Isoprenylation of Rab1B is impaired by mutations in its effector domain (original) (raw)
Related papers
Journal of Biological Chemistry
~21'"" 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 carboxylterminal sequence motif, Cuu(M/S/L), where u is an aliphatic amino acid. Proteins ending with M or S are substrates for farnesyltransferase, whereas those ending with L are modifed preferentially by geranylgeranyltransferase. We recently reported that GTP-binding proteins encoded by rublB (GGCC), rub2 (GGCC), and rub5 (CCSN) are modified by 20-carbon isoprenyl derivatives of [3H]mevalonate when translated in vitro, despite having carboxyl-terminal sequences distinct from the Cuu(M/S/L) motif. We now show that
Isoprenylation of rab proteins on structurally distinct cysteine motifs
Journal of Cell Science, 1992
rab proteins are low molecular weight GTP-binding proteins highly related to Ypt1p and Sec4p, which are involved in the control of secretion in yeast Saccharomyces cerevisiae. Morphological and biochemical studies have shown that rab proteins are membrane associated and are localized to specific subcompartments along the exocytic and endocytic pathway. Membrane association requires the presence of C-terminal cysteine residues. The present report indicates that the structurally distinct cysteine motifs of rab proteins are subjected to isoprenylation both in vitro and in vivo. Studies on deletion mutants suggest that an intact C-terminal end is required for the association of rab proteins with the membrane and is necessary for the post-translational modification. Finally, we show that the isoprenoid transferase which modifies rab termini is different from the enzyme which farnesylates nuclear lamins and ras proteins in vitro.
Isoprenoid modification of rab proteins terminating in CC or CXC motifs
Proceedings of the National Academy of Sciences, 1991
Mevalonate starvation of hamster fibroblasts resulted in a shift of rab1b from the membrane to the cytosolic fraction, suggesting that rab1b depends upon an isoprenoid modification for its membrane localization. rab1b and rab3a proteins expressed in insect cells incorporated a product of [3H]mevalonate, and gas chromatography analysis of material released by Raney nickel cleavage demonstrated that rab1b and rab3a are modified by geranylgeranyl groups. Additionally, in vitro prenylation analysis demonstrated farnesyl modification of H-ras but geranylgeranyl modification of five rab proteins (1a, 1b, 2, 3a, and 6). Together, these results suggest that the carboxyl-terminal CC/CXC motifs (X = any amino acid) specifically signal for addition of geranylgeranyl, but not farnesyl, groups. A rab1b mutant protein lacking the two carboxyl-terminal cysteine residues was not prenylated in vitro. However, since a mutant H-ras protein that terminates with tandem cysteine residues was also not mod...
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 ...
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
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).
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
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 .