In vitro assembly,purification, and crystallization of the rab geranylgeranyl transferase:substrate complex

Posttranslational modification with the geranygeranyl moiety is essential for the ability of Rab GTPases to control processes of membrane docking and fusion. This modification is conferred by Rab geranylgeranyltransferase (RabGGTase), which catalyzes the transfer of two 20-carbon geranylgeranyl groups from geranylgeranyl pyrophosphate onto C-terminal cysteine residues of Rab proteins. The enzyme consists of a catalytic alpha/beta heterodimer and an accessory protein termed Rab escort protein (REP-1) that delivers the newly prenylated Rab proteins to their target membrane. In order to understand the structural basis of Rab prenylation, we have investigated in vitro assembly and crystallization of the RabGGTase:REP-1:Rab complex. In order to ensure maximal stability of the ternary complex, we generated its monoprenylated form, which corresponds to a reaction intermediate and displays the highest affinity between the components. This was achieved by expressing the individual components in baculovirus and Escherichia coli systems with subsequent purification followed by in vitro monoprenylation of Rab7 with immobilized recombinant RabGGTase. Purified monoprenylated REP-1:Rab7 was complexed with recombinant RabGGTase and crystallized in hanging drops. The crystals obtained initially diffract to 8 A on an in-house X-ray source.     

Allosteric regulation of substrate binding and product release in geranylgeranyltransferase type II

GTPases of the Rab family are key components of vesicular transport in eukaryotic cells. Posttranslational attachment of geranylgeranyl moieties is essential for Rab function. Geranylgeranyltransferase type II (GGTase-II) catalyzes the modification of Rab proteins once they are in complex with their escort protein (REP). Upon completion of prenylation, REP and modified Rab leave the enzyme, enabling a new round of catalysis. We have studied the mechanism underlying substrate binding and product release in the geranylgeranylation of Rab proteins. Binding of the Rab7:REP-1 complex to GGTase-II was found to be strongly modulated by geranylgeranyl pyrophosphate (GGpp). The affinity of GGTase-II for the Rab7:REP-1 complex increases from ca. 120 nM to ca. 2 nM in the presence of GGpp. To study the effect of GGpp on interaction of the enzyme with its product, we generated semisynthetic doubly prenylated Rab7 bearing a fluorescent reporter group. Using this novel compound, we demonstrated that the affinity of doubly prenylated Rab7:REP-1 complex for GGTase-II was 2 and 18 nM in the absence and presence of GGpp, respectively. The difference in affinities originates mainly from a difference in the dissociation rates. Thus, binding of the new isoprenoid substrate molecule facilitates the product release by GGTase-II. The affinity of GGpp for the prenylated Rab7:REP-1:GGTase-II was K(d) = 22 nM, with one molecule of GGpp binding per molecule of prenylated ternary complex. We interpreted this finding as an indication that the geranylgeranyl moieties transferred to Rab protein do not occupy the GGpp binding site of the GGTase-II. In summary, these results demonstrate that GGpp acts as an allosteric activator that stabilizes the Rab7:REP-1:GGTase-II complex and triggers product release upon prenylation, preventing product inhibition of the enzyme.     

Thematic review series: lipid posttranslational modifications. 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.     

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 alpha/beta 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 A resolution at the ESRF. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 68.7, b = 197.7, c = 86.1 A, beta = 113.4 degrees. Preliminary structural analysis revealed the presence of one molecule in the asymmetric unit.     

Structure of the Rab7:REP-1 complex: insights into the mechanism of Rab prenylation and choroideremia disease

Members of the RabGDI/REP family serve as multifunctional regulators of the Rab family of GTP binding proteins. Mutations in members of this family, such as REP-1, lead to abnormalities, including progressive retinal degradation (choroideremia) in humans. The crystal structures of the REP-1 protein in complex with monoprenylated or C-terminally truncated Rab7 proteins revealed that Rab7 interacts with the Rab binding platform of REP-1 via an extended interface involving the Switch 1 and 2 regions. The C terminus of the REP-1 molecule functions as a mobile lid covering a conserved hydrophobic patch on the surface of REP-1 that in the complex coordinates the C terminus of Rab proteins. Using semisynthetic fluorescent Rab27A, we demonstrate that although Rab27A can be prenylated by REP-2, this reaction can be effectively inhibited by other Rab proteins, providing a possible explanation for the accumulation of unprenylated Rab27A in choroideremia.     

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.     

A cell-specific,prenylation-independent mechanism regulates targeting of type II RACs

The RHO proteins, which regulate numerous signaling cascades, undergo prenylation, facilitating their interaction with membranes and with proteins called RHO.GDP dissociation inhibitors. It has been suggested that prenylation is required for RHO function. Eleven RHO-related proteins were identified in Arabidopsis. Eight of them are putatively prenylated. We show that targeting of the remaining three proteins, AtRAC7, AtRAC8, and AtRAC10, is prenylation independent, requires palmitoylation, and occurs by a cell-specific mechanism. AtRAC8 and AtRAC10 could not be prenylated by either farnesyltransferase or geranylgeranyltransferase I, whereas AtRAC7 could be prenylated by both enzymes in yeast. The association of AtRAC7 with the plasma membrane in plants did not require farnesyltransferase or a functional CaaX box. Recombinant AtRAC8 was palmitoylated in vitro, and inhibition of protein palmitoylation relieved the association of all three proteins with the plasma membrane. Interestingly, AtRAC8 and a constitutively active mutant, Atrac7mV(15), were not associated with the plasma membrane in root hair cells, whose elongation requires the localization of prenylated RHOs in the plasma membrane at the cell tip. Moreover, Atrac7mV(15) did not induce root hair deformation, unlike its prenylated homologs. Thus, AtRAC7, AtRAC8, and AtRAC10 may represent a group of proteins that have evolved to fulfill unique functions.     

Phosphoisoprenoids modulate association of Rab geranylgeranyltransferase with REP-1.

Rab geranylgeranyltransferase (RabGGTase or GGTase-II) catalyzes the post-translational prenylation of Rab proteins. Rab proteins are recognized as substrates only when they are complexed to Rab Escort Protein (REP). The classical model of prenylation complex assembly assumes initial formation of the Rab.REP binary complex, which subsequently binds to RabGGTase loaded with the isoprenoid donor geranylgeranyl pyrophosphate (GGpp). We demonstrate here that REP-1 can also associate with RabGGTase in the absence of Rab protein and that this interaction is dramatically strengthened by the presence of phosphoisoprenoids such as GGpp. The GGpp-dependent interaction between RabGGTase and REP-1 was observed using affinity precipitations and gel filtration and was quantitated on the basis of fluorescence assays. In the presence of GGpp, REP-1 binds to RabGGTase with a K(d) value of approximately 10 nm, while in its absence the affinity between the two proteins is in the micromolar range. We further demonstrate that binding of Rab7 to the RabGGTase.GGpp.REP-1 complex occurs without prior dissociation of REP-1. Analysis of binding and prenylation rate constants indicate that the RabGGTase.GGpp.REP-1 complex can function as a kinetically competent intermediate of the prenylation reaction. We conclude that, depending on the prevailing concentrations, binding of REP-1 to RabGGTase in the presence of GGpp may serve as an alternative pathway for the assembly of the prenylation machinery in vivo. Implications of these findings for the role of REP-1 in the prenylation reaction are discussed.     

Expression of mammalian geranylgeranyltransferase type-II in Escherichia coli and its application for in vitro prenylation of Rab proteins

Mammalian geranylgeranyltransferase type II (GGTase-II) is a 100-kDa heterodimer that catalyzes the transfer of two 20-carbon geranylgeranyl groups from geranylgeranyl pyrophosphate onto C-terminal cysteine residues of Rab GTPases. This modification is essential for the biological activity of Rab proteins. Geranylgeranylation can be performed in vitro using recombinant GGTase-II but so far large-scale production of the enzyme was challenging. We report here the design of a two plasmid expression system that will produce GGTase-II at levels as high as 15 mg/L in Escherichia coli. The protein was produced as a heterodimer with the alpha subunit bearing a cleavable tandem 6His-glutathione S-transferase (GST) tag that was used for two-step purification of the enzyme. Purified enzyme was functionally active as determined by in vitro prenylation and phosphoisoprenoid binding assay. Furthermore, the GST-tagged GGTase-II was used for preparative in vitro prenylation of the Rab7:REP-1 complex. Using this procedure, 10 mg of doubly prenylated Rab7:REP-1 complex were obtained.     

Functional aspects of polyisoprenoid protein substituents: roles in protein-protein interaction and trafficking

There are now numerous examples of post-translational modification with geranylgeranyl or farnesyl substituents. Once thought of as solely a mechanism for association of proteins with membranes, other functional aspects of protein prenylation have come to be appreciated. Although, in almost all instances, such proteins are membrane associated, they are often found to also engage in protein-protein interactions. In some instances, such interactions are critical aspects of prenylated protein trafficking. In this review, the role of prenylation in mediating protein-protein interactions will be considered. The hypothesis will be developed that such interactions occur through recognition of the prenyl group and a second domain, on the prenylated protein, by a heterodimeric protein partner.     

Expression and localization of rab escort protein isoforms in parotid acinar cells from rat

Rab proteins are geranylgeranylated on their carboxyl terminal cysteine motifs by geranylgeranyltransferase II (GGTase). Rab escort protein (REP) is required to present Rab proteins to GGTase. REP may remain bound to newly isoprenylated Rab proteins and present them to their target membrane. Other studies have shown that Rab proteins cycle between the membrane and cytosolic compartments and that cytosolic Rab proteins are complexed with rab-GDI. In the present study, we examined the expression and localization of REP isoforms in parotid acinar cells. Although both REP isoforms, REP-1 and REP-2, were detected in parotid cytosol, REP-2 was the predominant isoform. Subcellular fractionation revealed that approximately 42% of cellular REP-2 is membrane-associated. REP-2 was partially removed from parotid membranes with 1 M NaCl or Na(2)CO(3), indicating that REP-2 is a peripheral membrane protein. Membrane-associated REP-2 did not colocalize with Rab3D on secretory granule membranes. However, density gradient centrifugation revealed that membrane-associated REP-2 and Rab3D colocalize on low- and high-density membrane fractions in parotid acinar cells. Isoproterenol, an agent which induces amylase release from parotid glands, caused a shift in both REP-2 and Rab3D to less dense membrane fractions. When acinar cell cytosol was fractionated by gel filtration chromatography, Rab3D eluted exclusively with REP, not rab-GDI. In contrast, Rab1B and Rab5 eluted with both REP and Rab-GDI. Colocalization of Rab3D and REP-2 on acinar cell membranes suggests that REP-2 plays a role in delivering Rab3D to parotid membranes and may regulate guanine nucleotide binding to membrane-associated Rab3D. In addition, unlike other Rab proteins, cytosolic Rab3D appears to associate exclusively with REP, not rab-GDI in parotid acinar cells.     

Rab GTPase Prenylation Hierarchy and Its Potential Role in Choroideremia Disease

Protein prenylation is a widespread post-translational modification in eukaryotes that plays a crucial role in membrane targeting and signal transduction. RabGTPases is the largest group of post-translationally C-terminally geranylgeranylated. All Rabs are processed by Rab geranylgeranyl-transferase and Rab escort protein (REP). Human genetic defects resulting in the loss one of two REP isoforms REP-1, lead to underprenylation of RabGTPases that manifests in retinal degradation and blindness known as choroideremia. In this study we used a combination of microinjections and chemo-enzymatic tagging to establish whether Rab GTPases are prenylated and delivered to their target cellular membranes with the same rate. We demonstrate that although all tested Rab GTPases display the same rate of membrane delivery, the extent of Rab prenylation in 5 hour time window vary by more than an order of magnitude. We found that Rab27a, Rab27b, Rab38 and Rab42 display the slowest prenylation in vivo and in the cell. Our work points to possible contribution of Rab38 to the emergence of choroideremia in addition to Rab27a and Rab27b.     

Rab4 function in membrane recycling from early endosomes depends on a membrane to cytoplasm cycle

The monomeric GTPase rab4 is associated with early endosomes and regulates recycling vesicle formation. Because the function of rab proteins in the biosynthetic pathway does not appear to depend on cycling between membranes and cytosol, we were interested to investigate whether or not this holds true for rab function in the endocytic pathway. We created a chimeric rab4 protein (NHrab4cbvn) in which the carboxyl-terminal prenylation motif was replaced by the transmembrane domain of cellubrevin. The chimeric protein was permanently attached to membranes, properly targeted to early endosomes, and bound guanine nucleotide to the same extent as wild type rab4. However, in transport assays we found that basolaterally endocytosed transferrin was less efficiently transported to the apical cell surface in Madin-Darby canine kidney cells transfected with NHrab4cbvn than in cells expressing wild type rab4. Hence, rab4 function requires ongoing cycles of association and dissociation from early endosomes. This cycle is altered during mitosis when rab4 accumulates in the cytoplasm through phosphorylation by a mitotic kinase. We show here, using a rab4 construct that is permanently hooked onto membranes, that the membrane-bound pool of rab4 is targeted by a mitotic kinase.     

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.     

Rab geranylgeranylation occurs preferentially via the pre-formed REP-RGGT complex and is regulated by geranylgeranyl pyrophosphate

Prenylation (or geranylgeranylation) of Rab GTPases is catalysed by RGGT (Rab geranylgeranyl transferase) and requires REP (Rab escort protein). 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. In the present paper we show that REP mutants defective in RGGT binding (REP1 F282L and REP1 F282L/V290F) 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 (geranylgeranyl pyrophosphate) acts as an allosteric regulator of the prenylation reaction. We observed that REP-RGGT complexes are formed in vivo and are unstable in the absence of intracellular GGPP. RGGT increases the ability of REP to extract endogenous prenylated Rabs from membranes in vitro by stabilizing a soluble REP-RGGT-Rab-GG (geranylgeranylated Rab) 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.     

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.     

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.     

Double prenylation by RabGGTase can proceed without dissociation of the mono-prenylated intermediate

Rab geranylgeranyltransferase (RabGGTase) catalyzes the prenylation of Rab proteins. Despite possessing a single active site, RabGGTase is able to add geranylgeranyl moieties onto each of the two C-terminal cysteine residues of Rab. We have studied the kinetics of Rab double prenylation employing a combination of a novel high pressure liquid chromatography (HPLC)-based in vitro prenylation assay and fluorescence spectroscopy. Transfer of the first geranylgeranyl group proceeds with a k(1) = 0.16 s(-1), while the conversion from singly to double prenylated Rab is 4-fold slower (k(2) = 0.039 s(-1)). We found that following the first transfer reaction, the conjugated lipid is removed from the active site of RabGGTase but mono-prenylated Rab.REP complex remains bound to RabGGTase with a K(d) < 1 nm. In contrast to the doubly prenylated Rab7.REP dissociation of the mono-prenylated species from RabGGTase was only weakly stimulated by phosphoisoprenoid. Based on the obtained rate constants we calculated that at least 72% of mono-prenylated Rab molecules proceed to double prenylation without dissociating from RabGGTase. The obtained data provides an explanation of how RabGGTase discriminates between mono-prenylated intermediate and double prenylated reaction product. It also indicates that the phosphoisoprenoid acts both as a substrate and as a sensor governing the kinetics of protein.protein interactions in the double prenylation reaction.     

Rab9 functions in transport between late endosomes and the trans Golgi network.

Rab proteins represent a large family of ras-like GTPases that regulate distinct vesicular transport events at the level of membrane targeting and/or fusion. We report here the primary sequence, subcellular localization and functional activity of a new member of the rab protein family, rab9. The majority of rab9 appears to be located on the surface of late endosomes. Rab9, purified from Escherichia coli strains expressing this protein, could be prenylated in vitro in the presence of cytosolic proteins and geranylgeranyl diphosphate. In vitro-prenylated rab9 protein, but not C-terminally truncated rab9, stimulated the transport of mannose 6-phosphate receptors from late endosomes to the trans Golgi network in a cell-free system that reconstitutes this transport step. Rab7, a related rab protein that is also localized to late endosomes, was inactive in the in vitro transport assay, despite its efficient prenylation and capacity to bind and hydrolyze GTP. These results strongly suggest that rab9 functions in the transport of mannose 6-phosphate receptors between late endosomes and the trans Golgi network. Moreover, our results confirm the observation that a given organelle may bear multiple rab proteins with different biological functions.     

Post-translational processing of Schizosaccharomyces pombe YPT proteins.

ras proteins are post-translationally processed at their carboxyl-terminal CAAX motif by a triplet of modifications: prenylation of C with farnesyl, proteolytic trimming of AAX, and carboxyl-methylation. These modifications co-operate with palmitoylation of nearby sites or a polybasic region to target plasma membrane localization. The related YPT/rab proteins in contrast are localized to compartments of the endo-membrane system and may be involved in directing membrane traffic. These proteins end in XCC or CXC motifs. We have analyzed the processing of members of this subfamily form the fission yeast Schizosaccharomyces pombe. We find using in vitro translation in reticulocyte lysates that YPT1, -3, and -5 are prenylated with geranylgeranyl and that they incorporate label from [3H]mevalonic acid when expressed in transfected COS cells in vivo. Furthermore, prenylation was necessary for membrane binding in vivo. The CXC protein YPT5, but neither of the two XCC proteins YPT1 and YPT3, was carboxyl-methylated in S. pombe and in COS cells in vivo. However, YPT5 was not carboxyl-methylated in vitro in lysates which were able to methylate ras protein. YPT3 was detectably palmitoylated when expressed in COS cells, though at a much lower level than ras.