Rab Geranylgeranyl Transferase Research Articles

Crystallization and Preliminary X-ray Diffraction Analysis of the Rab Escort Protein-1 in Complex with Rab Geranylgeranyltransferase

Posttranslational prenylation of proteins is a widespread phenomenon and the majority of prenylated proteins are geranylgeranylated members of the Rab GTPase family. Geranylgeranylation is catalyzed by Rab geranylgeranyltransferase (RabGGTase) and is critical for the ability of Rab protein to mediate vesicular docking and fusion of various intracellular vesicles. RabGGTase consists of a catalytic α/β heterodimer and an accessory protein termed Rab escort protein (REP-1) that delivers the newly prenylated Rab proteins to their target membrane. Mutations in the REP-1 gene in humans lead to an X-chromosome-linked defect known as choroideremia—a debilitating disease that inevitably culminates in complete blindness. Here we report in vitro assembly and purification of the stoichiometric ternary complex of RabGGTase with REP-1 stabilized by a hydrolysis-resistant phosphoisoprenoid analog—farnesyl phosphonyl(methyl)phoshonate. The complex formed crystals of extended plate morphology under low ionic-strength conditions. X-ray diffraction data were collected to 2.8 Å resolution at the ESRF. The crystals belong to the monoclinic space group P21, with unit-cell parameters a = 68.7, b = 197.7, c = 86.1 $$A, β = 113.4°. Preliminary structural analysis revealed the presence of one molecule in the asymmetric unit.

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.

Interaction of Farnesylated PRL-2, a Protein-tyrosine Phosphatase, with the β-Subunit of Geranylgeranyltransferase II

Protein of regenerating liver (PRL)-1, -2, and -3 comprise a subgroup of closely related protein-tyrosine phosphatases featuring a C-terminal prenylation motif conforming to either the consensus sequence for farnesylation, CAAX, or geranylgeranylation, CCXX. Yeast two-hybrid screening for PRL-2-interacting proteins identified the beta-subunit of Rab geranylgeranyltransferase II (betaGGT II). The specific interaction of betaGGT II with PRL-2 but not with PRL-1 or -3 occurred in yeast and HeLa cells. Chimeric PRL-1/-2 molecules were tested for their interaction with betaGGT II, and revealed that the C-terminal region of PRL-2 is required for interaction, possibly the PRL variable region immediately preceeding the CAAX box. Additionally, PRL-2 prenylation is prequisite for betaGGT II binding. As prenylated PRL-2 is localized to the early endosome, we propose that this is where the interaction occurs. PRL-2 is not a substrate for betaGGT II, as isoprenoid analysis showed that PRL-2 was solely farnesylated in vivo. Co-expression of the alpha-subunit (alpha) of GGT II, betaGGT II, and PRL-2 resulted in alpha/betaGGT II heterodimer formation and prevented PRL-2 binding. Expression of PRL-2 alone inhibited the endogenous alpha/betaGGT II activity in HeLa cells. Together, these results indicate that the binding of alphaGGT II and PRL-2 to betaGGT II is mutually exclusive, and suggest that PRL-2 may function as a regulator of GGT II activity.

Prenylation of Rab GTPases: molecular mechanisms and involvement in genetic disease

Small GTPases of the Rab family regulate membrane transport pathways. More than 50 mammalian Rab proteins are known, many with transport step-specific localisation. Rabs must associate with cellular membranes for activity and membrane attachment is mediated by prenyl (geranylgeranyl) post-translational modification. Mutations in genes encoding proteins essential for the geranylgeranylation reaction, Rab escort protein and Rab geranylgeranyl transferase, underlie genetic diseases. Choroideremia patients have loss of function mutations in REP1 and the murine Hermansky–Pudlak syndrome model gunmetal possesses a splice-site mutation in the α-subunit of RGGT. Here we discuss recent insights into Rab prenylation and advances towards our understanding of both diseases.

Rab27a is required for regulated secretion in cytotoxic T lymphocytes.

Rab27a activity is affected in several mouse models of human disease including Griscelli (ashen mice) and Hermansky-Pudlak (gunmetal mice) syndromes. A loss of function mutation occurs in the Rab27a gene in ashen (ash), whereas in gunmetal (gm) Rab27a dysfunction is secondary to a mutation in the alpha subunit of Rab geranylgeranyl transferase, an enzyme required for prenylation and activation of Rabs. We show here that Rab27a is normally expressed in cytotoxic T lymphocytes (CTLs), but absent in ashen homozygotes (ash/ash). Cytotoxicity and secretion assays show that ash/ash CTLs are unable to kill target cells or to secrete granzyme A and hexosaminidase. By immunofluorescence and electron microscopy, we show polarization but no membrane docking of ash/ash lytic granules at the immunological synapse. In gunmetal CTLs, we show underprenylation and redistribution of Rab27a to the cytosol, implying reduced activity. Gunmetal CTLs show a reduced ability to kill target cells but retain the ability to secrete hexosaminidase and granzyme A. However, only some of the granules polarize to the immunological synapse, and many remain dispersed around the periphery of the CTLs. These results demonstrate that Rab27a is required in a final secretory step and that other Rab proteins also affected in gunmetal are likely to be involved in polarization of the granules to the immunological synapse.

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.

5′-UTR Structural Organization, Transcript Expression, and Mutational Analysis of the Human Rab Geranylgeranyl Transferase α-Subunit (RABGGTA) Gene

Hermansky-Pudlak syndrome (HPS) is a recessively inherited disease with dysfunction of several related subcellular organelles including platelet-dense granules, melanosomes, and lysosomes. Our recent identification of the mutation in murine Rab geranylgeranyl transferase α-subunit gene (Rabggta) in one mouse model of HPS, the gunmetal mouse, suggested that human patients with similar phenotypes might have mutations in the human orthologous RABGGTA gene. This prompted reanalysis of the 5′-untranslated structure of the human RABGGTA gene in normal individuals and in patients with deficiencies of platelet-dense granules (αδ-SPD), alpha granules (α-SPD or gray platelet syndrome, GPS) or alpha plus dense granules (αδ-SPD). We report the complete sequence of intron α of RABGGTA and demonstrate that exon α is immediately upstream of intron α. The exon/intron structural organization of the 5′-untranslated region (UTR) of human RABGGTA was found to be similar to that of the mouse Rabggta gene. However, exons α and introns α are not homologous between mouse and human. Features of the 5′-UTR of RABGGTA suggest it is a housekeeping gene. While obvious disease-causing mutations of human RABGGTA were not found in our existing SPD patients by sequencing its entire coding region, several polymorphisms of RABGGTA including a putative cryptic splicing mutation in intron 4 were identified. Knowledge of the 5′-UTR structure of RABGGTA and its common polymorphisms will be useful for mutation screening or linkage analysis in patients with albinism, thrombocytopenia, or platelet SPD.

Rab geranylgeranyltransferase in wheat seedlings

Rab geranylgeranyltransferase in wheat seedlings

Molecular basis for Rab prenylation.

Rab escort proteins (REP) 1 and 2 are closely related mammalian proteins required for prenylation of newly synthesized Rab GTPases by the cytosolic heterodimeric Rab geranylgeranyl transferase II complex (RabGG transferase). REP1 in mammalian cells is the product of the choroideremia gene (CHM). CHM/REP1 deficiency in inherited disease leads to degeneration of retinal pigmented epithelium and loss of vision. We now show that amino acid residues required for Rab recognition are critical for function of the yeast REP homologue Mrs6p, an essential protein that shows 50% homology to mammalian REPs. Mutant Mrs6p unable to bind Rabs failed to complement growth of a mrs6Delta null strain and were found to be dominant inhibitors of growth in a wild-type MRS6 strain. Mutants were identified that did not affect Rab binding, yet prevented prenylation in vitro and failed to support growth of the mrs6Delta null strain. These results suggest that in the absence of Rab binding, REP interaction with RabGG transferase is maintained through Rab-independent binding sites, providing a molecular explanation for the kinetic properties of Rab prenylation in vitro. Analysis of the effects of thermoreversible temperature-sensitive (mrs6(ts)) mutants on vesicular traffic in vivo showed prenylation activity is only transiently required to maintain normal growth, a result promising for therapeutic approaches to disease.

Abnormal vesicular trafficking in mouse models of Hermansky-Pudlak syndrome.

Hermansky-Pudlak Syndrome (HPS) is a group of related multigenic recessively inherited disorders which causes abnormalities in the biosynthesis and/or function of three related organelles; melanosomes, platelet-dense granules and lysosomes. These lead, in turn, to hypopigmentation, prolonged bleeding and ceroid deposition. Positional cloning strategies have identified five mouse HPS genes. Two orthologous human diseases (HPS1 and HPS2) have likewise been identified. At least four of the five mouse genes encode proteins involved in the regulation of intracellular vesicle trafficking. The pearl (HPS2) and mocha genes encode the beta3A and delta subunits, respectively, of the AP-3 adaptor complex, which captures organelle membrane proteins at the trans-Golgi apparatus. The protein products of the pallid and gunmetal genes are also important components of the vesicular trafficking machinery. The former interacts with a t-SNARE, syntaxin13, and the latter is the alpha subunit of Rab geranylgeranyltransferase, which renders Rab proteins sufficiently lipophilic to function at their target membranes. The pale ear (HPS1) gene encodes a ubiquitously expressed protein of unknown function. Recent physiological studies have shown that mouse HPS mutants, like their human HPS counterparts, have variably reduced lifespans and may have lung abnormalities.

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.

Crystal structure of Rab geranylgeranyltransferase at 2.0 Å resolution

Background: Rab geranylgeranyltransferase (RabGGT) catalyzes the addition of two geranylgeranyl groups to the C-terminal cysteine residues of Rab proteins, which is crucial for membrane association and function of these proteins in intracellular vesicular trafficking. Unlike protein farnesyltransferase (FT) and type I geranylgeranyltransferase, which both prenylate monomeric small G proteins or short peptides, RabGGT can prenylate Rab only when Rab is in a complex with Rab escort protein (REP).Results: The crystal structure of rat RabGGT at 2.0 Å resolution reveals an assembly of four distinct structural modules. The β subunit forms an α–α barrel that contains most of the residues in the active site. The α subunit consists of a helical domain, an immunoglobulin (Ig)-like domain, and a leucine-rich repeat (LRR) domain. The N-terminal region of the α subunit binds to the active site in the β subunit; residue His2α directly coordinates a zinc ion. The prenyl-binding pocket of RabGGT is deeper than that in FT.Conclusions: LRR and Ig domains are often involved in protein–protein interactions; in RabGGT they might participate in the recognition and binding of REP. The binding of the N-terminal peptide of the α subunit to the active site suggests an autoinhibition mechanism that might contribute to the inability of RabGGT to recognize short peptides or Rab alone as its substrate. Replacement of residues Trp102β and Tyr154β in FT by Ser48β and Leu99β, respectively, in RabGGT largely determine the different lipid-binding specificities of the two enzymes.

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.

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.

Single prenyl-binding site on protein prenyl transferases.

Three distinct protein prenyl transferases, one protein farnesyl transferase (FTase) and two protein geranylgeranyl transferases (GGTase), catalyze prenylation of many cellular proteins. One group of protein substrates contains a C-terminal CAAX motif (C is Cys, A is aliphatic, and X is a variety of amino acids) in which the single cysteine residue is modified with either farnesyl or geranylgeranyl (GG) by FTase or GGTase type-I (GGTase-I), respectively. Rab proteins constitute a second group of substrates that contain a C-terminal double-cysteine motif (such as XXCC in Rab1a) in which both cysteines are geranylgeranylated by Rab GG transferase (RabGGTase). Previous characterization of CAAX prenyl transferases showed that the enzymes form stable complexes with their prenyl pyrophosphate substrates, acting as prenyl carriers. We developed a prenyl-binding assay and show that RabGGTase has a prenyl carrier function similar to the CAAX prenyl transferases. Stable RabGGTase:GG pyrophosphate (GGPP), FTase:GGPP, and GGTase-I:GGPP complexes show 1:1 (enzyme:GGPP) stoichiometry. Chromatographic analysis of prenylated products after single turnover reactions by using isolated RabGGTase:GGPP complex revealed that Rab is mono-geranylgeranylated. This study establishes that all three protein prenyl transferases contain a single prenyl-binding site and suggests that RabGGTase transfers two GG groups to Rabs in independent and consecutive reactions.

Mechanism of Rab geranylgeranylation: formation of the catalytic ternary complex.

Rab proteins are geranylgeranylated on one or two C-terminal cysteines by Rab geranylgeranyl transferase (RabGGTase). The reaction is dependent on a Rab-binding protein, termed Rab escort protein (REP). Here, we studied the role of REP in the geranylgeranylation reaction. We first characterized the interaction between REP and ungeranylgeranylated Rab using analytical ultracentrifugation and a fluorescence-based assay. We measured an equilibrium dissociation constant of 0.2 microM for the formation of a 1:1 REP-Rab complex and showed that this interaction relies mostly on ionic bonds and does not involve the two C-terminal cysteine residues. Second, we show that REP is required for recognition of Rab by RabGGTase and therefore that the REP-Rab complex is the true substrate for RabGGTase. Third, we show that free REP inhibits the geranylgeranylation reaction, suggesting that the complex is recognized by RabGGTase primarily via a REP-binding site. Our data suggest a model whereby REP behaves kinetically as an essential activator of the reaction.

The yeast Rab escort protein binds intracellular membranes in vivo and in vitro.

In both mammals and yeast, intracellular vesicular transport depends on the correct shuttling between membrane and cytosol of the Rab/Ypt small G proteins. Membrane association of these proteins requires prenylation by the Rab geranylgeranyl transferase that recognizes a complex formed by the Rab/Ypt protein and the Rab escort protein (REP). After prenylation the Rab/Ypt protein is delivered to the target membranes by REP. Little is known about the early steps of the Rab-REP complex formation and where this association occurs in the cell. Although prenylation is believed to take place in the cytosol, we show that the yeast Rab escort protein Mrs6 is present in both soluble and particulate fractions of cell extracts. Mrs6p is associated with the heavy microsomal fraction that contains endoplasmic reticulum-Golgi membranes but is absent in the plasma membrane, vacuoles, mitochondria, and microsomal subfraction associated with mitochondria. The solubilization pattern of the particulate pool of Mrs6p implies that this protein is peripherally but tightly associated with membranes via hydrophobic interactions and metal ions. We also report that the C terminus of Mrs6p is important for maintaining the solubility of the protein because its deletion or replacement with the C terminus of RabGDI results in a protein that localizes only to membranes.

The Genes Encoding Geranylgeranyl Transferase α-Subunit and Transglutaminase 1 Are Very Closely Linked but Not Functionally Related in Terminally Differentiating Keratinocytes

We report here the entire exon/intron structure of the gene encoding the α-subunit of human Rab geranylgeranyl transferase (RABGGTA) gene, which is positioned in a tandem head-to-tail arrangement with the transglutaminase 1 (TGM1) gene, and its polyadenylation signal sequence is located just 2.3 kbp upstream of the capsite ofTGM1.Even thoughTGM1andRABGGTAhave different functions, their close localization raised the question as to whether they are functionally related in the epidermis. To address this question, we have studied the expression of the two genes by RT-PCR in normal human epidermal keratinocytes cultured under various differentiation conditions. While the expression of theTGM1gene is markedly affected by the calcium concentration of the medium, all trans retinoic acid, vitamin D3, and TPA treatment, the expression of theRABGGTAgene was unaffected by these reagents. Taken together, even though these two genes are unusually closely linked, they are not functionally related in the terminal differentiation program of epidermal keratinocytes.

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.