Rab GGTase Research Articles

Effects of Isoprenoid Biosynthetic Pathway Inhibition on Glucose Uptake in Myeloma Cells

Multiple myeloma (MM) is a plasma cell malignancy characterized by the synthesis and secretion of high levels of monoclonal protein (MP). MM cells are dependent on glucose consumption and GLUT transporter activity and there has been interest in targeting glucose uptake as a therapeutic strategy. Our work has shown that inhibitors of the isoprenoid biosynthetic pathway (IBP) disrupt Rab GTPase geranylgeranylation thus impairing MP trafficking in MM cells, leading to an accumulation of MP in the ER and induction of the unfolded protein response and apoptosis. However, there are over 60 different Rab GTPases in mammalian cells involved in regulating a wide range of membrane trafficking events including exocytic, endocytic and lysosomal pathways, whose disruption may also be contributing to MM cell death. Global gene expression analysis of MM cells treated with the Rab geranylgeranyl transferase (GGTase) inhibitor, 3-PEHPC, revealed novel pathways impacted by Rab GTPase inhibition, including genes involved in glucose metabolism. As previous work with statins have suggested that statin-induced inhibition of glucose uptake may be a consequence of either blocking protein geranylgeranylation or cholesterol depletion, we were interested in determining whether disruption of isoprenoid synthesis and/or Rab geranylgeranylation has an impact on glucose uptake in MM cells. Notably, MM cells are unique in their dependence on additional members of the GLUT family including GLUT4 whose cell surface expression is regulated by Rabs. We therefore examined the effects of IBP inhibitors and direct prenyl transferase inhibitors on glucose uptake in MM cells (RPMI-8226, U266 and MM.1S) using flow cytometric analysis of the uptake of 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose), a fluorescent analog of D-glucose. The IBP inhibitors lovastatin (an inhibitor of HMG-CoA reductase) and digeranyl bisphosphonate (DGBP) (an inhibitor of geranylgeranyl diphosphate synthase) reduced glucose uptake in a concentration- and time-dependent manner in all cell lines (except U266 which we have previously shown to be resistant to DGBP) by 20-50% compared to untreated control cells. To determine whether the effects of these drugs on glucose uptake are a consequence of depletion of a specific isoprenoid species, experiments were performed in which cells were incubated in the presence or absence of the IBP inhibitors and intermediates in the IBP, namely mevalonate, FPP or GGPP. Co-incubation of lovastatin with mevalonate or GGPP completely prevented the effects of lovastatin on glucose uptake in MM.1S and U266 cells while GGPP prevented the block in glucose uptake induced by DGBP in MM.1S cells. However, co-incubation of lovastatin with FPP partially prevented the effects of lovastatin in U266 cells but had no effect in MM.1S cells. Treatment of U266 cells, but not RPMI-8226 or MM.1S cells, with the squalene synthase inhibitor zaragozic acid resulted in a significant decrease in glucose uptake. MM.1S and U266 cell lines were treated with individual prenyltransferase inhibitors (FTI-277, GGTI-2133 or 3-PEHPC) to distinguish between the effects of altering cellular isoprenoid levels versus directly protein prenylation. Whereas none of the prenyltransferase inhibitors had significant effects on the U266 cells, only inhibition of Rab GGTase by 3-PEHPC resulted in a significant decrease in glucose uptake in MM.1S cells. In aggregate these studies demonstrate that Rab inhibition via disruption of geranylgeranylation diminishes glucose uptake in MM cells. In addition, a cholesterol-dependent effect related to IBP inhibition was observed in the U266 cells but not in the other tested MM cells, suggesting that MM cells may vary in their dependence on glucose uptake by distinct GLUT transporters. Future studies will investigate these findings in primary MM cells and identify the specific GLUT family members which are affected by direct Rab GGTase inhibitors as well as by IBP inhibitors. Improved understanding of the mechanisms underlying the effects of these inhibitors will inform further development of therapies targeting glucose uptake in MM. DisclosuresHolstein:Amgen: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Abstract 4768: Determination of Rab GTPase-mediated pathways critical for the antimyeloma activity of Rab GGTase inhibitors

Abstract Multiple myeloma (MM) is a plasma cell malignancy characterized by the production of high levels of monoclonal protein (MP). This leads to an increased protein folding burden in the endoplasmic reticulum (ER) in MM cells, resulting in a constitutively upregulated unfolded protein response (UPR) and a lower threshold for ER stress-mediated apoptosis. We have previously shown that inhibitors of Rab GGTase, the enzyme responsible for Rab GTPase geranylgeranylation, disrupt MP trafficking in MM cells, leading to an accumulation of MP in the ER and induction of the UPR and apoptosis. There are over 60 different Rab GTPases in mammalian cells involved in regulating a wide range of membrane trafficking events including exocytic, endocytic, and lysosomal pathways. In this study, we aimed to further define how disruption of RabGTPase function in MM cells leads to apoptosis by knocking down individual Rab proteins. Given the accumulation of MP and induction of UPR, our initial focus was on secretory Rabs. Knockdown of Rab1A/B (>50%) using siRNA in RPMI 8226 cells lead to moderate increases in intracellular lambda light chain in Rab1A/B knockdowns after 72 hours (∼120-140% of control levels by ELISA). However, despite the increase in intracellular MP, no increases in UPR or apoptosis markers were seen by western blot analysis or annexin-V/propidium iodide flow cytometry. Extended time course experiments (up to 120 hours) also did not exhibit any increase in UPR or apoptosis. Knockdown of Rab6A (>60%), another RabGTPase associated with the secretory pathway, did not lead to increases in intracellular lambda light chain or induction of apoptosis. These results suggest that although individual knockdown of Rab1 is sufficient to partially disrupt MP trafficking, either more complete knockdown of the individual Rabs or knockdown of multiple Rabs is necessary for induction of the UPR and apoptosis. Additional optimization of the knockdowns will be performed in order to verify these results. These studies support the further development of Rab GGTase inhibitors that globally effect Rab GTPase geranylgeranylation as a novel anti-myeloma strategy. Citation Format: Kaitlyn M. Dykstra, Cheryl L. Allen, Sarah A. Holstein. Determination of Rab GTPase-mediated pathways critical for the antimyeloma activity of Rab GGTase inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4768.

Applying the prodrug strategy to α-phosphonocarboxylate inhibitors of Rab GGTase – synthesis and stability studies

Fourteen novel prodrug-like analogs of two highly ionic phosphonocarboxylate inhibitors of Rab geranylgeranyl transferase were synthesized and preliminary assessment of their chemical and enzymatic stability was evaluated in buffers (pH 6.5 and 7.4) and rat intestinal homogenate (pH 6.5). Both acidic groups in phosphonocarboxylates were subject to modification. Phosphonic acid was protected either as bis(acyloxyalkyl) ester or phosphonodiamidate derived from amino acids. The carboxylic acid group was either left unchanged or was studied as ethyl ester. The compounds exhibited favorable stability in physiologically relevant pH (t1/2 above 18 h), while in intestinal homogenate they showed a large variety of half-lives (from 5 minutes to over 150 hours). LC MS studies have shown that the main product of decomposition under studied conditions resulted from cleavage of one of the ester (for acyloxyalkyl analogs) or amide (for phosphonodiamidate) bonds with phosphorus.

Synthesis and Structure-Function Relationship Of Novel Triazole Phosphonates As Inhibitors Of Rab Geranylgeranyl Transferase

Multiple myeloma is characterized by the proliferation of malignant plasma cells which secrete large quantities of monoclonal protein (MP). As yet, no therapeutic strategies have been developed which directly target MP secretion. The Rab family of small GTPases plays key roles in intracellular vesicle trafficking. We have demonstrated that agents which impair the geranylgeranylation of Rab proteins induce apoptosis of myeloma cells by disrupting MP secretion, resulting in accumulation of MP within the cells and activation of the unfolded protein response pathway (UPR). Previously identified inhibitors of Rab geranylgeranyl transferase (Rab GGTase), the enzyme responsible for post-translational modification of Rab proteins, lack potency and have limited therapeutic potential. We hypothesized that potent and selective inhibitors of Rab GGTase could be designed based on available crystallographic data which reveals how the isoprenoid substrate interacts with the enzyme's active site. Families of compounds were therefore designed incorporating three motifs to allow for interaction with key components of the enzyme's active site: a non-hydrolyzable polar head group, a zinc-binding motif, and an isoprenoid or isoprenoid-like chain. The isoprenoid-based compounds were prepared using a divergent synthetic strategy based on azide-acetylene cycloadditions or “click” chemistry. We have previously demonstrated that direct use of an isoprenoid azide in a click reaction results in a triazole product that is a mixture of olefin isomers, due to an unavoidable [3,3] sigmatropic rearrangement that scrambles the stereochemistry of the first olefin prior to cycloaddition. This problem now has been circumvented by the development of a new synthetic strategy which is based on a regiospecific epoxidation of the C-2 olefin in the isoprenoid alcohol, followed by introduction of the azide and formation of the triazole. After completion of a click reaction the product can be reduced back to the parent olefin with good stereocontrol, to afford isoprenylated triazoles of the same chain length as those prepared directly from isoprenoid azides. Series of compounds were thus prepared in which chain length (including isoprenoid and isoprenoid-like), olefin stereochemistry, and polar head group (including bisphosphonic acids and carboxyphosphonic acids) were varied. All novel agents were subjected to in vitro enzyme assays for Rab GGTase as well as the related enzymes farnesyl transferase (FTase) and geranylgeranyl transferase I (GGTase I) and IC50 values were obtained. The cytotoxic activities of these agents were determined via MTT assays in human myeloma cell lines. Disruption of protein prenylation in myeloma cells was assessed via immunoblot analysis of farnesylated (Ras) and geranylgeranylated (Rap1a and Rab6) proteins. The results from these enzymatic and cell-based assays enabled the determination of a detailed structure-function relationship. These studies reveal that both chain length and olefin stereochemistry have a significant impact on both inhibitor potency and selectivity with respect to the target enzyme and the related prenyltransferases. For example, when the chain length was increased by just one methylene unit (farnesyl to homofarnesyl), the potency against Rab GGTase was enhanced by approximately 4-fold while changing the stereochemistry at the second olefin site from trans to cis diminished the potency by approximately 5-fold. These studies are guiding the design and synthesis of future generations of inhibitors with the ultimate goal of developing a potent and selective Rab GGTase inhibitor which can be utilized as a therapeutic agent for myeloma. Disclosures:No relevant conflicts of interest to declare.

Rab-geranylgeranyl transferase regulates glucose-stimulated insulin secretion from pancreatic β cells

A growing body of evidence implicates essential roles for small molecular weight G-proteins (e.g., Cdc42, Rac1, Arf6 and Rab3A and Rab27A) in islet β-cell function including glucose-stimulated insulin secretion (GSIS). One of the known mechanisms for optimal activation of small G-proteins involves post-translational prenylation, which is mediated by farnesyltransferase (FTase) and geranylgeranyl transferases (GGTases I and II). The FTase catalyzes incorporation of a 15-carbon farnesyl group while the GGTase mediates incorporation of a 20-carbon geranylgeranyl group into the C-terminal cysteines of G-proteins. The FTase, GGTase I and GGTase II prenylate Ras, Cdc42/Rac1, and Rab G-proteins, respectively. While considerable evidence exists on FTase/GGTase I-mediated regulation of GSIS, very little is known about GGTase II (also referred to as Rab GGTase; RGGT) and its regulatory proteins in the cascade of events leading to GSIS. Herein, we provide the first immunological evidence to suggest expression of α- and β-subunits of RGGT in clonal INS 832/13 β-cells, normal rat islets and human islets. Furthermore, Rab escort protein1 (REP1), which has been shown to be critical for prenylation of Rab G-proteins, is also expressed in these cells. Furthermore, evidence is presented to suggest that siRNA-mediated knockdown of α- or β-subunits of RGGT and REP1 markedly attenuates GSIS in INS 832/13 cells. These findings provide the first evidence in support of key roles for RGGT and its regulatory proteins in GSIS.

Identification of a Novel Phosphonocarboxylate Inhibitor of Rab Geranylgeranyl Transferase That Specifically Prevents Rab Prenylation in Osteoclasts and Macrophages

Nitrogen-containing bisphosphonate drugs inhibit bone resorption by inhibiting FPP synthase and thereby preventing the synthesis of isoprenoid lipids required for protein prenylation in bone-resorbing osteoclasts. NE10790 is a phosphonocarboxylate analogue of the potent bisphosphonate risedronate and is a weak anti-resorptive agent. Although NE10790 was a poor inhibitor of FPP synthase, it did inhibit prenylation in J774 macrophages and osteoclasts, but only of proteins of molecular mass approximately 22-26 kDa, the prenylation of which was not affected by peptidomimetic inhibitors of either farnesyl transferase (FTI-277) or geranylgeranyl transferase I (GGTI-298). These 22-26-kDa proteins were shown to be geranylgeranylated by labelling J774 cells with [(3)H]geranylgeraniol. Furthermore, NE10790 inhibited incorporation of [(14)C]mevalonic acid into Rab6, but not into H-Ras or Rap1, proteins that are modified by FTase and GGTase I, respectively. These data demonstrate that NE10790 selectively prevents Rab prenylation in intact cells. In accord, NE10790 inhibited the activity of recombinant Rab GGTase in vitro, but did not affect the activity of recombinant FTase or GGTase I. NE10790 therefore appears to be the first specific inhibitor of Rab GGTase to be identified. In contrast to risedronate, NE10790 inhibited bone resorption in vitro without markedly affecting osteoclast number or the F-actin "ring" structure in polarized osteoclasts. However, NE10790 did alter osteoclast morphology, causing the formation of large intracellular vacuoles and protrusion of the basolateral membrane into large, "domed" structures that lacked microvilli. The anti-resorptive activity of NE10790 is thus likely due to disruption of Rab-dependent intracellular membrane trafficking in osteoclasts.

Assay for Rab Geranylgeranyltransferase Using Size Exclusion Chromatography

A simple chromatographic assay for Rab geranylgeranyltransferase (Rab GGTase) has been developed. The method involves separation of the reaction mixture on a Sephadex G-25 superfine minicolumn. Addition of 2-propanol to the assay results in substantial (∼90%) decline of formation of noncovalent lipid–protein complexes, increasing reproducibility and reliability of the method. The activity of Rab prenyltransferase was measured in crude and partially purified enzyme preparations from wheat seedlings; measurements for several other plants and rat brain cytosol fractions are also presented. This method can be routinely applied to evaluate the activity of different protein prenyltransferases.

3] Fluorescence methods for monitoring interactions of rab proteins with nucleotides, rab escort protein, and geranylgeranyltransferase

This chapter describes several examples of fluorescence signals that can be used for monitoring interactions of Rab proteins with nucleotides, Rab escort protein, and geranylgeranyltransferase studies. As a model system Rab7 is chosen, a small GTPase involved in the biogenesis of late endosomes and lysosomes. Its interaction is investigated with the Rab escort protein 1 (REP-1) and Rab geranylgeranyltransferase (Rab GGTase). REP proteins are required to present Rab proteins to the enzyme geranylgeranyltransferase to allow their C-terminal prenylation (double geranylgeranylation). The details of the interactions involved in this process are poorly understood, but there is already an indication that REP has a higher affinity for the GDP than for the GTP form of Rab proteins. To quantitate this and other interactions more fully and to obtain information on the dynamics, fluorescence from different reporter groups associated with Rab7 is exploited.

Rab geranylgeranyl transferase alpha mutation in the gunmetal mouse reduces Rab prenylation and platelet synthesis.

Few molecular events important to platelet biogenesis have been identified. Mice homozygous for the spontaneous, recessive mutation gunmetal (gm) have prolonged bleeding, thrombocytopenia, and reduced platelet alpha- and delta-granule contents. Here we show by positional cloning that gm results from a G-->A substitution mutation in a splice acceptor site within the alpha-subunit of Rab geranylgeranyl transferase (Rabggta), an enzyme that attaches geranylgeranyl groups to Rab proteins. Most Rabggta mRNAs from gm tissues skipped exon 1 and lacked a start codon. Rabggta protein and Rab geranylgeranyl transferase (GGTase) activity were reduced 4-fold in gm platelets. Geranylgeranylation and membrane association of Rab27, a Rab GGTase substrate, were significantly decreased in gm platelets. These findings indicate that geranylgeranylation of Rab GTPases is critical for hemostasis. Rab GGTase inhibition may represent a new treatment for thrombocytosis and clotting disorders.

Rapid identification of cysteine-linked isoprenyl groups by metabolic labeling with [3H]farnesol and [3H]geranylgeraniol.

The posttranslational modification of proteins by the covalent attachment of farnesyl and geranylgeranyl groups to cysteine residues at or near the C-terminus via a thioether bond is now well established in mammalian cells (1–6). Most isoprenylated proteins are thought to serve as regulators of cell signaling and membrane trafficking. Farnesylation and geranylgeranylation of the cysteinyl residues have been shown to promote both protein–protein and protein–membrane interactions (6–8). Isoprenylation, and, in some cases, the subsequent palmitoylation, provide a mechanism for the membrane association of polypeptides, which lack a transmembrane domain, and appear to be prerequisite for their in vivo activity (6,9,10). Three distinct protein prenyltransferases catalyzing these modifications have been identified (1–5). Two geranylgeranyltransferases (GGTases) have been characterized, and are known to modify distinct protein substrates. The CaaX GGTase (also known as GGTase-1) geranylgeranylates proteins that end in a CaaL(F) sequence, where C is cysteine, a is usually an aliphatic amino acid, and the C-terminal amino acyl group is leucine (L) or phenylalanine (F). Rab GGTase (also known as GGTase-2) catalyzes the attachment of two geranylgeranyl groups to paired carboxyl-terminal cysteines in most members of the Rab family of GTP-binding proteins (11). These proteins terminate in a Cys-Cys, Cys-X-Cys or Cys-Cys-X-X motif, where X is a small hydrophobic amino acid. Another set

Studies of the mouse Rab geranylgeranyl transferase β subunit: gene structure, expression and regulation

The mouse Rab geranylgeranyl transferase β (Rab GGTase β) catalytic subunit gene was isolated and characterized. This gene ( Rabggtb) spans a distance of approx. 7 kb and is organized into eight exons. All the exon/intron junction sequences follow the GT/AG rule. Multiple transcription initiation sites are located within 384 bp upstream from the translation start codon. The 5′-flanking region contains several potential binding sites for transcription factors, but no TATA box is identified in this region. Expression of this gene was detected in all the major organs in adult animals. In mouse embryos, its expression was examined by in situ hybridization. Specific expression of this gene was elevated in mid-gestation stages, particularly developing liver and spinal cord. Northern blot analysis of an embryonic carcinoma cell line P19 showed that the steady-state level of Rabggtb mRNA expression was increased dramatically by cycloheximide (CHX) treatment as early as 2 h, suggesting a role of post-transcriptional regulation of Rab GGTase β gene expression. Actinomycin D was used to determine the half-life of Rab GGTase β transcripts. CHX treatment resulted in a dramatic increase of the half-life of Rab GGTase β transcripts, from 8 h to greater than 12 h.

CDNA Cloning and Chromosomal Localization of the Genes Encoding the α- and β-Subunits of Human Rab Geranylgeranyl Transferase: The 3′ End of the α-Subunit Gene Overlaps with the Transglutaminase 1 Gene Promoter

We have isolated and sequenced the complete coding sequences of the human genes for the α- and β-subunits of Rab geranylgeranyl transferase (Rab GGTase). The α- and β-subunit genes code for proteins of 567 and 331 amino acids, respectively, showing 91 and 95% amino acid identity to their rat counterparts. We employed fluorescencein situhybridization to map the β-subunit gene to human chromosome 1p31. The α-subunit gene could be assigned to 14q11.2, less than 2 kb upstream of the transcription initiation site of the gene for transglutaminase 1 (TGM1). The two genes are arranged in tandem in a head-to-tail orientation. The short intergenic sequence between the two loci contains several promoter elements that are involved in the induction ofTGM1gene expression in squamous cells. These results suggest thatcis-acting factors for cell-type-specific transcription of one gene are located within the transcribed region of a functionally unrelated gene.

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.

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.

Mechanism of Digeranylgeranylation of Rab Proteins: FORMATION OF A COMPLEX BETWEEN MONOGERANYLGERANYL-Rab AND Rab ESCORT PROTEIN

Rab proteins are Ras-related small GTPases that are digeranylgeranylated at carboxyl-terminal cysteines, a modification essential for their action as molecular switches regulating intracellular vesicular transport. Geranylgeranylation of Rabs is a complex reaction that requires a catalytic Rab geranylgeranyl transferase (GGTase) and a Rab escort protein (REP). REP binds unprenylated Rab and presents it to Rab GGTase. After GG transfer, REP remains associated with diGG-Rab, which leads to insertion of the Rab into a specific membrane. We used recombinant Rab1a single cysteine mutants that accept only one GG group to study the mechanism of the digeranylgeranylation reaction. Using the prenylation assay, gel filtration chromatography, and density ultracentrifugation, we show that REP, but not Rab GGTase, forms a stable complex with unprenylated, monoGG- and diGG-Rab1a. The REP.monoGG-Rab1a complex is stable in the presence of detergents or phospholipids, whereas the REP.diGG-Rab1a complex partially dissociates under these conditions. The stoichiometry of the REP.Rab complex appears to be 1:1 before prenylation. Prenylation induces a change in complex stoichiometry, with the formation of a 2:2 or 2:1 REP.Rab complex. A possible mechanism by which Rab proteins are digeranylgeranylated is suggested by the current studies. We propose that each geranylgeranyl addition is an independent reaction that leads to the production of monoGG-Rab and diGG-Rab, respectively. The stability of the REP.monoGG-Rab complex prevents monoGG-Rab from dissociating from REP prior to the second geranylgeranylation reaction, ensuring efficient digeranylgeranylation of Rab substrates.