geranylgeranylated rab in cys-ala-cys, cys-cys, carboxyl ... · recombinant rab proteins. wild-type...

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Proc. NatI. Acad. Sci. USA Vol. 91, pp. 10712-10716, October 1994 Biochemistry Geranylgeranylated Rab proteins terminating in Cys-Ala-Cys, but not Cys-Cys, are carboxyl-methylated by bovine brain membranes in vitro (protein prenylatl/Rab geranylgeranyltranfee/carboxyl hyansferae/membrane traffic) TOR E. SMELAND, MIGUEL C. SEABRA, JOSEPH L. GOLDSTEIN, AND MICHAEL S. BROWN Department of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235 Contributed by Joseph L. Goldstein, July 22, 1994 ABSTRACT Geraylgeranylated Rab proteins usually ter- minate In either Cys-Cys or Cys-Xaa-Cys, where Xaa is Ala, Ser, or Gly. In both cdases of proteins, the two cysteines are geranylgeranylated, but only the Cys-Xaa-Cys class has been shown to be carboxyl-methylated on the terminal cysteine in Wvo. In the current study, we used recombinant Rab gera- nylgeranyltransferase and a Rab escort protein (REP-1) to attach geranylgeranyl residues to the two cysteines at the carboxyl termius of Rab3A (Cys-Ala-Cys) and RablA (Cys- Cys). The geranylgeranylated proteins were then incubated with bovine cerebeflar membranes that contain an enzyme that trsfers [3Hlmethyl from S-[methyl-3PHadenosyl-L-methio- nine to geranylgeranylated cysteine. The enzyme transferred [3Hhmethyl to geranylgeranylated Rab3A but not to gera- nylgeranylated RablA. Replacement of the Cys-Ala-Cys ter- minus of Rab3A with Cys-Cys abolished methylatlon, and the opposite result was obtained when the Cys-Cys of RablA was replaced with Cys-Ala-Cys. When -the Cys-Cys terminus of RablA was changed to Ser-Cys, methylatlon was restored. These studies suggest that the carboxyl-terminal cysteine of Rab proteins terminating in Cys-Xaa-Cys but not Cys-Cys is methylated and that the resistance of Cys-Cys proteins to methylation is attributable to the vicinal geranylgeranylated yteines. Many proteins in eukaryotic cells contain prenyl groups, either farnesyl (C15) or geranylgeranyl (C20), attached in thioether linkage to cysteine residues at or near the carboxyl terminus (1). Two broad classes of prenylated proteins exist: (i) those that terminate in CAAX boxes, where C is cysteine, A is an aliphatic amino acid, and X is typically methionine, serine, or leucine, and (ii) those that terminate in either CC or CXC, where C is cysteine and X is typically alanine or serine. After prenylation with either farnesyl or geranylger- anyl groups, the CAAX proteins are cleaved by a protease that removes the three carboxyl-terminal amino acids, after which a methyltransferase carboxyl-methylates the farnesy- lated or geranylgeranylated cysteine (2). These reactions render the carboxyl terminus hydrophobic, facilitating its interaction with membranes or with other proteins. The CAAX proteins include GTP-binding proteins, such as Ras, Rho, and Rac, as well as the y subunits of heterotrimeric guanine nucleotide binding proteins and nuclear lamins, among others. All known CC- or CXC-terminated proteins belong to the Rab family, and all are modified by geranylgeranyl groups (3-5). The Rab family contains more than 30 low molecular weight GTP-binding proteins, each of which is attached to a specific set of membranous organelles. Rab proteins are required for the budding and fusion process by which mem- brane vesicles move from one organelle to another (6). Only fragmentary information currently exists regarding the methylation of Rab proteins. YPT5, a Rab protein from the yeast Schizosaccharomyces pombe, terminates in Cys- Ala-Cys (CAC) and was shown to be methylated (7, 8). Two other Rab proteins from the same organism, YPT1 and YPT3, which terminate in CC, were not methylated (8). Rab3A, a protein of brain synaptic vesicles, terminates in CAC. In vivo, both cysteines are geranylgeranylated, and the terminal cysteine is carboxyl-methylated (4). Similarly, Rab4, which terminates in CGC, is carboxyl-methylated (9). In contrast, Rab2, which terminates in CC, is geranylgeranylated but not carboxyl-methylated (10). Amembrane-boundenzymethatcarboxyl-methylatespren- ylated cysteine has been characterized biochemically in membranes from rat liver (11), bovine brain (12), rabbit brain (13), bovine retinal rod outer segments (14), and human neutrophils (15). The enzyme uses S-adenosyl-L-methionine (SAM) as a methyl donor, and it is inhibited by the end product S-adenosyl-L-homocysteine. It carboxyl-methylates either farnesylated or geranylgeranylated cysteine (16, 17). A cDNA encoding this carboxyl methyltransferase was cloned from yeast by complementation of a mutant that is defective in carboxyl methylation of geranylgeranylated and farnesy- lated cysteines (18-20). The cDNA encodes a protein that is predicted to have multiple membrane-spanning regions and has no sequence resemblance to other methyltransferases. Expression of the cDNA in Escherichia coli confers the ability to carboxyl-methylate geranylgeranylated cysteine and farnesylated cysteine (18). All of these data suggest that a single membrane-bound enzyme carboxyl-methylates pro- teins that terminate in either farnesyl- or geranylgeranylcys- teine. However, the lack of methylation of CC-terminated geranylgeranylated proteins in yeast and animal cells remains to be explained. In the current studies, we have sought to determine whether bovine brain carboxyl methyltransferase can meth- ylate geranylgeranylated proteins that terminate in CC. We have used recombinant Rab geranylgeranyltransferase (GG- Tase) (5, 21, 22) to geranylgeranylate Rab3A (terminating in CAC) and RablA (terminating in CC) in vitro. We have then subjected these proteins to methylation using a bovine brain membrane preparation. The results indicate that the meth- yltransferase can carboxyl-methylate the geranylgeranylated CAC protein but not the geranylgeranylated CC protein and that the block in the latter case is attributable to the imme- diately adjacent cysteine. Abbreviations: DTT, dithiothreitol; GGPP, geranylgeranyl pyro- phosphate; GGTase, geranylgeranyltransferase; SAM, S-adenosyl- L-methionine; [3H]SAM, S-[methyl-3H]adenosine-L-methionine; REP-1, Rab escort protein 1. 10712 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. NatI. Acad. Sci. USAVol. 91, pp. 10712-10716, October 1994Biochemistry

Geranylgeranylated Rab proteins terminating in Cys-Ala-Cys, butnot Cys-Cys, are carboxyl-methylated by bovine brain membranesin vitro

(protein prenylatl/Rab geranylgeranyltranfee/carboxyl hyansferae/membrane traffic)

TOR E. SMELAND, MIGUEL C. SEABRA, JOSEPH L. GOLDSTEIN, AND MICHAEL S. BROWNDepartment of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235

Contributed by Joseph L. Goldstein, July 22, 1994

ABSTRACT Geraylgeranylated Rab proteins usually ter-minate In either Cys-Cys or Cys-Xaa-Cys, where Xaa is Ala,Ser, or Gly. In both cdases of proteins, the two cysteines aregeranylgeranylated, but only the Cys-Xaa-Cys class has beenshown to be carboxyl-methylated on the terminal cysteine inWvo. In the current study, we used recombinant Rab gera-nylgeranyltransferase and a Rab escort protein (REP-1) toattach geranylgeranyl residues to the two cysteines at thecarboxyl termius of Rab3A (Cys-Ala-Cys) and RablA (Cys-Cys). The geranylgeranylated proteins were then incubatedwith bovine cerebeflar membranes that contain an enzyme thattrsfers [3Hlmethyl from S-[methyl-3PHadenosyl-L-methio-nine to geranylgeranylated cysteine. The enzyme transferred[3Hhmethyl to geranylgeranylated Rab3A but not to gera-nylgeranylated RablA. Replacement of the Cys-Ala-Cys ter-minus of Rab3A with Cys-Cys abolished methylatlon, and theopposite result was obtained when the Cys-Cys of RablA wasreplaced with Cys-Ala-Cys. When -the Cys-Cys terminus ofRablA was changed to Ser-Cys, methylatlon was restored.These studies suggest that the carboxyl-terminal cysteine ofRab proteins terminating in Cys-Xaa-Cys but not Cys-Cys ismethylated and that the resistance of Cys-Cys proteins tomethylation is attributable to the vicinal geranylgeranylatedyteines.

Many proteins in eukaryotic cells contain prenyl groups,either farnesyl (C15) or geranylgeranyl (C20), attached inthioether linkage to cysteine residues at or near the carboxylterminus (1). Two broad classes of prenylated proteins exist:(i) those that terminate in CAAX boxes, where C is cysteine,A is an aliphatic amino acid, and X is typically methionine,serine, or leucine, and (ii) those that terminate in either CCor CXC, where C is cysteine and X is typically alanine orserine. After prenylation with either farnesyl or geranylger-anyl groups, the CAAX proteins are cleaved by a proteasethat removes the three carboxyl-terminal amino acids, afterwhich a methyltransferase carboxyl-methylates the farnesy-lated or geranylgeranylated cysteine (2). These reactionsrender the carboxyl terminus hydrophobic, facilitating itsinteraction with membranes or with other proteins. TheCAAX proteins include GTP-binding proteins, such as Ras,Rho, and Rac, as well as the y subunits of heterotrimericguanine nucleotide binding proteins and nuclear lamins,among others.

All known CC- or CXC-terminated proteins belong to theRab family, and all are modified by geranylgeranyl groups(3-5). The Rab family contains more than 30 low molecularweight GTP-binding proteins, each of which is attached to aspecific set of membranous organelles. Rab proteins are

required for the budding and fusion process by which mem-brane vesicles move from one organelle to another (6).Only fragmentary information currently exists regarding

the methylation of Rab proteins. YPT5, a Rab protein fromthe yeast Schizosaccharomyces pombe, terminates in Cys-Ala-Cys (CAC) and was shown to be methylated (7, 8). TwootherRab proteins from the same organism, YPT1 and YPT3,which terminate in CC, were not methylated (8). Rab3A, aprotein of brain synaptic vesicles, terminates in CAC. Invivo, both cysteines are geranylgeranylated, and the terminalcysteine is carboxyl-methylated (4). Similarly, Rab4, whichterminates in CGC, is carboxyl-methylated (9). In contrast,Rab2, which terminates in CC, is geranylgeranylated but notcarboxyl-methylated (10).Amembrane-boundenzymethatcarboxyl-methylatespren-

ylated cysteine has been characterized biochemically inmembranes from rat liver (11), bovine brain (12), rabbit brain(13), bovine retinal rod outer segments (14), and humanneutrophils (15). The enzyme uses S-adenosyl-L-methionine(SAM) as a methyl donor, and it is inhibited by the endproduct S-adenosyl-L-homocysteine. It carboxyl-methylateseither farnesylated or geranylgeranylated cysteine (16, 17). AcDNA encoding this carboxyl methyltransferase was clonedfrom yeast by complementation of a mutant that is defectivein carboxyl methylation of geranylgeranylated and farnesy-lated cysteines (18-20). The cDNA encodes a protein that ispredicted to have multiple membrane-spanning regions andhas no sequence resemblance to other methyltransferases.Expression of the cDNA in Escherichia coli confers theability to carboxyl-methylate geranylgeranylated cysteineand farnesylated cysteine (18). All of these data suggest thata single membrane-bound enzyme carboxyl-methylates pro-teins that terminate in either farnesyl- or geranylgeranylcys-teine. However, the lack of methylation of CC-terminatedgeranylgeranylated proteins in yeast and animal cells remainsto be explained.

In the current studies, we have sought to determinewhether bovine brain carboxyl methyltransferase can meth-ylate geranylgeranylated proteins that terminate in CC. Wehave used recombinant Rab geranylgeranyltransferase (GG-Tase) (5, 21, 22) to geranylgeranylate Rab3A (terminating inCAC) and RablA (terminating in CC) in vitro. We have thensubjected these proteins to methylation using a bovine brainmembrane preparation. The results indicate that the meth-yltransferase can carboxyl-methylate the geranylgeranylatedCAC protein but not the geranylgeranylated CC protein andthat the block in the latter case is attributable to the imme-diately adjacent cysteine.

Abbreviations: DTT, dithiothreitol; GGPP, geranylgeranyl pyro-phosphate; GGTase, geranylgeranyltransferase; SAM, S-adenosyl-L-methionine; [3H]SAM, S-[methyl-3H]adenosine-L-methionine;REP-1, Rab escort protein 1.

10712

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Nadl. Acad. Sci. USA 91 (1994) 10713

EXPERIMENTAL PROCEDURESMaterials and Methods. We obtained [1-3H]geranylgeranyl

pyrophosphate ([3H]GGPP, 15 Ci/mmol; 1 Ci = 37 GBq) andunlabeled GGPP from American Radiolabeled Chemicals;S-[methyl-3H]adenosyl-L-methionine ([3H]SAM, 60-85 Ci/mmol) from DuPont/New England Nuclear; Bradford pro-tein assay reagent and molecular mass standards from Bio-Rad; farnesyl acetate from Aldrich; Nonidet P40 from Cal-biochem; Entensify from DuPont/New England Nuclear;and unlabeled SAM (iodide salt) and all other reagents fromSigma. N-Acetylfarnesylcysteine was synthesized by J. R.Falck (University of Texas Southwestern Medical Center,Dallas). Recombinant rat Rab escort protein 1 (REP-1) (5, 21)and recombinant rat Rab GGTase (a and subunits) (5, 22)were prepared in the baculovirus-5t9 insect cell system andpurified to apparent homogeneity as described in the indi-cated references. Phosphatidylcholine (Sigma) vesicles wereprepared by drying the desired amount of lipid under argon;adding buffer containing 50 mM sodium Hepes (pH 7.2), 0.1M NaCl, and 1 mM dithiothreitol (DTT) to a final concen-tration of 5 mg/ml; and sonicating for 10 win at roomtemperature in a bath sonicator immediately prior to use.Recombinant Rab Proteins. Wild-type canine RablA-CC

and human Rab3A-CAC proteins, as well as carboxyl-terminal mutant proteins RablA-CAC and Rab3A-CC, wereexpressed with six histidine residues inserted at the aminoterminus as described (5). Other carboxyl-terminal mutantRablA and Rab3A proteins were generated by PCR of 1 ngof DNA from the original pET14b-RablA-CC or pET14b-Rab3A-CAC clones, respectively. The sequence of the 5'oligonucleotide used in the PCRs was 5'-GTACTAGCATAT-GTCCAGCATGAATCCCGAATAT-3Y for RablA mutantproteins and 5'-GAGTGGCATATGGCATCGGCCACA-GACTCG-3' for Rab3A mutant proteins. The sequences ofthe 3' oligonucleotides used are listed in Table 1. Thecarboxyl-terminal amino acid sequence of all Rab proteinswas verified by nucleotide sequencing of the mutant plas-mids. All Rab proteins were expressed in BL21(DE3) E. coliand purified by Ni2+-Sepharose affinity chromatography asdescribed (5).Prepration of Mkrosomal Membranes. One bovine cere-

bellum (45 g) was minced, immersed in 5 vol ofbufferA [10o(wt/vol) sucrose/10 mM Tris chloride, pH 7.4/1 mM DTT],and homogenized in four aliquots at 4°C in a Waring blenderat low speed for four 20-sec intervals. The homogenate wasfiltered through four layers of cheesecloth, and the filtratewas centrifuged at 20,000 x g for 30 min at 4°C. Thesupernatant was recovered and centrifuged at 200,000 x g at40C for 1 h. Pellets were resuspended with a tight-fittingDounce homogenizer in buffer B (50 mM sodium Hepes, pH7.2/1 mM DTT) and dialyzed at 4°C against the same bufferovernight. The dialysate was frozen in multiple aliquots andstored at -70°C.

Geranylgeranylation of Rab Proteins. Rab proteins weregeranylgeranylated as described (5) except that phosphati-dylcholine replaced Nonidet P-40. Briefly, each 50-p reac-tion mixture contained 50mM sodium Hepes (pH 7.2), 1 mM

DTT, 5 mM MgCI2, phosphatidylcholine vesicles (0.5 mg/ml), 2 ptM [3H]GGPP (33,000 dpm/pmol), 10 .uM Rab protein,100 ng of recombinant rat REP-1, and 100 ng of recombinantrat Rab GGTase. After incubation at 3TC for 15 min,reactions were stopped with SDS/sample buffer, and thismixture was loaded onto 12.5% SDS/PAGE gels (23). Gelswere treated with Entensify, dried, and exposed to KodakXAR film at -700C for the indicated times.

Methylation of Rab Proteins. A two-stage assay was per-formed. In the first stage, Rab proteins were geranylgerany-lated at 370C in a reaction mixture containing 50 mM sodiumHepes (pH 7.2), 5 mM MgCl2, 1 mM DTT, phosphatidylcho-line (0.5 mg/ml), and 10 pM Rab protein. Repeated additionsofunlabeled GGPP (each at 50 ng), recombinant REP-i (eachat 100 ng), and recombinant Rab GGTase (each at 100 ng)were made at 0, 15, 30, and 45 min. The final concentrationsof GGPP, REP-1, and Rab GGTase were 16 pM, 16 pg/ml,and 16 pg/ml, respectively. The final volume of the reactionmixture after the additions at 45 min was 25 p1. After anadditional 15-min incubation at 37°C (total incubation time of60 min), the mixtures were placed on ice. In the second stageof the assay, each 25-pi mixture received 2-5 1Ad of cerebellarmicrosomal membranes (25 pg) in buffer B, after which thevolume of the reaction mixture was adjusted to 46 id withbuffer B followed by a 4-,l addition of [3H]SAM (170,000dpm/pmol) to achieve a final concentration of 4 pM. Afterincubation for 30 win at 370C, each reaction was stopped byaddition ofSDS/sample buffer, and the mixtures were loadedonto a SDS/12.5% polyacrylamide gel. Gels were treated andfluorographed as described above.

RESULTSTo establish an assay for carboxyl-methyltransferase activ-ity, we produced Rab proteins in E. coli and geranylgerany-lated them by incubation with recombinant rat Rab GGTaseplus recombinant REP-i in the presence of GGPP. Underthese conditions the enzyme attaches geranylgeranyl groupsto both cysteines in Rab3A (CAC) and in RablA (CC) (24).After 60 min, we added bovine cerebellar membranes as asource of methyltransferase activity and [3H]SAM as amethyl donor. After a further 30 min, the reaction wasstopped by addition of SDS, and the mixture was subjectedto polyacrylamide gel electrophoresis and fluorography toidentify the 3H-labeled Rab protein. Fig. 1 shows that meth-ylation of Rab3A required all of the components of thegeranylgeranylation reaction mixture. Omission of REP-1/Rab GGTase (lane 3) or GGPP (lane 4) in the first incubationprevented methylation, as did omission ofbovine membranesin the second incubation (lane 1). Thus, methylation requiresprior geranylgeranylation of the Rab protein.The properties of the methylating enzyme in bovine cere-

beilar membranes resembled those previously described forthe prenylated cysteine methyltransferase of bovine brain(12), bovine rod outer segments (14), and human neutrophils(15). Methylation of Rab3A was blocked by N-acetylfarne-sylcysteine, but not by farnesyl acetate (Fig. 2). The sensi-tivity to farnesylated cysteine is compatible with previous

Table 1. DNA sequences of 3' oligonucleotides used for producing mutant RablA and Rab3A proteins by PCRMutant Rab protein DNA sequence of 3' oligonucleotide

RablA-CS 5'-GCCAGTGGATCCAAGCTTTTAGCTGCAACCTCCACCTGACTGCTTGAC-3'RablA-SC 5'-GCCAGTGGATCCAAGCTTTTAGCAGCTACCTCCACCTGACTGCTTGAC-3'RablA-SS 5'-TATGAGCTCGAGTTAGGAGGAACCTCCACCTGACTGCTTGAC-3'Rab3A-CAS 5'-GCCATCGGATCCTCTAGACTAGCTTGCGCAGTCCTGGTGCGGTGGCAC-3'Rab3A-SAC 5'-GCCATCGGATCCTCTAGACTAGCATGCGCTGTCCTGGTGCGGTGGCAC-3'Rab3A-SAS 5'-GCCATCGGATCCCTAGCTTGCGCTGTCCTGGTGCGGTGGCACCTG-3'

Carboxyl-terminal mutants ofcanine RablA and human Rab3A were produced by PCR. The wild-type carboxyl-tenminalsequence of RablA is CC and of Rab3A is CAC. The mutant carboxyl-terminal amino acid sequences are indicated.

Biochemistry: Smeland et al.

10714 Biochemistry: Smeland et al.

Rab 3A +_ _ _ + +

REP-1/Rab GGTasel + _+ +GGPP, + + _

Membranes _ + + I+45-

c] 31-

14-

1 2 3 4 5

kDa45-

< ~ <1ct Rab3A - Rab1A

c'lI CcI_

-4--IC) 6.

-

1

31 -

FIG. 1. Methylation of Rab3A-CAC requires prior geranylgera-nylation. A two-stage reaction (prenylation followed by methylation)was carried out. In the first stage, Rab3A-CAC was incubated for 60min at 37C in the presence (+) or absence (-) of recombinantREP-1/Rab GGTase and unlabeled GGPP as indicated. In the secondstage, bovine cerebellar membranes and 4 M [3HISAM (170,000dpm/pmol) were added. After 30 min at 37TC, the reaction mixturewas subjected to SDS/PAGE. The gels were enhanced with Enten-sify and exposed to Kodak XAR film for 18 h at -70TC. Molecularmass standards are indicated.

suggestions that the same enzyme methylates geranylgera-nylated and farnesylated cysteine (16, 17). The apparent Kmof the carboxyl methyltransferase for SAM was 2 b&M, andthe reaction was inhibited by S-adenosyl-L-homocysteine(50%o inhibition at 1.8 uM), but not by 5'-methylthioadeno-sine at concentrations up to 1 mM (data not shown).

Fig. 3 compares the ability of the methyltransferase tomethylate Rab3A and RablA. Both proteins were geranylger-anylated by the Rab GGTase, as indicated by a controlexperiment performed with VH]GGPP (lanes 1 and 4). How-ever, only Rab3A was methylated (lanes 2 and 5). To deter-mine whether this difference related to the carboxyl-terminalsequence, we used in vitro mutagenesis techniques to changethe Rab3A carboxyl terminus to CC and the RablA carboxylterminus to CAC. This maneuver reversed the specificity for

100 N

co 80- Famesyl Acetate

.20

N-Acetyl20 -Famesylcysteine

O Il0 20 40 60 80 100

Compound (pM)

FIG. 2. Inhibition ofRab3A methylation by N-acetylfarnesylcys-teine. Wild-type Rab3A-CAC was geranylgeranylated with unlabeledGGPP and then methylated by bovine cerebellar membranes in thepresence of [3H]SAM (170,000 dpm/pmol) in a two-stage reaction. Inthe second stage of the reaction, each tube received 1 Al of dimethylsulfoxide [final concentration, 2.5% (vol/vol)] containing the indi-cated concentration of N-acetylfarnesylcysteine (e) or farnesyl ac-etate (o). After 30 min at 37C, the reaction mixture was subjectedto SDS/PAGE. The gel was enhanced with Entensify and exposedto Kodak XAR film for 16 h at -70TC. The resultant fluorograph wasscanned with a densitometer, and the intensities of the Rab3A bandwere quantified with IMAGEQUANT software (Molecular Dynamics).The "100% of control value" refers to the band intensity in tubescontaining neither N-acetylfamesylcysteine nor farnesyl acetate.

21 3 4 516

:ft40.4~ jo.- *

_~ _m i.A

14-

FIG. 3. Prenylation and methylation of wild-type and carboxyl-terminal mutant forms ofRab3A and RablA. Wild-type Rab3A-CAC(lane 2), mutant Rab3A-CC (lane 3), wild-type RablA-CC (lane 5),and mutant RablA-CAC (lane 6) were geranylgeranylated withunlabeled GGPP and then methylated by bovine cerebeliar mem-branes in the presence ofpHISAM in a two-stage reaction. Reactionmixtures were subjected to SDS/PAGE, and the gel was enhancedwith Entensify and exposed to Kodak XAR film for 18 h at -70TC.Lanes 1 and 4 contain wild-type Rab3A-CAC and wild-typeRablA-CC that were prenylated in the presence of[3H]GGPP and notsubjected to methylation. Molecular mass standards are indicated.The arrow denotes the position ofmigration ofwild-type Rab3A. Theasterisk (*) denotes a contaminating protein in the membrane prep-aration whose methylation is not dependent on prior geranylgera-nylation.

methylation. Mutant RablA-CAC was methylated (lane 6),but mutant Rab3A-CC was not (lane 3).As shown in Fig. 4A, Cys -- Ser substitutions in either

position of the CAC or CC sequence did not prevent pren-ylation of the remaining cysteine, whereas replacement ofboth cysteines abolished prenylation. A different result wasobtained in the methylation reaction (Fig. 4B). Replacementof the upstream cysteine of the CAC sequence had no effecton methylation of Rab3A (lane 10), but replacement of thecarboxyl-terminal cysteine abolished methylation (ane 11).The result with RablA was strikingly different. The nativeprotein, terminating in CC, was not methylated (lane 13), butthe mutant protein terminating in SC was methylated (lane14). These data indicate that the failure of methylation of thecarboxyl-terminal cysteine ofthe CC sequence is attributableto interference from the immediately adjacent geranylgera-nylated cysteine.To determine whether a geranylgeranylated CC-termi-

nated protein can bind to the carboxyl methyltransferase, wetested the ability of geranylgeranylated RablA-CC to com-pete with geranylgeranylated Rab3A-CAC for methylation.The assay took advantage of the observation that RablAmigrates faster on SDS/polyacrylamide gels than doesRab3A, thus allowing methylation of the two proteins to beassessed in the same incubation. As shown in Fig. 5, increas-ing amounts of geranylgeranylated RablA-CC inhibited themethylation of Rab3A-CAC (lanes 2-4). Fifty percent inhi-bition was achieved by 6 ,ug ofRablA-CC, as determined bydensitometric analysis of the fluorograph. The RablA-CCwas not methylated. As a positive control for this experi-ment, we tested the ability of RablA-CAC to complete withRab3A-CAC. The RablA-CAC was methylated (lower bandin lanes 5-7), and it competed with Rab3A-CAC (note theprogressive decline in intensity of the upper band in lanes5-7). These data indicate that the CC-terminated proteinretains its ability to bind to the carboxyl methyltransferase,but it cannot accept a methyl group.

Proc. Nad. Acad. Sci. USA 91 (1994)

Proc. Nadl. Acad. Sci. USA 91 (1994) 10715

A [3H]GGiPP

A Rab3A RablA j

OU n cn0 O U) coI< < <0U /)0U) C/)U

kDa 12 4 516 1718145 -

31

B Unlabeled GGPP-* [3H]SAM

lRab3A Rab1A

L) 0C)fC) U) C Cl) C)COC)C'9 1 _101_1 12 13 14 116 kDa

45

- 31

- 1414 --

FIG. 4. Prenylation and methylation of serine-substituted mutant forms of Rab3A-CAC and RablA-CC. (A) Wild-type Rab3A-CAC andRablA-CC and the indicated Cys -. Ser substitution mutants were geranylgeranylated with [3H]GGPP. The reaction mixtures were subjectedto SDS/PAGE, and the gel was enhanced with Entensify and exposed to Kodak XAR film for 18 h at -70TC. (B) Wild-type Rab3A-CAC andRablA-CC and the indicated Cys -- Ser substitution mutants were geranylgeranylated with unlabeled GGPP and then methylated with bovunecerebellar membranes in the presence of [3H]SAM in a two-stage reaction. Reaction mixtures were treated as above. Molecular mass standardsare indicated.

DISCUSSIONThe current data suggest a fundamental difference betweenRab proteins that terminate in CXC and those that terminatein CC. Whereas both classes of proteins are doubly gem-nylgeranylated by Rab GGTase (24), only the CAC protein ismethylated by the carboxyl methyltransferase of bovinecerebeliar membranes. These data provide a probable expla-nation for the previously published findings showing thatYPT5, Rab3A, and Rab4, which terminate in CAC, aremethylated, whereas YPT1, YPT3, and Rab2, which termi-nate in CC, are not methylated (7, 8).The failure to methylate the carboxyl-terminal cysteine of

the CC sequence of RablA is attributable to the cysteineimmediately upstream. Replacement of this cysteine with aserine converted RablA into an efficient substrate for themethyltransferase. It is unlikely that this interference ismediated by the cysteine itself, but rather it occurs becausethe cysteine is geranylgeranylated. The vicinal geranylgera-nyl groups do not appear to prevent the binding of the Rabprotein to the methyltransferase as shown by the competitionexperiment in Fig. 5. However, the vicinal geranylgerany-lated cysteines preclude methyl transfer once binding has

RablA-CC RF1A4A O<n! (pg/,g/tube)

kDa 1 2 13 61 7 8

45

31 Rab3A-CAC

* RablA-CAC

14

FIG. 5. Methylation of geranylgeranylated Rab3A-CAC in thepresence of geranylgeranylated wild-type and carboxyl-terminalmutant forms of RablA. Wild-type Rab3A-CAC (1.3 jg) was gera-nylgeranylated with unlabeled GGPP and then incubated with(3H]SAM and bovine cerebellar membranes in the absence (lane 1)or presence of the indicated amount of either unlabeled geranylger-anylated wild-type RablA-CC (lanes 2-4) or mutant RablA-CAC(lanes 5-7). Lane 8 contains 1.3 ug of the mutant RablA-CAC thathad been geranylgeranylated with unlabeled GGPP and incubatedwith bovine cerebellar membranes and [3HJSAM in the absence ofwild-type Rab3A-CAC. Reaction mixtures were subjected to SDS/PAGE, and the gel was enhanced with Entensify and exposed toKodak XAR film for 12 h at -700C. Molecular mass standards areindicated.

occurred. Moving the geranylgeranylated cysteine one resi-due upstream, as in the CAC sequence, abolishes this inter-ference.Although the CAC-terminated Rab proteins appear to be

carboxyl-methylated in vivo and in vitro, carboxyl methyl-ation may not be necessary for function. Thus, the stel4mutants of Saccharomyces cerevisiae are unable to methyl-ate geranylgeranylated cysteine or farnesylated cysteine (18).However, the sole phenotype is a deficiency of mating as aresult of the failure to secrete the a mating factor, which isnormally farnesylated and carboxyl-methylated (18-20). Al-though the Rab family proteins presumably are not methyl-ated in these cells, the cells show no secretory defect. Thisdiffers from the case ofthe bet2 mutant (25) in which a defectin geranylgeranyltransferase leads to a block in membranemovement in the secretory pathway. Thus, geranylgerany-lation, but not carboxyl methylation, of Rab proteins isnecessary for function.

We thank Dr. Frans Cremers for providing the Rab3A-SAS mutantconstruct, Jeff Cormier for DNA sequencing, Richard Gibson forinvaluable help in preparing bovine brain membranes, and KelliDietel and John Dawson for technical assistance. This work wassupported by research grants from the National Institutes of Health(HL 20948) and the Perot Family Foundation. T.E.S. is the recipientof a postdoctoral fellowship from the American Cancer Society(PF-3951).

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