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.—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 . GDP dissociation inhibitor . geranylgeranyl Members of the small GTPase Ras superfamily perform important regulatory functions, from cell growth to cytoskeleton dynamics to membrane trafficking. With the exception of Ran, Ras-like proteins undergo cotranslational or posttranslational lipid modifications, which act as hydrophobic membrane anchors, interacting with the cytoplasmic leaflet of cellular membranes and/or participating in protein-protein interactions. The most common lipid modification affecting small GTPases is protein prenylation, which involves the covalent addition of either farnesyl (15 carbon) or geranylgeranyl (20 carbon) pyrophosphate to proteins via thioether linkages catalyzed by protein prenyl transferases (1). Prenylation of Ras, Rho/ Rac, and Rab is absolutely critical for the proper function of the modified protein in cellular processes (reviewed in the other reviews in this series). The importance of protein prenylation first gained focus when it was found that oncogenic forms of Ras proteins required prenylation for their ability to transform cells (2, 3). Since then, the search for inhibitors of prenylation has been an active area of research [reviewed in this series (4)]. Three distinct protein prenyl transferases have been identified and can be classified into two main functional classes: the CAAX prenyl transferases, consisting of farnesyl transferase (FT) and geranylgeranyl transferase type I (GGT-I) [reviewed in this series by Lane and Beese (4a)], and the Rab geranylgeranyl transferase (RGGT, also known as GGT-II) (1). Substrates for the first class include CAAXcontaining farnesylated proteins (Ras, nuclear lamins, and others) and geranylgeranylated proteins of the Rho/Rac families and others. Rab protein family members are exclusive substrates of RGGT. THE RAB PROTEIN FAMILY: STRUCTURE/FUNCTION The Rab proteins (ras genes from rat brain), comprising .60 proteins, form the largest family of the Ras superfamily of small GTPases and are important regulators of organelle biogenesis and vesicle transport (5). They are conserved throughout evolution, from yeast to mammals (6). Some Rab proteins are ubiquitously expressed, whereas others are expressed in a tissue-specific or developmentally regulated manner. For example, Rab1 is found in all cell types, whereas others, such as Rab27a, are found in melanocytes and secretory cell types (7). Analysis of the budding yeast Saccharomyces cerevisiae genome indicates that there are 11 Rab genes called Ypt (yeast protein involved in transport), some of which are redundant Manuscript received 14 December 2005 and in revised form 5 January 2006. Published, JLR Papers in Press, January 9, 2006. DOI 10.1194/jlr.R500017-JLR200 1 To whom correspondence should be addressed. e-mail: [email protected] Copyright I 2006 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at http://www.jlr.org Journal of Lipid Research Volume 47, 2006 467 by gest, on A uust 8, 2017 w w w .j.org D ow nladed fom in their function (8). In mammals, .60 Rab proteins have been identified, which is not surprising considering the increase in complexity of trafficking pathways required to carry out diverse functions in a variety of different cell types. The evolutionary conservation of Rabs is highlighted by the fact that mouse Rab1a can compensate for the loss of Ypt1p in yeast (9). Studies of Rab protein function suggest that they are important in vesicular membrane transport (10, 11). Eukaryotic cells possess an elaborate internal membrane system composed of different intracellular compartments, each serving a different function. These compartments are highly dynamic and communicate with each other. Each Rab protein has a specific intracellular localization and thus regulates a specific membrane trafficking step. However, transport between two membrane compartments may be governed by more than one Rab member; thus, some Rab proteins may exhibit redundancy in their roles. All members of the Ras superfamily have conserved regions that are involved in binding guanine nucleotide and phosphate/Mg; these have been referred to previously as G1–G3 and PM1–PM3, respectively (12). There are two regions that undergo a significant conformational change upon GTP binding and hydrolysis: the switch I domain, which lies in the loop 2 region; and the switch II domain, which resides in the loop 4/a2/loop 5 region. Although the presence of a dicysteine prenylation motif at the C terminus is generally considered a good defining feature of a Rab protein, it is not absolute, as a few Rab proteins, such as Rab8 and Rab13, contain only a single cysteine motif. More recently, diagnostic feature distinguishing to distinguish Rabs from other Ras-like GTPases has been proposed based on sequence alignments of Rab proteins (5). Using this approach, five Rab family regions (RabF) were identified that were conserved only in Rab proteins, thus distinguishing them from other Ras-like proteins (Fig. 1). The RabF1 region is located in the socalled effector domain (loop 2/b2) in the switch I region. The remaining four regions, RabF2 (b3), RabF3 (loop 4), RabF4 (a2/loop 5), and RabF5 (b4/loop 6), all reside in and around the switch II region between b-sheets b3 and b4 (5). Because the RabF regions cluster around the two switch domains, which undergo changes in conformation on binding GDP or GTP, it has been suggested that these regions are involved in binding to general regulators of Rab function, such as Rab GDP dissociation inhibitor (RabGDI) protein and Rab escort protein (REP), as these regulatory proteins are nucleotide-sensitive (e.g., they associate better with the GDP form of Rab proteins) and recognize all Rabs (13, 14). In addition, four Rab subfamily regions (RabSF) were defined as regions of high conservation within subfamilies: RabSF1 (b1), RabSF2 (a1/loop 2), RabSF3 (a3/loop 7), and RabSF4 (a5) (5). RabSF1, RabSF3, and RabSF4 Fig. 1. Crystal structure of the Rab7:Rab escort protein-1 (Rab7:REP-1) complex showing regions conserved within the Rab family. Ribbon representation of REP-1 (white) bound to Rab7 (grayish blue). The Rab family regions (RabF) and Rab subfamily regions (RabSF) are highlighted in red and yellow, respectively. The guanine nucleotide binding regions are shown in green. Geranylgeranyl diphosphate (GGpp; brown) located in the prenyl binding pocket of REP-1 is shown in a ball-and-stick representation. All crystal structures were generated using Accelerys DS ViewerPro 5.0. The Protein Data Bank identifier for Rab7GG:REP-1 is 1VG0. 468 Journal of Lipid Research Volume 47, 2006 by gest, on A uust 8, 2017 w w w .j.org D ow nladed fom correspond to three regions previously referred to as Rab complementary-determining regions I, II, and III, based on the crystal structure of Rab3a complexed with its effector Rabphilin3a (15). Thus, RabSF1, RabSF3, and RabSF4 of Rab3a form a surface that mediates binding to Rabphilin3a, whereas RabSF2 forms another surface on the opposite face of Rab3a and could interact with other effectors. Based on these findings, it was proposed that effectors bind to RabF regions to discriminate between the nucleotidebound states and to RabSF regions to confer specificity. RAB GERANYLGERANYLATION: TWO ALTERNATIVE PATHWAYS Like the CAAX prenyl transferases, RGGT is heterodimeric and consists of distinct aand b-subunits. However, its mechanism of action is distinct from that of the other prenyl transferases. The enzyme was first isolated from rat brain cytosol and purified as a multicomponent enzyme (components A and B) that was able to attach geranylgeranyl groups onto Rab proteins (16, 17). Component B represents the catalytic component, now called RGGT. Unlike the CAAX prenyl transferases, RGGT does not recognize short peptides containing the Rab C-terminal prenylation motif, nor does it recognize the Rab protein alone (17, 18). Instead, it binds component A, now called REP, which is a Rab binding protein (19). Several details concerning the mechanisms of Rab protein prenylation remain unclear. Biochemical assays have led to the proposal of two possible pathways (Fig. 2). The classical mechanism of Rab prenylation implicates first the association of an unprenylated Rab protein with REP (19). The equilibrium dissociation constant was measured to be 0.2 AM (although it varies between Rabs), and the interaction relies mostly on ionic bonds and does not involve the two C-terminal cysteine residues (18). The complex is then recognized by RGGT (Kd, 1 nM), which adds two geranylgeranyl moieties to the Rab protein without prior dissociation of REP (20, 21). After the transfer of the isoprenoids onto C-terminal cysteines, the ternary complex remains associated until the binding of a new geranylgeranyl diphosphate (GGpp) molecule, which stimulates the release of the Rab-GG:REP complex (20). REP is then believed to escort the prenylated Rab protein to its target membrane (22) (Fig. 2). An alternative pathway for Rab prenylation was also proposed (23). Solid phase precipitation assays demonstrated that REP-1 and RGGT can form a tight complex in the presence of GGpp (Kdz 10 nM) (Fig. 2). This complex could associate with Rab protein, but 10 times slower than REP:Rab to RGGT:GGpp. It was proposed that in vivo the pathway followed should depend on the concentrations of the proteins involved. At high concentrations of RGGT, REP, Rab, and GGpp, the association of Rab with the RGGT:GGpp:REP complex becomes rate-determining Fig. 2. Cartoon showing the two possible pathways for Rab protein prenylation. In the classical pathway, newly translated Rabs bind REP and the complex is recognized by GGpp-bound Rab geranylgeranyl transferase (RGGT). RGGT catalyzes the transfer of geranylgeranyl groups to C-terminal cysteines of the Rab protein. After prenylation, RGGT dissociates from REP, which remains bound to the prenylated Rab protein and delivers it to target membranes. REP is then released into the cytosol to take part in a new cycle of prenylation. In the alternative pathway, REP forms a complex with RGGT in the presence of GGpp under conditions in which these constituents are at higher concentrations relative to the Rab protein. The REP:RGGT:GGpp complex then binds newly translated Rab protein and the geranylgeranylation reaction takes place. RGGT dissociates as before, whereas REP escorts the prenylated Rab to membranes as in the classical pathway. Kd values of the Rab:REP:RGGT:GGpp complex for each pathway are indicated. Geranylgeranylation of Rab GTPases 469 by gest, on A uust 8, 2017 w w w .j.org D ow nladed fom and is favored, whereas at low concentrations, the classical pathway is preferred.