpp on interaction of the enzyme with its product, we generated semisynthetic doubly prenylated Rab7 bearing a fluorescent reporter group. Using this novel compound, we demonstrated that the affinity of doubly prenylated Rab7:REP-1 complex for GGTase-II was 2 and 18 nM in the absence and presence of GGpp, respectively. The difference in affinities originates mainly from a difference in the dissociation rates. Thus, binding of the new isoprenoid substrate molecule facilitates the product release by GGTase-II. The affinity of GGpp for the prenylated Rab7:REP-1:GGTase-II was K(d) = 22 nM, with one molecule of GGpp binding per molecule of prenylated ternary complex. We interpreted this finding as an indication that the geranylgeranyl moieties transferred to Rab protein do not occupy the GGpp binding site of the GGTase-II. In summary, these results demonstrate that GGpp acts as an allosteric activator that stabilizes the Rab7:REP-1:GGTase-II complex and triggers product release upon prenylation, preventing product inhibition of the enzyme.     

Ras (CXXX) and Rab (CC/CXC) prenylation signal sequences are unique and functionally distinct.

Rab proteins typically lack the consensus carboxyl-terminal CXXX motif that signals isoprenoid modification of Ras and other isoprenylated proteins and, instead, terminate in either CC or CXC sequences (C = cysteine, X = any amino acid). To compare the functional relationship between the Ras CXXX and the Rab CC/CXC motifs, we have generated chimeric Ras proteins terminating in Rab carboxyl-terminal CC or CXC sequences. These mutant Ras proteins were not isoprenylated in vitro or in vivo, demonstrating that the CC and CXC sequences alone are not sufficient to replace a CXXX sequence to signal Ras isoprenoid modification. Surprisingly, chimeric Ras/Rab proteins terminating in significant lengths of carboxyl-terminal sequences from Rab1b (7-139 residues), Rab2 (5-151 residues), or Rab3a (12 residues) were also not isoprenylated. These results demonstrate that the sequence requirements for isoprenoid modification of Rab proteins are more complex than the simple tetrapeptide CXXX sequence for isoprenoid modification of Ras proteins and suggest that the Rab geranylgeranyl transferase(s) requires recognition of protein conformation to signal the addition of geranylgeranyl groups. Finally, competition studies demonstrate that a common geranylgeranyl transferase activity is responsible for the modification of Rab proteins terminating in CC or CXC motifs.     

Low levels of Ypt protein prenylation cause vesicle polarization defects and thermosensitive growth that can be suppressed by genes involved in cell wall maintenance

The Rab/Ypt small G proteins are essential for intracellular vesicle trafficking in mammals and yeast. The vesicle-docking process requires that Ypt proteins are located in the vesicle membrane. C-terminal geranylgeranyl anchors mediate the membrane attachment of these proteins. The Rab escort protein (REP) is essential for the recognition of Rab/Ypt small G proteins by geranylgeranyltransferase II (GGTase II) and for their delivery to acceptor membranes. What effect an alteration in the levels of prenylated Rab/Ypt proteins has on vesicle transport or other cellular processes is so far unknown. Here, we report the characterization of a yeast REP mutant, mrs6-2, in which reduced prenylation of Ypt proteins occurs even at the permissive temperature. A shift to the restrictive temperature does not alter exponential growth during the first 3 h. The amount of Sec4p, but not Ypt1p, bound to vesicle membranes is reduced 2.5 h after the shift compared with wild-type or mrs6-2 cells incubated at 25 degrees C. In addition, vesicles fail to be polarized towards the bud and small budded binucleate cells accumulate at this time point. Growth in 1 M sorbitol or overexpression of MLC1, encoding a myosin light chain able to bind the unconventional type V myosin Myo2, or of genes involved in cell wall maintenance, such as SLG1, GFA1 and LRE1, suppresses mrs6-2 thermosensitivity. Our data suggest that, at least at high temperature, a critical minimal level of Ypt protein prenylation is required for maintaining vesicle polarization.     

Geranylgeranyl switching regulates GDI-Rab GTPase recycling

Rab GTPases, key regulators of membrane targeting and fusion, require the covalent attachment of geranylgeranyl lipids to their C terminus for function. To elucidate the role of lipid in Rab recycling, we have determined the crystal structure of Rab guanine nucleotide dissociation inhibitor (alphaGDI) in complex with a geranylgeranyl (GG) ligand (H(2)N-Cys-(S-GG)-OMe). The lipid is bound beneath the Rab binding platform in a shallow hydrophobic groove. Mutation of the binding pocket in the brain-specific alphaGDI leads to mental retardation. Strikingly, lipid binding acts through a conserved allosteric switching mechanism to promote release of the GDI-Rab[GDP] complex from the membrane.     

Inhibition of geranylgeranylation mediates sensitivity to CHOP-induced cell death of DLBCL cell lines

Prenylation is a post-translational hydrophobic modification of proteins, important for their membrane localization and biological function. The use of inhibitors of prenylation has proven to be a useful tool in the activation of apoptotic pathways in tumor cell lines. Rab geranylgeranyl transferase (Rab GGT) is responsible for the prenylation of the Rab family. Overexpression of Rab GGTbeta has been identified in CHOP refractory diffuse large B cell lymphoma (DLBCL). Using a cell line-based model for CHOP resistant DLBCL, we show that treatment with simvastatin, which inhibits protein farnesylation and geranylgeranylation, sensitizes DLBCL cells to cytotoxic treatment. Treatment with the farnesyl transferase inhibitor FTI-277 or the geranylgeranyl transferase I inhibitor GGTI-298 indicates that the reduction in cell viability was restricted to inhibition of geranylgeranylation. In addition, treatment with BMS1, a combined inhibitor of farnesyl transferase and Rab GGT, resulted in a high cytostatic effect in WSU-NHL cells, demonstrated by reduced cell viability and decreased proliferation. Co-treatment of BMS1 or GGTI-298 with CHOP showed synergistic effects with regard to markers of apoptosis. We propose that inhibition of protein geranylgeranylation together with conventional cytostatic therapy is a potential novel strategy for treating patients with CHOP refractory DLBCL.     

Dual prenylation is required for Rab protein localization and function

The majority of Rab proteins are posttranslationally modified with two geranylgeranyl lipid moieties that enable their stable association with membranes. In this study, we present evidence to demonstrate that there is a specific lipid requirement for Rab protein localization and function. Substitution of different prenyl anchors on Rab GTPases does not lead to correct function. In the case of YPT1 and SEC4, two essential Rab genes in Saccharomyces cerevisiae, alternative lipid tails cannot support life when present as the sole source of YPT1 and SEC4. Furthermore, our data suggest that double geranyl-geranyl groups are required for Rab proteins to correctly localize to their characteristic organelle membrane. We have identified a factor, Yip1p that specifically binds the di-geranylgeranylated Rab and does not interact with mono-prenylated Rab proteins. This is the first demonstration that the double prenylation modification of Rab proteins is an important feature in the function of this small GTPase family and adds specific prenylation to the already known determinants of Rab localization.     

Synthesis of a fluorescent analogue of geranylgeranyl pyrophosphate and its use in a high-throughput fluorometric assay for Rab geranylgeranyltransferase

Protein prenylation is one of the most common post-translational modifications affecting hundreds of eukaryotic proteins. Rab geranylgeranyl transferase prenylates exclusively the GTPases of Rab family, and inhibition of this enzyme induces apoptosis in cancer cells, making it an attractive anticancer target. To efficiently test for possible inhibitors of this enzyme, a robust high-throughput assay is required. Here, we present protocols for the synthesis of a fluorescent analogue of geranylgeranyl pyrophosphate NBD-FPP. We utilized this fluorescent probe to design a high-throughput fluorometric assay of Rab prenylation. This continuous fluorometric assay offers the advantage of being sensitive, cost-effective and amendable to miniaturization. The protocol includes the synthesis of the fluorescent substrate, setup of the assay, assay procedure and data analysis. The procedure for the Rab geranylgeranyl transferase (RabGGTase) plate assay depends on the number of compounds in the screen but generally can be performed within a day.     

Multiple factors contribute to inefficient prenylation of Rab27a in Rab prenylation diseases

Post-translational geranylgeranylation of Rab GT-Pases is essential for their membrane association and function as regulators of intracellular vesicular transport. The reaction is catalyzed by Rab geranylgeranyltransferase (RGGT) and is assisted by the Rab escort proteins (REP), which form stable complexes with newly synthesized GDP-bound Rabs. Two genetic diseases involve the Rab geranylgeranylation machinery: choroideremia, an X-linked retinal degeneration resulting from loss-of-function mutations in REP1, and gunmetal, a mouse model of Hermansky-Pudlak syndrome resulting from mutations in the alpha-subunit of RGGT. A small subset of Rab proteins is selectively under-prenylated in both diseases, most notably Rab27a. Here we analyze why Rab27a is selectively affected in diseases of Rab geranylgeranylation. Semi-quantitative immunoblotting suggests that mass action, i.e. the amount of Rab27a relative to other Rabs, is unlikely to be a factor as the expression level of Rab27a is similar to other Rabs not affected in these diseases. In vitro binding assays and fluorescence resonance energy transfer detected by fluorescence lifetime imaging microscopy in intact cells demonstrate that Rab27a binds equally well to both REP1 and REP2, suggesting differential affinity of Rab27a for REP isoforms is not an important factor. However, steady-state kinetic analysis of the geranylgeranylation reaction indicates that REP2-Rab27a has lower affinity for RGGT compared with REP1-Rab27a. Furthermore, we show that Rab27a has relatively low GTPase activity, presumably decreasing the affinity of the REP interaction in vivo. We suggest that the restricted phenotypes observed in these diseases result from multiple contributing factors.     

Tomato Rab1A homologs as molecular tools for studying Rab geranylgeranyl transferase in plant cells.

Rab proteins attach to membranes along the secretory pathway where they contribute to distinct steps in vesicle-mediated transport. To bind membranes, Rab proteins in fungal and animal cells must be isoprenylated by the enzyme Rab geranylgeranyl transferase (Rab GGTase). We have isolated three tomato (Lycopersicon esculentum, M.) cDNAs (LeRab 1A, B, and C) encoding Rab-like proteins and show here that all three are substrates for a Rab GGTase-like activity in plant cells. The plant enzyme is similar to mammalian Rab GGTase in that the plant activity (a) is enhanced by detergent and (b) is inhibited by mutant Rab lacking a prenylation consensus sequence. LeRab1B contains a rare prenylation target motif and was the best substrate for the plant, but not the yeast, Rab GGTase. LeRab1A, B, and C are functional homologs of the Saccharomyces cerevisiae Rab protein encoded by YPT1 and are differentially expressed in tomato. LeRab1A mRNA, but not that of LeRab1B or C, is induced by ethylene in tomato seedlings and is also upregulated in ripening fruit. The increase in LeRab1A mRNA expression in ripe fruit may be linked to increased synthesis and export of enzymes like polygalacturonase, pectin esterase, and other enzymes important in fruit softening.     

Modification of Rab5 with a photoactivatable analog of geranylgeranyl diphosphate

A photoprobe analog of geranylgeranyl diphosphate (2-diazo-3,3,3-trifluoropropionyloxy-farnesyl diphosphate or DATFP-FPP) inhibits mevalonate-dependent prenylation of in vitro translated Rab5 in rabbit reticulocyte lysate, suggesting that it competes for lipid binding to the Rab geranylgeranyl transferase. Modification of Rab5 with DATFP-FPP, demonstrated by gel mobility shift and Triton X-114 phase separation experiments, confirms that the enzyme uses the analog as a substrate. The sedimentation of DATFP-modified Rab5 as a larger mass complex on sucrose density gradients indicates that it binds to other factors in rabbit reticulocyte lysate. Most importantly, DATFP-Rab5 cross-links to these soluble factors upon exposure to UV light. Immunoprecipitation with antibodies raised against proteins known to interact with Rab5 reveals that the cross-linked complexes contain Rab escort protein and GDI-1. DATFP-Rab5 also associates with membranes in a guanosine-5'-O-(3-thiotriphosphate)-stimulated manner. However, although prenylated Rab5 can be cross-linked to two unknown membrane-associated factors by the chemical cross-linker disuccinimidyl suberate, these proteins fail to be UV cross-linked to membrane-bound DATFP-Rab5. These results strongly suggest that membrane-associated factors bind Rab5 through protein-protein interactions rather than protein-prenyl interactions. The modification of Rab5 with DATFP-FPP establishes a novel photoaffinity technique for the characterization of prenyl-binding sites.