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• W2033745197 abstract "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. 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. Protein prenylation involves the covalent addition of either farnesyl (15-carbon) or geranylgeranyl (20-carbon) isoprenoids onto C-terminal cysteines via thioether linkages and is necessary for proteins to associate with cellular membranes to carry out intracellular functions (1Lane K.T. Beese L.S. J. Lipid Res. 2006; 47: 681-699Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 2Leung K.F. Baron R. Seabra M.C. J. Lipid Res. 2006; 47: 467-475Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Among the proteins that are modified by prenylation are small GTPases such as Ras, Rho, and Rab family proteins, all of which require prenylation for activity. Three distinct protein prenyltransferases have been identified, all of which are heterodimeric enzymes made up of an α and β subunit (1Lane K.T. Beese L.S. J. Lipid Res. 2006; 47: 681-699Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 2Leung K.F. Baron R. Seabra M.C. J. Lipid Res. 2006; 47: 467-475Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). The CAAX 5The abbreviations used are: CAAX, C is cysteine, A is aliphatic residue, X is any amino acid; Icmt, isoprenylcysteine carboxyl methyltransferase; RBL, rat basophilic leukemia; RGGT, Rab geranylgeranyltransferase; REP, Rab escort protein; ER, endoplasmic reticulum; EGF, epidermal growth factor; GFP, green fluorescent protein; HEK, human embryonic kidney; AdoMet, S-adenosyl-l-methionine; MEF, mouse embryonic fibroblast; PBS, phosphate buffered saline; PNS, post-nuclear supernatant; PIPES, 1, 4-piperazinediethanesulfonic acid. prenyltransferases, consisting of farnesyltransferase and geranylgeranyltransferase type I, modify proteins with a C-terminal CAAX motif, where C is cysteine, A is usually an aliphatic residue, and X is any amino acid. When X is a methionine, serine, glutamine, or alanine, the substrate is farnesylated, whereas if it is a leucine or phenylalanine, the substrate is geranylgeranylated. Rab geranylgeranyltransferase (RGGT, also known as geranylgeranyltransferase type II) forms a different class of protein prenyltransferases. RGGT specifically modifies Rab proteins but only when they are in complex with an accessory protein known as Rab escort protein (REP) (2Leung K.F. Baron R. Seabra M.C. J. Lipid Res. 2006; 47: 467-475Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). There are two proposed mechanisms for Rab protein prenylation. In the classical pathway, newly synthesized Rab binds REP, which presents the Rab to RGGT (3Andres D.A. Seabra M.C. Brown M.S. Armstrong S.A. Smeland T.E. Cremers F.P. Goldstein J.L. Cell. 1993; 73: 1091-1099Abstract Full Text PDF PubMed Scopus (286) Google Scholar). Alternatively, REP can associate with RGGT and the complex can bind to unprenylated Rab (4Thoma N.H. Iakovenko A. Goody R.S. Alexandrov K. J. Biol. Chem. 2001; 276: 48637-48643Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The enzyme then catalyzes the sequential addition of geranylgeranyl groups onto two C-terminal cysteines of the Rab protein. Finally, RGGT dissociates and REP is thought to deliver the prenylated Rab protein to membranes (5Seabra M.C. Wasmeier C. Curr. Opin. Cell Biol. 2004; 16: 451-457Crossref PubMed Scopus (236) Google Scholar). Following prenylation, CAAX-containing Ras and Rho GTPases are targeted to the endoplasmic reticulum (ER) and undergo proteolytic cleavage of the AAX tripeptide, catalyzed by the CAAX protease, Ras, and a-factor converting enzyme (Rce1) (6Winter-Vann A.M. Casey P.J. Nat. Rev. Cancer. 2005; 5: 405-412Crossref PubMed Scopus (280) Google Scholar, 7Wright L.P. Philips M.R. J. Lipid Res. 2006; 47: 883-891Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). The newly exposed prenylated cysteine is then further modified by carboxyl methylation on the α-carboxyl group by isoprenylcysteine carboxyl methyltransferase (Icmt), which is also located on the ER (6Winter-Vann A.M. Casey P.J. Nat. Rev. Cancer. 2005; 5: 405-412Crossref PubMed Scopus (280) Google Scholar, 7Wright L.P. Philips M.R. J. Lipid Res. 2006; 47: 883-891Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Carboxyl methylation enhances the hydrophobicity of the C terminus of prenylated proteins, although this effect is more apparent in farnesylated proteins than in geranylgeranylated proteins (8Michaelson D. Ali W. Chiu V.K. Bergo M. Silletti J. Wright L. Young S.G. Philips M. Mol. Biol. Cell. 2005; 16: 1606-1616Crossref PubMed Scopus (129) Google Scholar, 9Silvius J.R. l'Heureux F. Biochemistry. 1994; 33: 3014-3022Crossref PubMed Scopus (240) Google Scholar). The importance of post-prenylation processing has been exemplified by studies using gene-targeted inactivation, where it was found that mice deficient in Rce1 (10Kim E. Ambroziak P. Otto J.C. Taylor B. Ashby M. Shannon K. Casey P.J. Young S.G. J. Biol. Chem. 1999; 274: 8383-8390Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) or Icmt (11Bergo M.O. Leung G.K. Ambroziak P. Otto J.C. Casey P.J. Gomes A.Q. Seabra M.C. Young S.G. J. Biol. Chem. 2001; 276: 5841-5845Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) are embryonic lethal. Interestingly, Icmt-/- mice exhibited a more severe phenotype, which could be explained by the fact that Icmt may have more substrates than Rce1. Indeed, Rab proteins with a CXC motif are methylated on the C-terminal prenylcysteine, although the role of methylation in Rab proteins is unclear (12Smeland T.E. Seabra M.C. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10712-10716Crossref PubMed Scopus (64) Google Scholar). Several studies have demonstrated the importance of methylation and its role in the membrane association of many CAAX proteins, in particular Ras proteins. In cells deficient in Rce1 and Icmt, Ras proteins exhibit a significant decrease in membrane association (10Kim E. Ambroziak P. Otto J.C. Taylor B. Ashby M. Shannon K. Casey P.J. Young S.G. J. Biol. Chem. 1999; 274: 8383-8390Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Furthermore, the absence of methylation results in mislocalization of Ras from the plasma membrane. Consistent with its role in membrane association, methylation appears to regulate downstream signaling pathways of Ras through its localization. The Icmt small substrate inhibitor, N-acetyl-S-farnesyl-l-cysteine, blocks EGF-stimulated extracellular signal-regulated kinase (ERK) phosphorylation, a downstream target of EGF signaling and the activation of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) and Raf-1 kinases (13Chiu V.K. Silletti J. Dinsell V. Wiener H. Loukeris K. Ou G. Philips M.R. Pillinger M.H. J. Biol. Chem. 2004; 279: 7346-7352Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), although N-acetyl-S-farnesyl-l-cysteine-mediated effects have to be interpreted carefully given that some effects are unrelated to Icmt inhibition (6Winter-Vann A.M. Casey P.J. Nat. Rev. Cancer. 2005; 5: 405-412Crossref PubMed Scopus (280) Google Scholar). Another Icmt-specific inhibitor, cysmethynil, also leads to mislocalization of Ras proteins and blocks EGF-induced stimulation of MAPK and Akt (6Winter-Vann A.M. Casey P.J. Nat. Rev. Cancer. 2005; 5: 405-412Crossref PubMed Scopus (280) Google Scholar). The majority of Rab proteins possess a di-cysteine motif such as CC, CXC, or CCXX, and both cysteines are modified by geranylgeranyl lipid groups. However, a few possess a CAAX motif, such as Rab8 and Rab13, and are modified by a single geranylgeranyl moiety (14Joberty G. Tavitian A. Zahraoui A. FEBS Lett. 1993; 330: 323-328Crossref PubMed Scopus (43) Google Scholar). The reason why some Rabs are monoprenylated is not known, but the presence of a CAAX motif suggests that they have the potential to be processed by CAAX proteolysis and carboxyl methylation. In this study, we addressed the post-prenylation processing of single cysteine Rabs. We reveal for the first time that Rab-CAAX proteins are carboxyl methylated both in vitro and in vivo. In the absence of CAAX processing by Rce1 or Icmt, the localization of Rab-CAAX proteins is unaffected. However, the cycle of membrane association and retrieval is affected with decreased levels of membrane-associated Rabs, suggesting a role for methylation in regulating Rab activity. Plasmid Constructs—pEGFP-Rab11a was a kind gift of James Goldenring (Vanderbilt University School of Medicine, Nashville, TN). pEGFP-mRab23 was a kind gift from Carol Wicking (University of Queensland, Australia) (from here on, mRab23 will be referred to as Rab23). pEGFP-Rab8aGGCC, pEGFPRab8aGCSC, pEGFP-Rab38CALS, and pEGFP-Rab38CAVS were generated using the Stratagene QuikChange site-directed mutagenesis system, as described previously (15Gomes A.Q. Ali B.R. Ramalho J.S. Godfrey R.F. Barral D.C. Hume A.N. Seabra M.C. Mol. Biol. Cell. 2003; 14: 1882-1899Crossref PubMed Scopus (116) Google Scholar). pGEX-4T-1-Rab13, Rab18, and Rab23 were generated by PCR amplification of the Rab cDNA of interest and cloned into pGEX-4T-1 vector using EcoRI-SalI, EcoRI-XhoI, and EcoRI-XhoI, respectively. Human RabGDIβ was amplified by PCR from a cDNA library (MHS1011, Invitrogen) and cloned into pFastBac-HTB using NcoI-XbaI. The sequences of all plasmid constructs used were confirmed by DNA sequencing. Recombinant Proteins—Recombinant GST-Rab5a, GST-Rab13, GST-Rab18, GST-Rab23, and GST-Rab38 were expressed in BL21 cells and purified on glutathione-agarose beads (Sigma). Recombinant RGGT and REP1 were prepared by infection of Sf9 cells with recombinant baculoviruses encoding each subunit of the desired enzyme and purified by nickel-Sepharose affinity chromatography as described previously (16Armstrong S.A. Brown M.S. Goldstein J.L. Seabra M.C. Methods Enzymol. 1995; 257: 30-41Crossref PubMed Scopus (28) Google Scholar, 17Seabra M.C. James G.L. Methods Mol. Biol. 1998; 84: 251-260PubMed Google Scholar). Recombinant human RabGDIβ was prepared by infection of Sf9 cells with recombinant baculovirus using standard procedures. Briefly, baculoviruses were generated following subcloning of RabGDIβ into pFastBac-HTB using the Bac-to-Bac® system according to the manufacturer's instructions (Invitrogen). Recombinant histidine (His)-tagged RabGDIβ was produced as follows. After a 96-h infection with P4 viral stock, Sf9 cells were centrifuged at 800 × g and resuspended in lysis buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 5 mm MgCl2, 1 mm β-mercaptoethanol, 0.2% Triton X-100, Roche complete protease inhibitor mixture). After sonication, the solution was clarified by ultracentrifugation at 100,000 × g for 1 h at 4°C.The supernatant was incubated for 90 min with nickel-nitrilotriacetic acid beads (Qiagen) at 4 °C. The beads (3 ml) were first washed with 100 ml of lysis buffer and then with 100 ml of the same buffer without detergent. Recombinant His-RabGDIβ was eluted from the beads with a gradient of imidazole (0-250 mm). The eluate was dialyzed overnight in buffer containing 50 mm Tris, pH 7.5, 150 mm NaCl, 5 mm MgCl2, 1 mm β-mercaptoethanol and purity judged by SDS-PAGE. All recombinant proteins were more than 70% pure, snap frozen in small aliquots, and stored at -80 °C until use. Antibodies—Anti-GFP polyclonal rabbit antibody (Ab290, Abcam) was used at 1-2 μl/tube for immunoprecipitation. Texas Red-X phalloidin (T-7471, Molecular Probes) was used at 1:1000 dilution for immunofluorescence according to the manufacturer's instructions. Anti-human Rab8 and Rab11 monoclonal antibodies (numbers 610844 and 610656, BD Transduction Laboratories) for immunoblotting were used at 1:1000 according to the manufacturer's instructions. Polyclonal anti-Rab7 was a gift of J. Gruenberg (University of Geneva, Switzerland). Anti-RabGDI antibody was obtained after purification of rabbit serum raised against full-length rat RabGDIβ. The antibody recognizes both RabGDI isoforms with a higher potency against the β-isoform. Cell Culture and Transfection—HeLa and human embryonic kidney (HEK) 293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mm l-glutamine, 100 units/ml penicillin G, and 100 units/ml streptomycin at 37 °C with 10% CO2. Wild type and mouse embryonic fibroblasts (MEFs) null for Rce1 and Icmt were kind gifts from Steve Young (UCLA) and were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, 100 units/ml streptomycin, 2 mm l-glutamine, 1 mm sodium pyruvate, 0.1 mm β-mercaptoethanol and minimal essential medium non-essential amino acids. Rat basophilic leukemia (RBL) cells were cultured in Iscove's modified Dulbecco's medium containing 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin G, 100 units/ml streptomycin, and 2 mm l-glutamine. Cells used in immunofluorescence experiments were grown on 24-well plated coverslips for 24 h, transfected, and fixed after 24 h (HeLa and HEK 293 cells). Cells used for subcellular fractionation were grown in 10-cm dishes, transfected, and homogenized 24 h after transfection. HeLa cells and HEK 293 cells were transfected with FuGENE 6 (Roche Diagnostics) according to the manufacturer's instructions. RBL cells were transfected by electroporation. Briefly, following trypsinization, cells were resuspended at 107 cells in 250 μl and placed in an electrocuvette with 10 μg of plasmid DNA. After incubation for 10 min at 4 °C, cells were electroporated at 250 mV, 960 microfarads using a Bio-Rad Gene-Pulser and returned to 4 °C for a further 10 min. Electroporated cells were then cultured on coverslips in a 10-cm dish containing 10 ml of medium. Cells were then fixed 24-48 h later using 3% (w/v) paraformaldehyde. In Vitro Methylation Assay—In vitro prenylation of GST-Rab proteins was performed in 25-μl reaction volumes in buffer containing 50 mm HEPES, pH 7.5, 150 mm NaCl, 5 mm MgCl2, 1mm dithiothreitol, and 0.5 μl of cold geranylgeranyl pyrophosphate (GGpp) to a final concentration of 20 μm. Each reaction contained 10 μm GST-Rab protein substrate and prenylation was initiated by the addition of 2 μm REP1 and 50 nm RGGT recombinant and incubated for 30 min at 37 °C. Following prenylation, 62.5 μg of Rce1 and/or 15 μg of Icmt Sf9 membranes (kind gifts from Patrick Casey, Duke University) were added to each condition, together with 0.5 μl of the methyl donor [3H]S-adenosyl-l-methionine (AdoMet) (700 cpm/pmol) and cold AdoMet to a final concentration of 10 μm, in a final volume of 30 μl. Reactions were incubated at 37 °C for 40 min and terminated by the addition of 50 μl of 10% Triton X-100 in PBS. 15 μl of glutathione beads in 500 μl of PBS were added to each condition and allowed to bind on a rotator for 1 h at room temperature. Next, the samples were centrifuged at 10,000 × g for 15 s, the supernatant was discarded, and the beads were washed three times in PBS. Finally, the beads were resuspended in 100 μl of PBS, transferred directly into a scintillation vial containing 4 ml of scintillation fluid, and the disintegrations per minute (dpm) were counted using a scintillation counter. In Vivo Carboxyl Methylation of Proteins in Cultured Cells—In vivo methylation assay was performed as described previously (18Newman C.M. Giannakouros T. Hancock J.F. Fawell E.H. Armstrong J. Magee A.I. J. Biol. Chem. 1992; 267: 11329-11336Abstract Full Text PDF PubMed Google Scholar). HEK 293 cells transfected with pEGFP plasmids for 8 h were first incubated in methionine-free medium (R7513, Sigma) for 1 h. They were then incubated in 1:9 complete medium:methionine-free medium made up to 5% fetal bovine serum, along with 200 μCi of l-[methyl-3H]methionine (Amersham Biosciences) overnight. The following day, cells were harvested mechanically, transferred to Eppendorf tubes, and washed twice with ice-cold PBS. The cells were resuspended in 200 μl of RIPA buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 0.5% Triton X-100, 0.5% sodium deoxycholate) containing complete protease inhibitor mixture (Roche) for 10 min to allow lysis to occur. This was followed by centrifugation at 10,000 × g and the post-nuclear supernatant (PNS) was transferred to a fresh tube. 2 μl of anti-GFP antibody (Abcam) was added to the PNS and the solution was mixed on a rotator at 4 °C for 1 h. Then, 10 μl of Protein G-Sepharose beads (in a 1:1 slurry of in RIPA buffer) were added to the PNS at 4 °C for 1 h. The beads were washed three times with RIPA buffer, and once with 50 mm Tris-HCl, pH 6.8, to remove excess detergents. The protein-antibody complexes were subjected to electrophoresis on 12.5% SDS-PAGE gels. Gels were then immersed in a solution containing 10% acetic acid and 45% methanol for 5 min to ensure fixation of the proteins, and then re-hydrated in water. This was followed by immersion in 1 m sodium salicylate for 20 min, after which the gels were immediately vacuum-dried on blotting paper. Detection of radiolabeled proteins was performed using autoradiographic film and exposed after 3-4 days. The radioactive bands of interest were excised and methyl-esterified proteins were detected by an alkali hydrolysis/diffusion assay as described previously (19Chelsky D. Gutterson N.I. Koshland Jr., D.E. Anal. Biochem. 1984; 141: 143-148Crossref PubMed Scopus (38) Google Scholar). Open tubes containing the gel piece were lowered into scintillation vials containing scintillation fluid. 1 m NaOH was added to each tube to immerse the gel piece and the vial was capped immediately, leaving the tube open inside. The vials were incubated at 37 °C overnight. Ester-linked methyl groups were hydrolytically cleaved by alkali, releasing [3H]methanol, which is distilled into the scintillation fluid. Following treatment with alkali, each Eppendorf tube was carefully removed and the vial containing alkali-labile methanol was capped. An equal volume of 1 m HCl was added to the Eppendorf tube to neutralize the alkali and the contents were transferred to a fresh vial containing scintillation fluid. This latter vial contained alkali-stable [3H]methionine that was incorporated into the peptide backbone. The amount of radioactivity in the alkali-labile and alkali-stable samples was measured using a scintillation counter with a tritium channel. To determine whether a protein was methylated in vivo, the methylation stoichiometry was calculated by the following equation: (alkali-labile dpm × number of methionine residues in the protein)/alkali-stable dpm. Immunofluorescence and Confocal Microscopy—After transfection with pEGFP plasmids, cells were washed with PBS and then incubated in permeabilization buffer (80 mm K-PIPES, pH 6.8, 5 mm EGTA, 1 mm MgCl2, 0.05% (w/v) saponin) for 5 min and then fixed in 3% (w/v) paraformaldehyde in PBS for 15 min. Excess fixative was removed by repeated washing in PBS. When Texas Red phalloidin was used, cells were further incubated for 15 min in PBS containing 0.5% bovine serum albumin and 0.05% saponin. The subsequent steps were performed in this solution. The cells were incubated with Texas Red phalloidin for 30 min and washed three times. The coverslips were mounted in ImmunoFluor medium (ICN, Basingstoke, Hants, United Kingdom) and the fluorescence was visualized using a DM-IRBE Leica confocal microscope. Images were processed using TCS-NT software associated with the microscope and Adobe Photoshop 5.5 software. All images presented are single sections in the z-plane and are representative of at least 80% of the transfected cells in the coverslip. Temperature Block Experiments—Temperature block experiments were performed as described previously (15Gomes A.Q. Ali B.R. Ramalho J.S. Godfrey R.F. Barral D.C. Hume A.N. Seabra M.C. Mol. Biol. Cell. 2003; 14: 1882-1899Crossref PubMed Scopus (116) Google Scholar). HeLa cells were seeded in 24-well plates and grown overnight as described above. The following day, cells were transfected and 4 h later, the medium was replaced with complete medium supplemented with 20 mm HEPES buffer (15630-056; Invitrogen) and cells were placed at 20 °C for 3 h to block exit of proteins from the Golgi apparatus. Cells were fixed at this point or further incubated at 37 °C for 1 h. All cells were permeabilized and fixed as described above. Subcellular Fractionation—After transfection, HEK 293 cells were harvested mechanically, transferred to 15-ml tubes, and centrifuged at 1000 × g for 5 min at 4 °C. Cells were washed with PBS and centrifuged once more. Cells were then resuspended in hypotonic lysis buffer (20 mm Tris, pH 7.5, 5 mm MgCl2, 1 mm dithiothreitol, Roche complete protease inhibitor mixture) and lysed by sonication, followed by centrifugation at 800 × g for 10 min at 4 °C. The PNS was transferred to a Beckman centrifuge tube and subjected to ultracentrifugation at 100,000 × g for 1 h at 4 °C using a TLA45 Beckman rotor. The supernatant (S100) containing the cytosolic fraction was transferred to a fresh tube and the pellet (P100) containing the membrane fraction was resuspended in an equivalent volume of lysis buffer. The fractions were subjected to electrophoresis on 12.5% SDS-PAGE gels, transferred to polyvinylidene difluoride membranes, and proteins were detected by Western immunoblotting. Densitometry quantification was achieved using Fuji Film Intelligent Dark Box LAS-3000 and Aida Image Analyze 3.52 software. RabGDI Extraction Assay—Membrane proteins (30 μg) prepared from MEFs in buffer containing 50 mm HEPES, pH 7.5, 150 mm NaCl, 5 mm MgCl2, 1 mm dithioerythritol, 1 mm GDP, and Roche complete protease inhibitor mixture, were incubated with increasing amounts of purified His-RabGDIβ (0-8 μm) for 20 min at 37 °C. The extracted Rab proteins in complex with RabGDIβ were separated from membrane proteins by ultracentrifugation at 100,000 × g for 1 h at 4°C. The soluble fraction (S100) and the membrane fraction (P100) were resolved on 12.5% SDS-PAGE. Gel Filtration Chromatography—S100 (100 μg) fractions prepared as above were loaded onto a Superdex 200 3.2/30 column using a SMART system (GE Healthcare). The column was equilibrated in buffer containing 50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 8 mm MgCl2, 2 mm EDTA, 1 mm dithiothreitol, and 10 μm GDP, at a flow rate of 40 μl/min. The samples (50 μl) were injected, and the material eluting between 0.8 and 2 ml was collected in 50-μl fractions. In Vitro Carboxyl Methylation of Rab-CAAX Proteins—To determine whether Rab proteins that naturally possess a CAAX motif are substrates for Rce1 and Icmt in vitro, recombinant GST-Rab fusion proteins were produced after expression in Escherichia coli. Because prenylation and proteolysis are required for Icmt-dependent methylation, the in vitro assay was designed as a coupled prenylation/proteolysis/methylation assay. Geranylgeranylation reactions of GST-Rab substrates were performed in the presence of recombinant RGGT and recombinant REP1, followed by the addition of Rce1 and Icmt-enriched membrane fractions to initiate AAX proteolysis and carboxyl methylation, respectively. Subsequently, proteins were isolated using glutathione beads and the amount of [3H]AdoMet incorporated was quantified by liquid scintillation counting. Rab18 was used initially as a model Rab protein with a CAAX motif for the in vitro methylation assays to determine optimal conditions. We found that GST-Rab18 methylation was strictly dependent on REP1 and RGGT (Fig. 1A). This is consistent with the fact that both Rce1 and Icmt only modify prenylated substrates. The incorporation of [3H]AdoMet increased with time to over 14 pmol after 60 min following the addition of Rce1, Icmt, and [3H]AdoMet. The yield was calculated to be ∼5%, taking into account the estimate that 10-30% of the total protein was prenylated. 6K. F. Leung, R. Baron, B. R. Ali, A. I. Magee, and M. C. Seabra, unpublished observations. GST-Rab18 showed an attenuated level of methylation in the presence of either Rce1 or Icmt alone. The reduced methylation is most likely due to the endogenous levels of both proteins present in the enriched membrane preparations used in the assay. The methylation status of GST-Rab18 was compared with that of other candidate Rab proteins possessing a CAAX motif (GST-Rab13, GST-Rab23, and GST-Rab38) (Fig. 1B). The results suggest that GST-Rab13, GST-Rab18, GST-Rab23, and GST-Rab38 were all carboxyl methylated to the same extent, suggesting that they are all substrates for Rce1 and Icmt (Fig. 1B). As a negative control, GST-Rab18 was mixed with Triton X-100 prior to addition of Rce1 and Icmt, which solubilizes these integral membrane proteins and abolishes their activity. As expected, no methylation was observed for GST-Rab18 when Triton X-100 was included prior to addition of Rce1 and Icmt membranes. GST-Rab5a was used as a control because it is doubly geranylgeranylated on adjacent cysteines, and therefore expected not to be a substrate for either Rce1 or Icmt (12Smeland T.E. Seabra M.C. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10712-10716Crossref PubMed Scopus (64) Google Scholar). As expected, GST-Rab5a showed no significant methylation under the same conditions in vitro. We conclude that GST-Rab13, GST-Rab18, GST-Rab23, and GST-Rab38 are carboxyl methylated in vitro. In Vivo Carboxyl Methylation of Rab Proteins—To investigate whether carboxyl methylation of CAAX-containing Rab proteins occurred in vivo, HEK 293 cells were transfected with EGFP-Rab constructs and labeled with l-[methyl-3H]methionine. The EGFP-tagged proteins were immunoprecipitated and subjected to SDS-PAGE and autoradiography. Because l-[methyl-3H]methionine is used to synthesize both the AdoMet pool and the tRNA-methionine pool in cells, an alkali hydrolysis assay was used to differentiate between methylated versus non-methylated proteins (18Newman C.M. Giannakouros T. Hancock J.F. Fawell E.H. Armstrong J. Magee A.I. J. Biol. Chem. 1992; 267: 11329-11336Abstract Full Text PDF PubMed Google Scholar). Methyl esters are subject to hydrolysis in the presence of alkali, releasing vapor phase [3H]methanol. Labeled methionines in the protein backbone are insensitive to this treatment and thus are differentiated from the labeled methyl groups. As a positive control, we used EGFP-Rac1 (C-terminal sequence, CLLL) which is known to be methylated (20Ando S. Kaibuchi K. Sasaki T. Hiraoka K. Nishiyama T. Mizuno" @default.
• W2033745197 created "2016-06-24" @default.
• W2033745197 creator A5009932874 @default.
• W2033745197 creator A5035635727 @default.
• W2033745197 creator A5057216989 @default.
• W2033745197 creator A5083002376 @default.
• W2033745197 creator A5090216858 @default.
• W2033745197 date "2007-01-01" @default.
• W2033745197 modified "2023-10-06" @default.
• W2033745197 title "Rab GTPases Containing a CAAX Motif Are Processed Post-geranylgeranylation by Proteolysis and Methylation" @default.
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