Thematic review series: Lipid Posttranslational Modifications. Farnesyl transferase inhibitors (original) (raw)
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Thematic review series: Lipid Posttranslational Modifications. Geranylgeranylation of Rab GTPases
J Lipid Res, 2005
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.-Leung, K. F., R. Baron, and M. C. Seabra. Geranylgeranylation of Rab GTPases. J. Lipid Res. 2006. 47: 467-475. Supplementary key words prenyl transferases . Rab escort protein .
Prenylation of Rab8 GTPase by type I and type II geranylgeranyl transferases
The Biochemical journal, 1998
Rab GTPases are post-translationally modified by addition of geranylgeranyl moieties to carboxyl-terminal cysteine residues. For Rab proteins ending with xxCC xCxC and CCxx motifs this modification is catalysed by geranylgeranyltransferase type II (GGTaseII), and is entirely dependent on the Rab substrate being bound to Rab escort protein (REP). Several Rab proteins contain carboxyl-terminal CaaL prenylation motifs typical of members of the Rho family, which are modified in a REP-independent manner by geranylgeranyltransferase type I (GGTaseI). The present studies show that one such Rab protein (Rab8), which ends with a CVLL motif, is uniquely able to serve as a substrate for either REP/GGTaseII or GGTaseI in cell-free assays. The modification of Rab8 by GGTaseI did not require REP, indicating that a REP-induced conformational change is not essential for exposure of the Rab carboxyl-terminal cysteine prenylation site. To determine whether one enzyme plays a predominant role in Rab8 ...
Membrane targeting of Rab GTPases is influenced by the prenylation motif
Molecular Biology of the Cell, 2003
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 (Rab27a ash ) 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 RabGDI␣ and propose that the mistargeting of Rabs to the ER results from loss of targeting information.
Biochemical Journal, 2008
Prenylation (or Geranylgeranylation, GG) of Rab GTPases is catalysed by Rab Geranylgeranyl Transferase (RGGT) and requires Rab Escort Protein (REP). In the classical pathway, REP associates first with unprenylated Rab which is then prenylated by RGGT. In the alternative pathway, REP associates first with RGGT; this complex then binds and prenylates Rab proteins. Here we show that REP mutants (REP1 F282L and REP1 F282L/V290F ) defective in RGGT binding are unable to compete with wild-type REP in the prenylation reaction in vitro. When over-expressed in cells, REP wild type and mutants are unable to form stable cytosolic complexes with endogenous unprenylated Rabs. These results suggest that the alternative pathway may predominate in vivo. We also extend previous suggestions that GGPP acts as an allosteric regulator of the reaction. We observed that REP:RGGT complexes are formed in vivo and are unstable in absence of intracellular GGPP. RGGT increases the ability of REP to extract endogenous prenylated Rabs from membranes in vitro by stabilising a soluble REP:RGGT:Rab-GG complex. This effect is regulated by GGPP, which promotes the dissociation of RGGT and REP:Rab-GG to allow delivery of prenylated Rabs to membranes.
Structure of Rab Escort Protein-1 in Complex with Rab Geranylgeranyltransferase
Molecular Cell, 2003
In contrast to other known prenyltransferases, 1 Department of Biomolecular Mechanisms RabGGTase does not recognize its protein substrate Max-Planck-Institute for Medical Research directly, but functions in concert with a 75 kDa protein Jahnstrasse 29 termed REP (Rab escort protein) (Andres et al., 1993). 69120 Heidelberg REP protein displays several stretches of high sequence 2 Max-Planck-Institute for Molecular Physiology homology (known as sequence conserved regions, or Otto-Hahn-Strasse 11 SCRs, 1A, 1B, 2, 3A, and 3B) to GDP-dissociation inhibi-44227 Dortmund tor (RabGDI). RabGDIs are the central regulators of Rab Germany protein function that are responsible for delivery and 3 Department of Molecular Biology removal of Rab proteins to and from membranes (re-Moscow State University viewed in Alory and Balch, 2001). Disruption of either of Moscow, 119899 the proteins leads to abnormalities in humans (reviewed Russia in Pereira-Leal et al., 2001; Alory and Balch, 2001) Based on this homology, the RabGDI and REP molecules were proposed to form a RabGDI/REP(CHM) fam-Summary ily of structurally and functionally related proteins (Alory and Balch, 2001). Crystal structure analysis of mamma-Posttranslational geranylgeranylation of Rab GTPases lian ␣RabGDI revealed a two-domain structure with a is catalyzed by Rab geranylgeranyltransferase (RabGGTmultisheet domain I composed of SCR1 and 3B and a ase), which consists of a catalytic ␣/ heterodimer and smaller ␣-helical domain II composed of SCR 2 and an accessory Rab escort protein (REP). The crystal 3A (Schalk et al. , 1996). Mutagenesis experiments have structure of isoprenoid-bound RabGGTase complexed defined a set of closely positioned conserved residues to REP-1 has been solved to 2.7 Å resolution. The in domain I, which comprise a putative Rab binding complex interface buries a surprisingly small surface platform that appears to be conserved between REPs area of ca. 680 Å and is unexpectedly formed by helices and GDIs (Wu et al., 1998; Alory and Balch, 2000). Later, 8, 10, and 12 of the RabGGTase ␣ subunit and helices an additional structural element, termed the GDI mobile D and E of REP-1. We demonstrate that the affinity of effector loop, was identified and shown to be involved RabGGTase for REP-1 is allosterically regulated by in retrieval of Rab proteins from the membrane, presumphosphoisoprenoid via a long-range trans-domain ably via interaction with a putative membrane receptor signal transduction event. Comparing the structure of (Luan et al., 2000).
Characterization of the ternary complex between Rab7, REP-1 and Rab geranylgeranyl transferase
European Journal of Biochemistry, 1999
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 K d value of about 120 nm. Association of RabGGTase with the Rab7±REP complex occurs with a rate constant of < 10 8 m 21´s21. 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.
Geranylgeranylation of Rab proteins
Biochemical Society Transactions, 1996
Protein prenylation is a type of lipid modification that affects about 0.5% of cellular proteins [l]. Prenylated proteins are covalently modified with either farnesyl or geranylgeranyl (GG) via thioether bonds to C-terminal cysteine residues
Prenylation of Rab GTPases: molecular mechanisms and involvement in genetic disease
Febs Letters, 2001
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 K K-subunit of RGGT. Here we discuss recent insights into Rab prenylation and advances towards our understanding of both diseases. ß
Protein Expression and Purification, 2001
protein trafficking in eukaryotic cells. In mammalian Mammalian geranylgeranyltransferase type II cells more than 40 Rabs have been cloned and this (GGTase-II) is a 100-kDa heterodimer that catalyzes the number is increasing (1, 2). Individual Rab proteins transfer of two 20-carbon geranylgeranyl groups from have been implicated as essential mediators of vesicle geranylgeranyl pyrophosphate onto C-terminal cystargeting and fusion events (3, 4). For their function, teine residues of Rab GTPases. This modification is Rab proteins require modification by geranylgeranyl essential for the biological activity of Rab proteins. isoprenoids which allows them to reversibly associate Geranylgeranylation can be performed in vitro using with membrane in a tightly controlled fashion. Covalent recombinant GGTase-II but so far large-scale producattachment of geranylgeranyl groups to two C-terminal tion of the enzyme was challenging. We report here the cysteines is catalyzed by geranylgeranyltransferase design of a two plasmid expression system that will type II (GGTase-II). GGTase-II is a heterodimer comproduce GGTase-II at levels as high as 15 mg/L in Escheposed of tightly associated 68-kDa ␣ and 45-kDa  subrichia coli. The protein was produced as a heterodimer units and belongs to the family of protein prenyltransfwith the ␣ subunit bearing a cleavable tandem 6Hiserases together with farnesyl transferase (FTase) and glutathione S-transferase (GST) tag that was used for geranylgeranyl transferase I (GGTase-I) (for review 5). two-step purification of the enzyme. Purified enzyme In contrast to other known prenyltransferases, was functionally active as determined by in vitro pre-GGTase-II does not recognize its protein substrate dinylation and phosphoisoprenoid binding assay. Furrectly, but functions in a concert with a protein chaperthermore, the GST-tagged GGTase-II was used for one termed REP (Rab escort protein) (6). preparative in vitro prenylation of the Rab7:REP-1 Posttranslational geranylgeranylation is crucial for complex. Using this procedure, 10 mg of doubly prenylated Rab7:REP-1 complex were obtained. ᭧ 2001 Aca-biological activity of Rab proteins since it ensures not demic Press only membrane association but also modulates the interaction of Rab proteins with their effectors and regulatory molecules. Traditionally, prenylated Rab proteins were either purified from tissue sources or from The low molecular mass GTP 2-binding proteins encoded by the Rab gene family play important roles in