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.     

Photoreceptor cGMP phosphodiesterase delta subunit (PDEdelta) functions as a prenyl-binding protein

Bovine PDEdelta was originally copurified with rod cGMP phosphodiesterase (PDE) and shown to interact with prenylated, carboxymethylated C-terminal Cys residues. Other studies showed that PDEdelta can interact with several small GTPases including Rab13, Ras, Rap, and Rho6, all of which are prenylated, as well as the N-terminal portion of retinitis pigmentosa GTPase regulator and Arl2/Arl3, which are not prenylated. We show by immunocytochemistry with a PDEdelta-specific antibody that PDEdelta is present in rods and cones. We find by yeast two-hybrid screening with a PDEdelta bait that it can interact with farnesylated rhodopsin kinase (GRK1) and that prenylation is essential for this interaction. In vitro binding assays indicate that both recombinant farnesylated GRK1 and geranylgeranylated GRK7 co-precipitate with a glutathione S-transferase-PDEdelta fusion protein. Using fluorescence resonance energy transfer techniques exploiting the intrinsic tryptophan fluorescence of PDEdelta and dansylated prenyl cysteines as fluorescent ligands, we show that PDEdelta specifically binds geranylgeranyl and farnesyl moieties with a Kd of 19.06 and 0.70 microm, respectively. Our experiments establish that PDEdelta functions as a prenyl-binding protein interacting with multiple prenylated proteins.     

Prenylated Rab acceptor protein is a receptor for prenylated small GTPases

Localization of Ras and Ras-like proteins to the correct subcellular compartment is essential for these proteins to mediate their biological effects. Many members of the Ras superfamily (Ha-Ras, N-Ras, TC21, and RhoA) are prenylated in the cytoplasm and then transit through the endomembrane system on their way to the plasma membrane. The proteins that aid in the trafficking of the small GTPases have not been well characterized. We report here that prenylated Rab acceptor protein (PRA1), which others previously identified as a prenylation-dependent receptor for Rab proteins, also interacts with Ha-Ras, RhoA, TC21, and Rap1a. The interaction of these small GTPases with PRA1 requires their post-translational modification by prenylation. The prenylation-dependent association of PRA1 with multiple GTPases is conserved in evolution; the yeast PRA1 protein associates with both Ha-Ras and RhoA. Earlier studies reported the presence of PRA1 in the Golgi, and we show here that PRA1 co-localizes with Ha-Ras and RhoA in the Golgi compartment. We suggest that PRA1 acts as an escort protein for small GTPases by binding to the hydrophobic isoprenoid moieties of the small GTPases and facilitates their trafficking through the endomembrane system.     

RhoGDI facilitates geranylgeranyltransferase-I-mediated RhoA prenylation

Protein prenylation is a post-translational modification where farnesyl or geranylgeranyl groups are enzymatically attached to a C-terminal cysteine residue. This modification is essential for the activity of small cellular GTPases, as it allows them to associate with intracellular membranes. Dissociated from membranes, prenylated proteins need to be transported through the aqueous cytoplasm by protein carriers that shield the hydrophobic anchor from the solvent. One such carrier is Rho GDP dissociation inhibitor (RhoGDI). Recently, it was shown that prenylated Rho proteins that are not associated with RhoGDI are subjected to proteolysis in the cell. We hypothesized that the role of RhoGDI might be not only to associate with prenylated proteins but also to regulate the prenylation process in the cell. This idea is supported by the fact that RhoGDI binds both unprenylated and prenylated Rho proteins with high affinity in vitro, and hence, these interactions may affect the kinetics of prenylation. We addressed this question experimentally and found that RhoGDI increased the catalytic efficiency of geranylgeranyl transferase-I in RhoA prenylation. Nevertheless, we did not observe formation of a ternary RhoGDI∗RhoA∗GGTase-I complex, indicating sequential operation of geranylgeranyltransferase-I and RhoGDI. Our results suggest that RhoGDI accelerates Rho prenylation by kinetically trapping the reaction product, thereby increasing the rate of product release.     

Postprenylation CAAX processing is required for proper localization of Ras but not Rho GTPases

The CAAX motif at the C terminus of most monomeric GTPases is required for membrane targeting because it signals for a series of three posttranslational modifications that include isoprenylation, endoproteolytic release of the C-terminal- AAX amino acids, and carboxyl methylation of the newly exposed isoprenylcysteine. The individual contributions of these modifications to protein trafficking and function are unknown. To address this issue, we performed a series of experiments with mouse embryonic fibroblasts (MEFs) lacking Rce1 (responsible for removal of the -AAX sequence) or Icmt (responsible for carboxyl methylation of the isoprenylcysteine). In MEFs lacking Rce1 or Icmt, farnesylated Ras proteins were mislocalized. In contrast, the intracellular localizations of geranylgeranylated Rho GTPases were not perturbed. Consistent with the latter finding, RhoGDI binding and actin remodeling were normal in Rce1- and Icmt-deficient cells. Swapping geranylgeranylation for farnesylation on Ras proteins or vice versa on Rho proteins reversed the differential sensitivities to Rce1 and Icmt deficiency. These results suggest that postprenylation CAAX processing is required for proper localization of farnesylated Ras but not geranygeranylated Rho proteins.     

Isoprenylation of Rab proteins possessing a C-terminal CaaX motif

Rab proteins are small GTPases highly related to the yeast Ypti and Sec4 proteins involved in secretion. The Rab proteins were found associated with membranes of different compartments along the secretory and endocytic pathways. They share distinct C-terminal cysteine motifs required for membrane association. Unlike the other Rab proteins, Rab8, Rab11 and Rab13 terminate with a C-terminal CaaX motif similar to those of Ras/Rho proteins. This report demonstrates that Rab8 and Rab13 proteins are isoprenylated in vivo and geranylgeranylated in vitro. Rab11 associates in vitro geranylgeranylpyrophosphate and farnesylpyrophosphate. Our study shows that the CaaX motif is required for isoprenylation.     

Splice Variants of SmgGDS Control Small GTPase Prenylation and Membrane Localization

Ras and Rho small GTPases possessing a C-terminal polybasic region (PBR) are vital signaling proteins whose misregulation can lead to cancer. Signaling by these proteins depends on their ability to bind guanine nucleotides and their prenylation with a geranylgeranyl or farnesyl isoprenoid moiety and subsequent trafficking to cellular membranes. There is little previous evidence that cellular signals can restrain nonprenylated GTPases from entering the prenylation pathway, leading to the general belief that PBR-possessing GTPases are prenylated as soon as they are synthesized. Here, we present evidence that challenges this belief. We demonstrate that insertion of the dominant negative mutation to inhibit GDP/GTP exchange diminishes prenylation of Rap1A and RhoA, enhances prenylation of Rac1, and does not detectably alter prenylation of K-Ras. Our results indicate that the entrance and passage of these small GTPases through the prenylation pathway is regulated by two splice variants of SmgGDS, a protein that has been reported to promote GDP/GTP exchange by PBR-possessing GTPases and to be up-regulated in several forms of cancer. We show that the previously characterized 558-residue SmgGDS splice variant (SmgGDS-558) selectively associates with prenylated small GTPases and facilitates trafficking of Rap1A to the plasma membrane, whereas the less well characterized 607-residue SmgGDS splice variant (SmgGDS-607) associates with nonprenylated GTPases and regulates the entry of Rap1A, RhoA, and Rac1 into the prenylation pathway. These results indicate that guanine nucleotide exchange and interactions with SmgGDS splice variants can regulate the entrance and passage of PBR-possessing small GTPases through the prenylation pathway.     

Rab GTPases containing a CAAX motif are processed post-geranylgeranylation by proteolysis and methylation

Post-translational modification by protein prenylation is required for membrane targeting and biological function of monomeric GTPases. Ras and Rho proteins possess a C-terminal CAAX motif (C is cysteine, A is usually an aliphatic residue, and X is any amino acid), in which the cysteine is prenylated, followed by proteolytic cleavage of the AAX peptide and carboxyl methylation by the Rce1 CAAX protease and Icmt methyltransferase, respectively. Rab GTPases usually undergo double geranylgeranylation within CC or CXC motifs. However, very little is known about processing and membrane targeting of Rabs that naturally contain a CAAX motif. We show here that a variety of Rab-CAAX proteins undergo carboxyl methylation, both in vitro and in vivo, with one exception. Rab38(CAKS) is not methylated in vivo, presumably because of the inhibitory action of the lysine residue within the AAX motif for cleavage by Rce1. Unlike farnesylated Ras proteins, we observed no targeting defects of overexpressed Rab-CAAX proteins in cells deficient in Rce1 or Icmt, as reported for geranylgeranylated Rho proteins. However, endogenous geranylgeranylated non-methylated Rab-CAAX and Rab-CXC proteins were significantly redistributed to the cytosol at steady-state levels and redistribution correlates with higher affinity of RabGDI for non-methylated Rabs in Icmt-deficient cells. Our data suggest a role for methylation in Rab function by regulating the cycle of Rab membrane recruitment and retrieval. Our findings also imply that those Rabs that undergo post-prenylation processing follow an indirect targeting pathway requiring initial endoplasmic reticulum membrane association prior to specific organelle targeting.     

Analysis of the small GTPase gene superfamily of Arabidopsis

Small GTP-binding proteins regulate diverse processes in eukaryotic cells such as signal transduction, cell proliferation, cytoskeletal organization, and intracellular membrane trafficking. These proteins function as molecular switches that cycle between "active" and "inactive" states, and this cycle is linked to the binding and hydrolysis of GTP. The Arabidopsis genome contains 93 genes that encode small GTP-binding protein homologs. Phylogenetic analysis of these genes shows that plants contain Rab, Rho, Arf, and Ran GTPases, but no Ras GTPases. We have assembled complete lists of these small GTPases families, as well as accessory proteins that control their activity, and review what is known of the functions of individual members of these families in Arabidopsis. We also discuss the possible roles of these GTPases in relation to their similarity to orthologs with known functions and localizations in yeast and/or animal systems.     

Rho guanine dissociation inhibitors: pivotal molecules in cellular signalling.

The small G proteins of the Ras family act as bimodal relays in the transfer of intracellular signals. This is a dynamic phenomenon involving a cascade of protein-protein interactions modulated by chemical modifications, structural rearrangements and intracellular relocalisations. Most of the small G proteins could be operationally defined as proteins having two conformational states, each of which interacts with different cellular partners. These two states are determined by the nature of the bound nucleotide, GDP or GTP. This capacity to cycle between a GDP-bound conformation and a GTP-bound conformation enables them to filter, to amplify or to temporise the upstream signals that they receive. Thus the control of this cycle is crucial. Membrane anchoring of the proteins in the Ras family is a prerequisite for their activity. Most of the proteins in the Rho/Rac and Rab subfamilies of Ras proteins cycle between cytosol and membranes. Then the control of membrane association/dissociation is an other important regulation level. This review will describe one family of crucial regulators acting on proteins in the Rho/Rac family-the Rho guanine nucleotide dissociation inhibitors, or RhoGDIs. As yet, only three RhoGDIs have been described: RhoGDI-1, RhoGDI-2 (or D4/Ly-GDI) and RhoGDI-3. RhoGDI 1 and 2 are cytosolic and participate in the regulation of both the GDP/GTP cycle and the membrane association/dissociation cycle of Rho/Rac proteins. The non-cytosolic RhoGDI-3 seems to act in a slightly different way.     

Cell migration and signaling specificity is determined by the phosphatidylserine recognition motif of Rac1

The Rho guanosine triphosphatases (GTPases) control cell shape and motility and are frequently overexpressed during malignant growth. These proteins act as molecular switches cycling between active GTP- and inactive GDP-bound forms. Despite being membrane anchored via their isoprenylated C termini, Rho GTPases rapidly translocate between membrane and cytosolic compartments. Here, we show that the Rho GTPase Rac1 preferentially interacts with phosphatidylserine (PS)-containing bilayers through its polybasic motif (PBM). Rac1 isoprenylation contributes to membrane avidity but is not critical for PS recognition. The similar protein Cdc42 (cell division cycle 42), however, only associates with PS when prenylated. Conversely, other Rho GTPases such as Rac2, Rac3, and RhoA do not bind to PS even when they are prenylated. Cell stimulation with PS induces translocation of Rac1 toward the plasma membrane and stimulates GTP loading, membrane ruffling, and filopodia formation. This stimulation also promotes Cdc42 activation and phosphorylation of mitogen-activated protein kinase through Rac1/PS signaling. Consequently, the PBM specifically directs Rac1 to effect cytoskeletal rearrangement and cell migration by selective membrane phospholipid targeting.     

Targeting Rab GTPases to distinct membrane compartments

Rab GTPases are key to membrane-trafficking events in eukaryotic cells, and human cells contain more than 60 Rab proteins that are localized to distinct compartments. The recent determination of the structure of a monoprenylated Rab GTPase bound to GDP-dissociation inhibitor provides new molecular details that are relevant to models of Rab delivery. The further discovery of an integral membrane protein that can dissociate prenylated Rab proteins from GDP-dissociation inhibitor gives new insights into the mechanisms of Rab localization.     

ExoS Rho GTPase-activating protein activity stimulates reorganization of the actin cytoskeleton through Rho GTPase guanine nucleotide disassociation inhibitor

ExoS is a bifunctional Type III cytotoxin of Pseudomonas aeruginosa with N-terminal Rho GTPase-activating protein (RhoGAP) and C-terminal ADP-ribosyltransferase domains. Although the ExoS RhoGAP inactivates Cdc42, Rac, and RhoA in vivo, the relationship between ExoS RhoGAP and the eukaryotic regulators of Rho GTPases is not clear. The present study investigated the roles of Rho GTPase guanine nucleotide disassociation inhibitor (RhoGDI) in the reorganization of actin cytoskeleton mediated by ExoS RhoGAP. A green fluorescent protein-RhoGDI fusion protein was engineered and found to elicit actin reorganization through the inactivation of Rho GTPases. Green fluorescent protein-RhoGDI and ExoS RhoGAP cooperatively stimulated actin reorganization and translocation of Cdc42 from membrane to cytosol, and a RhoGDI mutant, RhoGDI(I177D), that is defective in extracting Rho GTPases off the membrane inhibited the actions of RhoGDI and ExoS RhoGAP on the translocation of Cdc42 from membrane to cytosol. A human RhoGDI small interfering RNA was transfected into HeLa cells to knock down 90% of the endogenous RhoGDI expression. HeLa cells with knockdown RhoGDI were resistant to the reorganization of the actin cytoskeleton elicited by type III-delivered ExoS RhoGAP. This indicates that ExoS RhoGAP and RhoGDI function in series to inactivate Rho GTPases, in which RhoGDI extracting GDP-bound Rho GTPases off the membrane and sequestering them in cytosol is the rate-limiting step in Rho GTPase inactivation. A eukaryotic GTPase-activating protein, p50RhoGAP, showed a similar cooperativity with RhoGDI on actin reorganization, suggesting that ExoS RhoGAP functions as a molecular mimic of eukaryotic RhoGAPs to inactivate Rho GTPases through RhoGDI.     

The Chaperone Protein SmgGDS Interacts with Small GTPases Entering the Prenylation Pathway by Recognizing the Last Amino Acid in the CAAX Motif

Ras family small GTPases localize at the plasma membrane, where they can activate oncogenic signaling pathways. Understanding the mechanisms that promote membrane localization of GTPases will aid development of new therapies to inhibit oncogenic signaling. We previously reported that SmgGDS splice variants promote prenylation and trafficking of GTPases containing a C-terminal polybasic region and demonstrated that SmgGDS-607 interacts with nonprenylated GTPases, whereas SmgGDS-558 interacts with prenylated GTPases in cells. The mechanism that SmgGDS-607 and SmgGDS-558 use to differentiate between prenylated and nonprenylated GTPases has not been characterized. Here, we provide evidence that SmgGDS-607 associates with GTPases through recognition of the last amino acid in the CAAX motif. We show that SmgGDS-607 forms more stable complexes in cells with nonprenylated GTPases that will become geranylgeranylated than with nonprenylated GTPases that will become farnesylated. These binding relationships similarly occur with nonprenylated SAAX mutants. Intriguingly, farnesyltransferase inhibitors increase the binding of WT K-Ras to SmgGDS-607, indicating that the pharmacological shunting of K-Ras into the geranylgeranylation pathway promotes K-Ras association with SmgGDS-607. Using recombinant proteins and prenylated peptides corresponding to the C-terminal sequences of K-Ras and Rap1B, we found that both SmgGDS-607 and SmgGDS-558 directly bind the GTPase C-terminal region, but the specificity of the SmgGDS splice variants for prenylated versus nonprenylated GTPases is diminished in vitro. Finally, we present structural homology models and data from functional prediction software to define both similar and unique features of SmgGDS-607 when compared with SmgGDS-558.     

Self-assemblies of Rab- and Arf-family small GTPases on lipid bilayers in membrane tethering

Small GTPases of the Ras superfamily, which include Ras-, Rho-, Rab-, Arf-, and Ran-family isoforms, are generally known to function as a nucleotide-dependent molecular switch in eukaryotic cells. In the GTP-loaded forms, they selectively recruit their cognate interacting proteins or protein complexes, termed “effectors,” to the cytoplasmic face of subcellular membrane compartments, thereby switching on the downstream effector functions, which are vital for fundamental cellular events, such as cell proliferation, cytoskeletal organization, and intracellular membrane trafficking. Nevertheless, in addition to acting as the classic nucleotide-dependent switches for the effectors, recent studies have uncovered that small GTPases themselves can be self-assembled specifically into homo-dimers or higher-order oligomers on membranes, and these assembly processes are likely responsible for their physiological functions. This Review focuses particularly on the self-assembly processes of Rab- and Arf-family isoforms during membrane tethering, the most critical step to ensure the fidelity of membrane trafficking. A summary of the current experimental evidence for self-assemblies of Rab and Arf small GTPases on lipid bilayers in chemically defined reconstitution system is provided     

A structural model of the GDP dissociation inhibitor rab membrane extraction mechanism

Rab GDP dissociation inhibitors (GDI)-facilitated extraction of prenylated Rab proteins from membranes plays an important role in vesicular membrane trafficking. The investigated thermodynamic properties of yeast Rab.GDI and Rab.MRS6 complexes demonstrated differences in the Rab binding properties of the closely related Rab GDI and MRS6 proteins, consistent with their functional diversity. The importance of the Rab C terminus and its prenylation for GDI/MRS6 binding was demonstrated using both biochemical and structural data. The presented structures of the apo-form yeast Rab GDI and its two complexes with unprenylated Rab proteins, together with the earlier published structures of the prenylated Ypt1.GDI, provide evidence of allosteric regulation of the GDI lipid binding site opening, which plays a key role in the proposed mechanism of GDI-mediated Rab extraction. We suggest a model for the interaction of GDI with prenylated Rab proteins that incorporates a stepwise increase in affinity as the three different partial interactions are successively formed.     

Analysis of Rab GTPase-activating proteins indicates that Rab1a/b and Rab43 are important for herpes simplex virus 1 secondary envelopment

Assembly of herpes simplex virus 1 (HSV-1) occurs in the cytoplasm, where the capsid and tegument bud into host cell membranes. It is at this point that the viral glycoproteins are incorporated into the virion, as they are located at the assembly site. We investigated the role of the Rab GTPases in coordinating the assembly process by overexpressing 37 human Rab GTPase-activating proteins (GAPs) and assessing infectious titers. Rab GTPases are key cellular regulators of membrane trafficking events that, by their membrane association and binding of effector proteins, ensure the appropriate fusion of membranes. We identified that TBC1D20 and RN-tre and their partner Rabs, Rab1a/b and Rab43, respectively, are important for virion assembly. In the absence of Rab1a/b, the viral glycoproteins are unable to traffic from the endoplasmic reticulum to the assembly compartment, and thus unenveloped particles build up in the cytoplasm. The defect resulting from Rab43 depletion is somewhat more complex, but it appears that the fragmentation and dispersal of the trans-Golgi network and associated membranes render these compartments unable to support secondary envelopment.     

Structure of doubly prenylated Ypt1:GDI complex and the mechanism of GDI-mediated Rab recycling

In eukaryotic cells Rab/Ypt GTPases represent a family of key membrane traffic controllers that associate with their targeted membranes via C-terminally conjugated geranylgeranyl groups. GDP dissociation inhibitor (GDI) is a general and essential regulator of Rab recycling that extracts prenylated Rab proteins from membranes at the end of their cycle of activity and facilitates their delivery to the donor membranes. Here, we present the structure of a complex between GDI and a doubly prenylated Rab protein. We show that one geranylgeranyl residue is deeply buried in a hydrophobic pocket formed by domain II of GDI, whereas the other lipid is more exposed to solvent and is skewed across several atoms of the first moiety. Based on structural information and biophysical measurements, we propose mechanistic and thermodynamic models for GDI and Rab escort protein-mediated interaction of RabGTPase with intracellular membranes.     

The polybasic region of Ras and Rho family small GTPases: a regulator of protein interactions and membrane association and a site of nuclear localization signal sequences

Many small GTPases in the Ras and Rho families have a C-terminal polybasic region (PBR) comprised of multiple lysines or arginines. The PBR controls diverse functions of these small GTPases, including their ability to associate with membranes, interact with specific proteins, and localize in subcellular compartments. Different signaling pathways mediated by Ras and Rho family members may converge when the small GTPases are directed by their PBRs to shared binding sites in specific proteins or at cell membranes. The PBR promotes the interactions of small GTPases with SmgGDS, which is a nucleocytoplasmic shuttling protein that stimulates guanine nucleotide exchange by small GTPases. The PBR of Rac1 was recently found to have a functional nuclear localization signal (NLS) sequence, which enhances the nuclear accumulation of protein complexes containing SmgGDS and Rac1. Sequence analysis demonstrates that canonical NLS sequences (K-K/R-x-K/R) are present in the PBRs of additional Ras and Rho family members, and are evolutionarily conserved across several phyla. These findings suggest that the PBR regulates the nucleocytoplasmic shuttling of some Ras and Rho family members when they are in protein complexes that are too large to diffuse through nuclear pores. These diverse functions of the PBR indicate its critical role in signaling by Ras and Rho family GTPases.