Purification of component A of Rab geranylgeranyl transferase: possible identity with the choroideremia gene product.

Rab geranylgeranyl transferase (GG transferase) from rat brain contains two components, A and B. Component B comprises polypeptides of 60 and 38 kd. Here we report the purification of component A, a single 95 kd polypeptide. The holoenzyme attaches 3H-geranylgeranyl to cysteines in two GTP-binding proteins, Rab3A and Rab1A. The reaction is abolished when both cysteines in the COOH-terminal CysCys sequence of Rab1A are mutated to serines. The mutant protein inhibits transfer of 3H-geranylgeranyl to wild-type Rab1A and Rab3A, suggesting that the enzyme recognizes conserved sequences distinct from the COOH-terminus. Six peptides from rat component A show striking similarity to the product of the defective gene in choroideremia, an X-linked retinal degeneration disease. The choroideremia protein resembles Rab3A GDI, which binds Rab3A. We hypothesize that component A binds conserved sequences in Rab and that component B transfers geranylgeranyl. A defect in this reaction may cause choroideremia.     

Properties of Rab5 N-terminal domain dictate prenylation of C-terminal cysteines.

Rab5 is a Ras-related GTP-binding protein that is post-translationally modified by prenylation. We report here that an N-terminal domain contained within the first 22 amino acids of Rab5 is critical for efficient geranylgeranylation of the protein's C-terminal cysteines. This domain is immediately upstream from the "phosphate binding loop" common to all GTP-binding proteins and contains a highly conserved sequence recognized among members of the Rab family, referred to here as the YXYLFK motif. A truncation mutant that lacks this domain (Rab5(23-215) fails to become prenylated. However, a chimeric peptide with the conserved motif replacing cognate Rab5 sequence (MAYDYLFKRab5(23-215) does become post-translationally modified, demonstrating that the presence of this simple six amino acid N-terminal element enables prenylation at Rab5's C-terminus. H-Ras/Rab5 chimeras that include the conserved YXYLFK motif at the N-terminus do not become prenylated, indicating that, while this element may be necessary for prenylation of Rab proteins, it alone is not sufficient to confer properties to a heterologous protein to enable substrate recognition by the Rab geranylgeranyl transferase. Deletion analysis and studies of point mutants further reveal that the lysine residue of the YXYLFK motif is an absolute requirement to enable geranylgeranylation of Rab proteins. Functional studies support the idea that this domain is not required for guanine nucleotide binding since prenylation-defective mutants still bind GDP and are protected from protease digestion in the presence of GTP gamma S. We conclude that the mechanism of Rab geranylgeranylation involves key elements of the protein's tertiary structure including a conserved N-terminal amino acid motif (YXYLFK) that incorporates a critical lysine residue.     

Membrane targeting of Rab GTPases is influenced by the prenylation motif

Rab GTPases are regulators of membrane traffic. Rabs specifically associate with target membranes via the attachment of (usually) two geranylgeranyl groups in a reaction involving Rab escort protein and Rab geranylgeranyl transferase. In contrast, related GTPases are singly prenylated by CAAX prenyl transferases. We report that di-geranylgeranyl modification is important for targeting of Rab5a and Rab27a to endosomes and melanosomes, respectively. Transient expression of EGFP-Rab5 mutants containing two prenylatable cysteines (CGC, CC, CCQNI, and CCA) in HeLa cells did not affect endosomal targeting or function, whereas mono-cysteine mutants (CSLG, CVLL, or CVIM) were mistargeted to the endoplasmic reticulum (ER) and were nonfunctional. Similarly, Rab27aCVLL mutant is also mistargeted to the ER and transgenic expression on a Rab27a null background (Rab27aash) did not rescue the coat color phenotype, suggesting that Rab27aCVLL is not functional in vivo. CAAX prenyl transferase inhibition and temperature-shift experiments further suggest that Rabs, singly or doubly modified are recruited to membranes via a Rab escort protein/Rab geranylgeranyl transferase-dependent mechanism that is distinct from the insertion of CAAX-containing GTPases. Finally, we show that both singly and doubly modified Rabs are extracted from membranes by RabGDIalpha and propose that the mistargeting of Rabs to the ER results from loss of targeting information.     

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.     

Thematic review series: lipid posttranslational modifications. geranylgeranylation of Rab GTPases

Rab GTPases require special machinery for protein prenylation, which include Rab escort protein (REP) and Rab geranylgeranyl transferase (RGGT). The current model of Rab geranylgeranylation proposes that REP binds Rab and presents it to RGGT. After geranylgeranylation of Rab C-terminal cysteines, REP delivers the prenylated protein to membranes. The REP-like protein Rab GDP dissociation inhibitor (RabGDI) then recycles the prenylated Rab between the membrane and the cytosol. The recent solution of crystal structures of the Rab prenylation machinery has helped to refine this model and provided further insights. The hydrophobic prenyl binding pocket of RGGT and geranylgeranyl transferase type-I (GGT-I) differs from that of farnesyl transferase (FT). A bulky tryptophan residue in FT restricts the size of the pocket, whereas in RGGT and GGT-I, this position is occupied by smaller residues. A highly conserved phenylalanine in REP, which is absent in RabGDI, is critical for the formation of the REP:RGGT complex. Finally, a geranylgeranyl binding site conserved in REP and RabGDI has been identified within helical domain II. The postprenylation events, including the specific targeting of Rabs to target membranes and the requirement for single versus double geranylgeranylation by different Rabs, remain obscure and should be the subject of future studies.     

Molecular Cloning and Characterization of Rab27a and Rab27b,Novel Human Rab Proteins Shared by Melanocytes and Platelets

Rabs are prenylated, membrane-bound proteins involved in vesicular fusion and trafficking. We isolated the complete cDNAs of two rab isoforms, rab27a and rab27b, from human melanoma cells and melanocytes. Rab27a is the human homolog of a rat megakaryocyte rab called ram p25. Rab27b corresponds to a small GTP-binding protein, c25KG, which was previously purified from platelets but whose cDNA had not been cloned. Sequence comparisons with known rabs indicate that rab27a and rab27b comprise a melanocyte/platelet subfamily within the rab family. In addition, rab27a was expressed in a large variety of cell and tissue types, excluding brain, and rab27b manifested itself primarily in testis. Bacterially expressed and purified rab27a and rab27b exhibited GTP-binding activity and can now be used for antibody production and studies of the substrate specificities of geranylgeranyl transferase. In addition, the expression of rab27a and rab27b in both melanocytes and platelets makes them candidates for involvement in mouse and human disorders characterized by the combination of pigment dilution and a platelet storage pool defect.     

Farnesyl transferase inhibitors block the farnesylation of CENP-E and CENP-F and alter the association of CENP-E with the microtubules

Human tumor cell lines that are sensitive to the effects of farnesyl transferase inhibitors accumulate in G(2) --> M (except for cells with an activated Ha-ras that accumulate in G(1)). A search for CAAX box proteins from Swiss-Prot revealed more than 300 peptides. Of these, the centromeric proteins CENP-E and CENP-F are preferentially expressed during mitosis and are implicated as mediators of the G(2) --> M checkpoint. Experiments performed here show that peptides from the COOH-terminal CAAX box of CENP-E and CENP-F are substrates for farnesyl transferase but not geranylgeranyl transferase-I. Although both proteins are prenylated in the human tumor cell line DLD-1, their prenylation is completely inhibited by the farnesyl transferase inhibitor, SCH 66336. Immunohistochemical data with the lung carcinoma cell line, A549, showed that preventing the farnesylation of CENP-E and CENP-F by treatment with the farnesyl transferase inhibitor SCH 66336 does not affect their localization to the kinetochores. However, the presence of farnesyl transferase inhibitors alters the association between CENP-E and the microtubules. Our results imply that the inhibition of CENP-E farnesylation results in the alteration of the microtubule-centromere interaction during mitosis and results in the accumulation of cells prior to metaphase.     

Structures of RabGGTase-substrate/product complexes provide insights into the evolution of protein prenylation

Post-translational isoprenylation of proteins is carried out by three related enzymes: farnesyltransferase, geranylgeranyl transferase-I, and Rab geranylgeranyl transferase (RabGGTase). Despite the fact that the last one is responsible for the largest number of individual protein prenylation events in the cell, no structural information is available on its interaction with substrates and products. Here, we present structural and biophysical analyses of RabGGTase in complex with phosphoisoprenoids as well as with the prenylated peptides that mimic the C terminus of Rab7 GTPase. The data demonstrate that, unlike other protein prenyl transferases, both RabGGTase and its substrate RabGTPases completely 'outsource' their specificity for each other to an accessory subunit, the Rab escort protein (REP). REP mediates the placement of the C terminus of RabGTPase into the active site of RabGGTase through a series protein-protein interactions of decreasing strength and selectivity. This arrangement enables RabGGTase to prenylate any cysteine-containing sequence. On the basis of our structural and thermodynamic data, we propose that RabGGTase has evolved from a GGTase-I-like molecule that 'learned' to interact with a recycling factor (GDI) that, in turn, eventually gave rise to REP.     

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.     

rab GTP-binding proteins with three different carboxyl-terminal cysteine motifs are modified in vivo by 20-carbon isoprenoids.

p21ras and several other ras-related GTP-binding proteins are modified post-translationally by addition of 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoids to cysteines within a conserved carboxyl-terminal sequence motif, Caa(M/S/L), where a is an aliphatic amino acid. Proteins ending with M or S are substrates for farnesyltransferase, whereas those ending with L are modified preferentially by geranylgeranyltransferase. We recently reported that GTP-binding proteins encoded by rab1B (GGCC), rab2 (GGCC), and rab5 (CCSN) are modified by 20-carbon isoprenyl derivatives of [3H]mevalonate when translated in vitro, despite having carboxyl-terminal sequences distinct from the Caa(M/S/L) motif. We now show that these proteins function as specific acceptors for geranylgeranyl in vitro and are modified by 20-carbon isoprenyl groups in COS cells metabolically labeled with [3H]mevalonate. Proteins encoded by rab4 and rab6, with yet another distinct carboxyl-terminal motif (xCxC), are similarly modified by 20-carbon isoprenoids in vitro and in vivo. The geranylgeranyl modification of rab5 protein (CCSN) is catalyzed by an enzyme in brain cytosol but not by a purified geranylgeranyltransferase that modifies GTP-binding proteins with the CaaL motif. Unlike the prenylation of proteins with Caa(M/S/L) termini, the prenylation of rab5 protein is not inhibited by a synthetic peptide based on its carboxyl-terminal sequence (TRNQCCSN). When cellular isoprenoid synthesis is blocked by treatment of cells with lovastatin, rab proteins that are normally localized in membranes of the endoplasmic reticulum, Golgi apparatus, and endosomes accumulate in the cytosol. This change in rab protein localization is reversed by providing cells with mevalonate. These findings suggest that geranylgeranyl modification underlies the ability of rab GTP-binding proteins to associate with intracellular membranes, where they are postulated to function as mediators of vesicular traffic.     

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.     

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.     

A geranylgeranyltransferase for rhoA p21 distinct from the farnesyltransferase for ras p21S

We have clarified that rhoA p21 purified from bovine aortic smooth muscle is geranylgeranylated at the cysteine residue in the C-terminal CAAX motif (A is an aliphatic amino acid and X is any amino acid). In this paper, a geranylgeranyltransferase for rhoA p21 (rhoA p21 GGT) was partially purified from bovine brain cytosol. This enzyme transferred a geranylgeranyl moiety from geranylgeranyl pyrophosphate to rhoA p21 having the CAAX motif (rhoA p21-CAAX) but not to rhoA p21 lacking the AAX portion. rhoA p21 GGT was separated from the previously reported farnesyltransferase for ras p21s (ras p21 FT) by column chromatographies and did not geranylgeranylate or farnesylate c-Ha-ras p21-CAAX. ras p21 FT did not geranylgeranylate or farnesylate rhoA p21-CAAX. These results indicate that rhoA p21 GGT distinct from ras p21 FT is present in bovine brain cytosol.     

Rab escort protein-1 is a multifunctional protein that accompanies newly prenylated rab proteins to their target membranes.

Rab proteins comprise a family of small GTPases that serve a regulatory role in vesicular membrane traffic. Geranylgeranylation of these proteins on C-terminal cysteine motifs is crucial for their membrane association and function. This post-translational modification is catalysed by rab geranylgeranyl transferase (Rab-GGTase), a multisubunit enzyme consisting of a catalytic heterodimer and an accessory component, named rab escort protein (REP)-1. Previous in vitro studies have suggested that REP-1 presents newly synthesized rab proteins to the catalytic component of the enzyme, and forms a stable complex with the prenylated proteins following the transfer reaction. According to this model, a cellular factor would be required to dissociate the rab protein from REP-1 and to allow it to recycle in the prenylation reaction. RabGDP dissociation inhibitor (RabGDI) was considered an ideal candidate for this role, given its established function in mediating membrane association of prenylated rab proteins. Here we demonstrate that dissociation from REP-1 and binding of rab proteins to the membrane do not require RabGDI or other cytosolic factors. The mechanism of REP-1-mediated membrane association of rab5 appears to be very similar to that mediated by RabGDI. Furthermore, REP-1 and RabGDI share several other functional properties, the ability to inhibit the release of GDP and to remove rab proteins from membranes; however, RabGDI cannot assist in the prenylation reaction. These data suggest that REP-1 is per se sufficient to chaperone newly prenylated rab proteins to their target membranes.     

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.     

A Salmonella typhimurium effector protein SifA is modified by host cell prenylation and S-acylation machinery

SifA is a Salmonella effector protein that is required for maintenance of the vacuolar membrane that surrounds replicating bacteria. It associates with the Salmonella-containing vacuole but how it interacts with the membrane is unknown. Here we show by immunofluorescence, S100 fractionation and Triton X-114 partitioning that the membrane association and targeting properties of SifA are influenced by a motif encoded within the C-terminal six amino acids. This sequence shares homology with both CAAX and Rab geranylgeranyl transferase prenylation motifs. We characterized the post-translational processing of SifA and showed that the cysteine residue within the CAAX motif is modified by isoprenoid addition through the action of protein geranylgeranyl transferase I. SifA was additionally modified by S-acylation of an adjacent cysteine residue. Similar modifications to host cell proteins regulate numerous functions including protein targeting, membrane association, protein-protein interaction, and signal transduction. This is the only known example of a bacterial effector protein that is modified both by mammalian cell S-acylation and prenylation machinery.     

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.     

A novel prenyltransferase for a small GTP-binding protein having a C-terminal Cys-Ala-Cys structure

smg p25A/rab3A p25 is a member of the small GTP-binding protein superfamily which is implicated in intracellular vesicle transport. smg p25A has a cDNA-predicted C-terminal structure of Cys-Ala-Cys. The protein purified from bovine brain membranes is geranylgeranylated at both the two cysteine residues and carboxyl-methylated at the C-terminal cysteine residue. Two types of prenyltransferase for small GTP-binding proteins have thus far been reported: ras p21 farnesyltransferase (ras p21 FT) and rhoA p21 geranylgeranyltransferase (rhoA p21 GGT). Neither of them geranylgeranylated smg p25A having a C-terminal Cys-Ala-Cys structure. In this paper, a smg p25A GGT was partially purified from bovine brain cytosol and separated from the ras p21 FT and rhoA p21 GGT by column chromatographies. smg p25A GGT transferred the geranylgeranyl moiety from geranylgeranyl pyrophosphate to both the two cysteine residues in the C-terminal Cys-Ala-Cys structure of smg p25A. smg p25A GGT did not use farnesyl pyrophosphate as a substrate and was also inactive on c-Ha-ras p21 and rhoA p21 with either farnesyl pyrophosphate or geranylgeranyl pyrophosphate as a substrate. These results indicate that there are at least three types of prenyltransferase for small GTP-binding proteins in mammalian tissues.     

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.     

Protein farnesyl and geranylgeranyl transferases]

Posttranslational prenylation of proteins synthesized as soluble precursors enhances their hydrophobicity and enables them to bind biological membranes. These modifications consist in the attachment of a C15 farnesyl or a C20 geranylgeranyl moiety to the cysteine residue(s) of proteins bearing CAAX, CC or CXC C-terminal sequences (where C = cysteine, A = aliphatic residue and X = any amino-acid), such as proteins of the ras superfamily, gamma subunits of heterotrimetric G proteins, lamin B as well as yeast mating factor a. A farnesyl transferase (FTase) and two distinct geranylgeranyl transferases (GGTases I and II) have been recently identified. FTase and GGTase I modify proteins containing a C-terminal CAAX motif; such a sequence is necessary and sufficient for recognition by the enzymes. The nature of the fourth residue determines the nature of the modification: when X is a serine, a methionine or a phenylalanine, the protein is farnesylated, whereas the presence of a leucine residue results in the attachment of a geranylgeranyl group. Both these enzymes are alpha beta heterodimers; their purification, molecular cloning of their coding sequences as well as mutational studies in yeast have shown that they share a common alpha subunit, and that their beta subunits exhibit a significant level of sequence similarity. GGTase II modifies ras-related proteins exhibiting CC and CXC C-terminal sequences; the enzyme as well as its recognition motif are yet largely uncharacterized.