©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CAAX Geranylgeranyl Transferase Transfers Farnesyl as Efficiently as Geranylgeranyl to RhoB (*)

(Received for publication, December 23, 1994)

Scott A. Armstrong(§)(¶) Voe C. Hannah(§)(¶) Joseph L. Goldstein Michael S. Brown

From the Department of Molecular Genetics, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9046

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

RhoB, a small GTP-binding protein, was shown previously to contain farnesyl (C-15) as well as geranylgeranyl (C-20) groups (Adamson, P., Marshall, C. J., Hall, A., and Tilbrook, P. A. (1992) J. Biol. Chem. 267, 20033-20038). The COOH-terminal sequence of the protein is CCKVL. According to current rules of prenylation, the COOH-terminal leucine should render the protein a substrate for CAAX geranylgeranyl transferase (GGTase-1), but not for CAAX farnesyltransferase (FTase). To determine the mechanism of farnesylation, we prepared recombinant RhoB and incubated it with recombinant preparations of either FTase or GGTase-1. RhoB was neither farnesylated nor geranylgeranylated efficiently by FTase, but it was farnesylated as well as geranylgeranylated by GGTase-1. The enzyme attached farnesyl more efficiently than geranylgeranyl to RhoB. Neither farnesylation nor geranylgeranylation required the cysteine at the fifth position from the COOH terminus. However, replacement of the cysteine at the fourth position abolished attachment of both prenyl groups. We conclude that the previously observed farnesylation of RhoB is attributable to the FTase activity of GGTase-1. These data, and other accumulating data, indicate that GGTase-1 is a highly unusual enzyme that efficiently transfers both farnesyl and geranylgeranyl groups and that the choice of prenyl group is dictated by the nature of the protein acceptor.


INTRODUCTION

Two enzymes transfer prenyl groups from prenyl pyrophosphates to cysteine residues at the fourth position from the COOH terminus of various proteins (reviewed in (1, 2, 3) ). These prenyltransferases recognize cysteine in the context of the CAAX consensus, where C is cysteine, A is an aliphatic amino acid, and X is a COOH-terminal amino acid that specifies which transferase will act. Early studies suggested simple rules for CAAX specificity(4, 5, 6, 7) . CAAX farnesyltransferase (FTase) (^1)prefers methionine or serine at the X position and transfers a 15-carbon farnesyl group. CAAX geranylgeranyl transferase (also known as GGTase-1) prefers leucine at the X position and transfers a 20-carbon geranylgeranyl (GG) group. These conclusions were based on studies in which CAAX-terminated peptides were used as substrates (4, 5, 6) or in which the COOH-terminal CAAX sequence of a known substrate was mutated at the X position so as to change enzyme specificity(7) . More recent studies suggest that the determinants of enzyme recognition may be much more complex, particularly for GGTase-1. (^2)

The earliest indication of this complexity was revealed by the finding that both CAAX prenyl transferases are alpha/beta heterodimers that share a single alpha-subunit(8, 9, 10) . Both prenyltransferases have the unusual ability to form stable noncovalent complexes with their respective prenyl pyrophosphates(5, 11, 12) . These complexes can be isolated by size exclusion chromatography, after which the prenyl pyrophosphate can be transferred to a CAAX-terminated protein. CAAX FTase binds geranylgeranyl pyrophosphate (GGPP) as well as farnesyl pyrophosphate (FPP), but only the FPP is transferred to CAAX boxes(11, 12) . The situation with GGTase-1 is even more complex. In the initial studies with a partially purified enzyme preparation, Yokoyama et al.(6) showed that this enzyme can transfer either farnesyl or GG to leucine-terminated CAAX boxes, although farnesyl transfer was much less efficient.

Although the two enzymes prenylate each other's substrates inefficiently in vitro, this cross-talk may be significant under certain conditions in vivo. Yeast with defects in the beta-subunits of FTase are partially viable because the beta-subunit of GGTase-1 can compensate for the defective beta-subunit of FTase (13) . On the other hand, yeast lacking either the common alpha-subunit or both beta-subunits are nonviable. Moreover, overexpression of the beta-subunit of GGTase-1 partially corrects the defective phenotype in yeast lacking the beta-subunit of FTase, and vice versa(13) .

In addition to the two CAAX prenyltransferases, cells contain a third prenyltransferase designated Rab GGTase (also known as GGTase-2)(3) . Instead of CAAX boxes, this enzyme recognizes proteins that terminate in CysCys or CysXCys. The protein substrates must also contain an upstream sequence, so far unidentified, that is common to all members of the Rab family of GTP-binding proteins (14) . The enzyme contains alpha- and beta-subunits that are related to, but distinct from, those of the CAAX prenyltransferases(15, 16) , and it also requires a third polypeptide, designated Rab escort protein (REP), that binds the upstream Rab sequence and presents the Rab to the catalytic heterodimer(17, 18) .

The complexity of prenylation in vivo is illustrated in a recent study of the p21 Rho proteins(19) . These proteins, in the molecular mass range of 21 kDa, bind GTP and are believed to function in the coupling of membrane and cytoskeletal events. RhoB, a member of this family, terminates in the sequence CCKVL. According to the simple rules outlined above, this protein should be geranylgeranylated by GGTase-1. Adamson et al.(19) showed, surprisingly, that preparations of this protein isolated from transfected simian COS cells radiolabeled with [^3H]mevalonate contained roughly equal portions of [^3H]farnesyl and [^3H]geranylgeranyl residues. Moreover, incubation of the nonprenylated protein with a crude reticulocyte lysate led to prenyl transfer from both [^3H]FPP and [^3H]GGPP. Incorporation of both prenyl groups was abolished by mutation of the C of the CAAX box. Replacement of the immediately upstream C (i.e. the fifth residue from COOH terminus) reduced, but did not abolish, geranylgeranyl transfer, and it did not affect the amount of farnesyl transfer. Adamson et al.(19) concluded that the prenylation of RhoB was complicated and not consistent with any simple notion of CAAX box specificity. However, without the availability of purified enzymes, it was impossible for them to determine which enzyme actually attached farnesyl or geranylgeranyl to RhoB.

In the current studies we have used recombinant FTase and GGTase-1 to prenylate recombinant RhoB in vitro. The data show that this protein will accept either a farnesyl or a GG from GGTase-1. Surprisingly, the efficiency of farnesyl transfer is as great as that of GG transfer. On the other hand, RhoB is not a substrate for FTase.


EXPERIMENTAL PROCEDURES

General Procedures

Standard molecular biology techniques were used(20) . Sequencing reactions were performed on an Applied Biosystems model 373A DNA sequenator. The protein content of the samples was determined by the method of Bradford(21) , using the Bio-Rad protein assay reagent (Bio-Rad).

Production of Recombinant CAAX GGTase-1 and FTase in Sf9 Cells

Recombinant FTase was prepared by coinfection of fall armyworm ovarian (Sf9) cells with recombinant baculoviruses encoding the alpha- and beta-subunits of rat FTase and purified from Sf9 cytosol as previously described(22) . Recombinant CAAX GGTase-1, a heterodimer of the alpha-subunit of FTase and the beta-subunit of GGTase-1, was produced and purified as follows. A cDNA clone that encodes the GGTase-1 beta-subunit (23) was kindly provided by Patrick Casey (Duke University Medical School). A PstI-EcoRI fragment that contains the GGTase-1 beta-subunit coding region was ligated into a PstI-EcoRI digested pVL-1392 baculovirus transfer vector (24) and designated pVL-CAAXGGT. Recombinant baculoviruses encoding the GGTase-1 beta-subunit were generated by cotransfection of Sf9 cells with pVL-CAAXGGT and linearized BacPAK6 viral DNA (Clontech) and plaque purified as described elsewhere (14) . To produce the recombinant heterodimeric enzyme, Sf9 cells (50 ml in suspension at 1 times 10^6 cells/ml) were grown as previously described (14) and infected with baculoviruses encoding the alpha-subunit of FTase (22) and the beta-subunit of GGTase-1 at a multiplicity of infection of 1 alpha-subunit and 1 beta-subunit encoding virus per cell. Twenty-four hours after infection, the cells were collected by centrifugation and washed once with ice-cold phosphate-buffered saline. Cells were lysed in 8 ml of buffer containing 50 mM Tris-HCl (pH 7.4), 50 µM ZnCl(2), 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml pepstatin, 5 µg/ml leupeptin, and 5 µg/ml aprotinin by passage through a 25-gauge needle 15 times. The lysate was then centrifuged at 10^5 times g for 1 h at 4 °C, and the supernatant was chromatographed on a Q-Sepharose 5/5 column that had been equilibrated in a buffer containing 50 mM Tris-HCl (pH 7.4), 50 µM ZnCl(2), and 1 mM dithiothreitol. The heterodimeric CAAX GGTase-1 was eluted with a 50-ml linear gradient from 0 to 400 mM NaCl. Fractions were analyzed by enzyme assay and SDS-polyacrylamide gel electrophoresis, and active fractions were stored in multiple aliquots at -70 C°.

Histidine-tagged RhoB

RhoB containing six histidine residues at the NH(2) terminus was prepared as follows. PCR primers RH-1 (5`-AGAGAATTCCATATGGCGGCCATCCGCAAGAAGCTGGTG-3`) and RH-3 (5`-AGAAAGCTTTCATAGCACCTTGCAGCAGTTGATGCAGC-3`) based on the NH(2)- and COOH-terminal sequences of rat RhoB, respectively (25) , were used in a PCR with 1 µg of DNA from an amplified rat brain cDNA library (Stratagene). The resulting PCR product was digested with EcoRI-HindIII and ligated into an EcoRI-HindIII- digested pBluescript KS vector (Stratagene). The resulting plasmid, designated pBS-RhoB, was sequenced. A NdeI-SalI fragment from pBS-RhoB that contained the RhoB coding region was ligated into a NdeI-XhoI-digested pET14b vector (Novagen), which contains the 6 histidine codons. The resulting plasmid, designated pET14b-RhoB, was transformed into BL21 (DE3) Escherichia coli cells.

E. coli cells containing pET14b-RhoB were grown and lysed as recommended by the manufacturer (Novagen). The His-tagged wild-type RhoB was purified by nickel column chromatography as described previously (14) to greater than 95% purity as judged by SDS-polyacrylamide gel electrophoresis. The protein was dialyzed against a buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 3 mM MgCl(2), 1 mM EDTA, 1 mM dithiothreitol, and 0.1 mM sodium GDP, and stored in multiple aliquots at -70 °C.

Mutations in the COOH-terminal sequence of rat RhoB were introduced by PCR using 1 ng of the original pET-14b-RhoB clone. In all reactions the 5`-oligonucleotide was RH-1 as described above. The 3`-oligonucleotides were as follows: 5`-AGAGTCGACTCATAGCACCTTGCAAGAGTTGATGCAGC-3` for RhoB-SCKVL and 5`-AGAGTCGACTCATAGCACCTTAGAGCAGTTGATGCAGC-3` for RhoB-CSKVL. The PCR products were digested with NdeI-SalI and introduced into an NdeI-XhoI-digested pET14b vector. The mutant proteins were expressed in BL21 (DE3) E. coli cells and purified as described above. The COOH-terminal amino acid sequences of the mutant RhoB proteins were verified by DNA sequencing of the mutant plasmids.

Recombinant Ras Proteins

Wild-type human H-Ras with six histidine residues at the NH(2) terminus (His-tagged H-RasCVLS) was produced as described previously.^2 To produce His-tagged H-RasCVLL, pRcCMV-H-Ras(26) was digested with XbaI, and the fragment containing the H-RasCVLL coding region was ligated into an XbaI-digested pUC18 plasmid. This plasmid was then digested with HindIII, and the H-RasCVLL coding region was introduced into a HindIII-digested pTrcHis-C vector (Invitrogen). The orientation of the resulting plasmid pTRC-RasCVLL was confirmed by restriction mapping. His-tagged H-RasCVLL was produced in E. coli cells and purified as described above for wild-type RhoB.

Assay for Prenyltransferase Activity

Unless otherwise stated, each reaction mixture contained the following components in a final volume of 50 µl: 50 mM Tris-HCl (pH 7.4), 20 µM ZnCl(2), 20 mM KCl, 5 mM MgCl(2), 5 mM dithioerythritol, 20 µM Zwittergent(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) , 100 ng of recombinant enzyme, the indicated amount of [^3H]FPP (49,500 dpm/pmol; DuPont NEN) or [^3H]GGPP (33,000 dpm/pmol; American Radiochemical Co.), and the indicated protein substrate. Following incubation for 15 min at 37 °C, the amount of [^3H]farnesyl or [^3H]GG transferred was measured by ethanol precipitation and filtration on glass fiber filters (7) with modification as previously described(27) .

Preparation of [^3H]Prenyl Pyrophosphate/GGTase-1 Complex

One µg of recombinant CAAX GGTase-1 was incubated for 10 min at 37 °C with 50 mM Hepes (pH 7.2), 0.1 M NaCl, 0.2% (w/v) beta-D-octylglucoside, 1 mM dithiothreitol, and the indicated amount of [^3H]FPP (49,500 dpm/pmol) or [^3H]GGPP (33,000 dpm/pmol) in a final volume of 50 µl. Each reaction mixture was then applied to a 1-ml Sephadex G-50 (Pharmacia Biotech Inc.) column (at 4 °C) that had been previously equilibrated with the same buffer(12) . The columns were eluted by centrifugation at 820 times g for 2 min. The amount or [^3H]FPP of [^3H]GGPP in the eluate was determined in a scintillation counter.

Transfer of [^3H]GG or [^3H]Farnesyl to RhoB or Ras from Isolated Complex

A 20-µl aliquot of the [^3H]prenyl pyrophosphate/enzyme complex eluted from the Sephadex G-50 column (see above) was counted in a scintillation counter. A second 20-µl aliquot was added to 10 µl of buffer adjusted to give a final concentration of 5 mM MgCl(2), 20 µM ZnCl(2), and 20 µM RhoB or H-Ras. The reaction mixture was incubated for the indicated time at 37 °C, after which the amount of [^3H]farnesyl or [^3H]GG transferred to RhoB or H-Ras was measured as described above under ``Assay for Prenyltransferase Activity.''


RESULTS

Purified recombinant CAAX FTase transferred [^3H]farnesyl from [^3H]FPP to H-Ras with its normal CAAX box (CVLS), but not to a mutant H-Ras in which X had been changed to leucine (Fig. 1A). The enzyme transferred a very small but detectable amount of [^3H]farnesyl to RhoB. It did not transfer [^3H]GG from [^3H]GGPP to any of the protein substrates (Fig. 1B).


Figure 1: Inability of FTase to prenylate RhoB in presence of [^3H]FPP (A) or [^3H]GGPP (B). Assays contained, in a final volume of 50 µl, 100 ng of recombinant FTase and the indicated amount of Ras-CVLS (box), Ras-CVLL (), or RhoB (bullet) in the presence of either 2 µM [^3H]FPP (A) or 2 µM [^3H]GGPP (B). After incubation for 15 min at 37 °C, the amount of [^3H]farnesyl (A) or [^3H]GG (B) transferred to the indicated protein substrate was determined in duplicate. Blank values carried out in parallel reactions in the absence of any protein substrate (0.08-0.13 pmol/tube) were subtracted from each value.



The results with recombinant GGTase-1 were quite different (Fig. 2). As expected, this enzyme transferred [^3H]GG to the leucine-terminated mutant form of H-Ras (H-Ras-CVLL), but there was no appreciable transfer to wild-type H-Ras (H-Ras-CVLS) (Fig. 2B). GGTase-1 also transferred [^3H]GG to RhoB, which terminates in CCKVL (Fig. 2B). As expected, GGTase-1 did not transfer [^3H]farnesyl to H-Ras-CVLS, and it had only a slight ability to farnesylate H-Ras-CVLL (Fig. 2A). Surprisingly, however, GGTase-1 had a robust ability to farnesylate RhoB (Fig. 2A). With RhoB as acceptor, the maximal velocity for the transfer of [^3H]farnesyl was more than 3-fold greater than the maximal velocity of transfer of [^3H]GG (compare Panels A and B).


Figure 2: Farnesylation (A) and geranylgeranylation (B) of RhoB by recombinant CAAX GGTase-1. Assays contained, in a final volume of 50 µl, 100 ng of recombinant GGTase-1 and the indicated amount of Ras-CVLS (box), Ras-CVLL (), or RhoB (bullet) in the presence of either 2 µM [^3H]FPP (A) or 2 µM [^3H]GGPP (B). After incubation for 15 min at 37 °C, the amount of [^3H]farnesyl (A) or [^3H]GG (B) transferred to the indicated protein substrate was determined in duplicate. Blank values carried out in parallel reactions in the absence of any protein substrate (0.15-0.23 pmol/tube) were subtracted from each value.



Fig. 3shows the prenyl pyrophosphate saturation curves for GGTase-1. With RhoB as acceptor, GGTase-1 showed a relatively high affinity for [^3H]FPP (Fig. 3A). The concentration of [^3H]FPP giving half maximal velocity (S(0.5)) was in the range of 0.5 µM. The enzyme had a similar high affinity for [^3H]GGPP when H-Ras-CVLL was used as an acceptor (S(0.5) 0.5 µM) (Fig. 3B). However, with RhoB as acceptor, the affinity for [^3H]GGPP was much lower. Saturation was not approached at 5 µM, which was the highest concentration tested (Fig. 3B). The enzyme also showed a low affinity for [^3H]GGPP when H-Ras-CVLS was the acceptor (Fig. 3B).


Figure 3: Prenyl pyrophosphate saturation curves for recombinant CAAX GGTase-1. Assays contained, in a final volume of 50 µl, 100 ng recombinant GGTase-1, 20 µM of the indicated RhoB or H-Ras protein, and varying concentrations of either [^3H]FPP (A) or [^3H]GGPP (B). After incubation for 15 min at 37 °C, the amount of [^3H]farnesyl (A) or [^3H]GG (B) transferred to the indicated protein substrate was determined in duplicate. Blank values carried out in parallel reactions in the absence of any prenyl pyrophosphate (0.005-0.01 pmol/tube) were subtracted from each value.



To explore the roles of the two cysteine residues in the CCKVL sequence of RhoB, we prepared recombinant RhoB with serine residues replacing either of the cysteines (Fig. 4). Replacement of the first cysteine (SCKVL) reduced slightly the ability of the protein to be farnesylated (Fig. 4A) and produced a 2-fold increase in the amount of [^3H]GG incorporated (Fig. 4B). Replacement of the second cysteine (CSKVL) eliminated the acceptance of [^3H]farnesyl (Fig. 4A) and severely reduced the ability of the protein to accept [^3H]GG (Fig. 4B).


Figure 4: Farnesylation (A) and geranylgeranylation (B) of COOH-terminal mutants of RhoB by recombinant CAAX GGTase-1. Assays contained, in a final volume of 50 µl, 100 ng of recombinant GGTase-1, either 2 µM [^3H]FPP (A) or 2 µM [^3H]GGPP (B), and varying concentrations of the indicated RhoB protein. After incubation for 15 min at 37 °C, the amount of [^3H]farnesyl (A) or [^3H]GG (B) transferred to the protein substrate was measured in duplicate. Blank values carried out in parallel reactions in the absence of any protein substrate (0.12-0.17 pmol/tube) were subtracted from each value.



Fig. 5shows an experiment designed to compare the carrier function of GGTase-1 with respect to the two prenyl pyrophosphates. The enzyme was incubated with either [^3H]FPP (Fig. 5A) or [^3H]GGPP (Fig. 5B), and the complexes were isolated by gel exclusion chromatography. We then incubated the complexes with RhoB and measured the amount of [^3H]prenyl that was transferred. GGTase-1 bound comparable amounts of [^3H]FPP and [^3H]GGPP. During the subsequent incubation, approximately 50% of both prenyl groups were transferred to RhoB.


Figure 5: Prenyl pyrophosphate saturation curves for formation of [^3H]prenyl pyrophosphate/CAAX GGTase-1 complex and transfer of [^3H]prenyl from isolated complex to RhoB. Assays contained, in final volume of 50 µl, 1 µg of recombinant GGTase-1, the indicated amount of [^3H]FPP (A) or [^3H]GGPP (B), and buffer components as described under ``Experimental Procedures.'' After incubation for 10 min at 37 °C, the [^3H]prenyl pyrophosphate/enzyme complex was isolated on a Sephadex G-50 column as described under ``Experimental Procedures'' and divided into equal aliquots. One aliquot was used to determine the amount of isolated [^3H]prenyl pyrophosphate/enzyme complex (bullet). The second aliquot was incubated with 20 µM RhoB for 5 min at 37 °C, after which the amount of [^3H]farnesyl or [^3H]GG transferred to RhoB was determined () as described under ``Experimental Procedures.'' Blank values carried out in parallel reactions in the presence of 20 µM unlabeled FPP or GGPP (0.002-0.04 pmol/tube) were subtracted from each value. Each value is the average of duplicate reactions.



The binding of [^3H]FPP to GGTase-1 was competitively inhibited by unlabeled FPP and by unlabeled GGPP (Fig. 6A). Surprisingly, the binding of [^3H]GGPP was not inhibited by unlabeled FPP, although it was inhibited by unlabeled GGPP (Fig. 6B). This striking result was repeated on several occasions with the same results.


Figure 6: Inhibition of formation of [^3H]FPP/GGTase-1 complex (A) and [^3H]GGPP/GGTase-1 complex (B) by unlabeled prenyl pyrophosphates. One µg of recombinant CAAX GGTase-1 was incubated in 50 µl of a solution containing 75 nM [^3H]FPP (A) or [^3H]GGPP (B) plus the indicated concentration of unlabeled FPP or GGPP. After incubation for 10 min at 37 °C, the [^3H]prenyl pyrophosphate/enzyme complex was isolated, and the amount of ^3H radioactivity was determined as described under ``Experimental Procedures.'' Each value is the average of duplicate incubations.



As expected from the steady state kinetic analysis, the [^3H]FPP bound to GGTase-1 was transferred efficiently to RhoB, but not efficiently to H-Ras-CVLL or H-Ras-CVLS (Fig. 7A). The bound [^3H]GGPP was transferred efficiently to RhoB and to H-Ras-CVLL, but not to H-Ras-CVLS (Fig. 7B).


Figure 7: Transfer of CAAX GGTase-1 bound [^3H]FPP (A) or [^3H]GGPP (B) to RhoB and Ras protein substrates. One µg of recombinant GGTase-1 was incubated with either 0.5 µM [^3H]FPP (A) or [^3H]GGPP (B) in a final volume of 50 µl for 10 min at 37 °C. The resulting [^3H]prenyl pyrophosphate/enzyme complex was isolated as described under ``Experimental Procedures.'' The isolated complex was divided into two equal aliquots. One aliquot was used to measure the amount of complex formed, and the second aliquot was incubated for 45 s at 37 °C with 20 µM of the indicated RhoB or Ras protein, after which the percentage of [^3H]farnesyl or [^3H]GG transferred to the indicated protein substrate was determined as described under ``Experimental Procedures.'' Each value is the average of duplicate incubations.




DISCUSSION

The results in this study, considered together with previous data, reveal that the CAAX prenyltransferases are highly unusual enzymes. In the absence of a protein acceptor, both enzymes form highly stable noncovalent complexes with FPP and with GGPP (5, 11, 12) (Fig. 5). Farnesyltransferase will efficiently transfer only the farnesyl group(11, 12) . GGTase-1 will transfer either the farnesyl or the GG group depending on which protein acceptor is presented (Fig. 7).

The peculiar properties of GGTase-1 are particularly apparent when RhoB is the substrate. In this case the enzyme will transfer either the farnesyl or the GG group. Farnesyl transfer is more efficient than GG transfer in terms of the maximal velocity of the enzyme (Fig. 2). Moreover, its affinity for FPP is greater than for GGPP (Fig. 3). We believe that these findings explain the previous observation of Adamson et al.(19) that RhoB can be farnesylated as well as geranylgeranylated in animal cells and in reticulocyte lysates.

What are the properties of RhoB that make it an efficient acceptor of farnesyl groups? RhoB terminates in CCKVL. The C at the fifth position from the COOH terminus is not crucial in directing the prenylation specificity. When this C was substituted with a serine, RhoB remained susceptible to farnesylation as well as geranylgeranylation by GGTase-1 (Fig. 4). As expected, the C at the fourth position from the COOH terminus was crucial for the acceptance of both prenyl groups. Based on these observations, we believe that each RhoB molecule accepts only a single prenyl group, either a farnesyl or a GG, which is attached to the cysteine of the CAAX box.

A prominent feature of the RhoB CAAX box is the lysine in place of the usual aliphatic amino acid at the first A position. This is not likely to be important in dictating prenylation specificity since Adamson et al.(19) changed this lysine to a leucine without any effect on either farnesylation or geranylgeranylation in crude systems. Thus, the CAAX box and the adjacent C are unlikely to account for the ability of RhoB to be farnesylated by GGTase-1. The responsible sequence must be on the NH(2)-terminal side of the CAAX box.

One other small GTP binding protein, K-RasB, is known to have a regulatory sequence upstream of a CAAX box. Recently, this protein was shown to be geranylgeranylated by GGTase-1 even though its CAAX box terminates in methionine, which is not the preferred substrate for geranylgeranylation.^2 Studies with chimeric proteins indicated that the high affinity of K-RasB for GGTase-1 was attributable in part to a polylysine sequence immediately upstream of the CAAX box.^2 Inasmuch as RhoB does not have a polylysine sequence, this explanation cannot suffice for this protein.

The unusual ability to transfer different prenyl groups to different protein acceptors renders GGTase-1 highly unusual among transferase enzymes. Further studies are clearly needed to define the mechanism for this selectivity, and eventually to find the structural basis.


FOOTNOTES

*
This work was supported by the National Institutes of Health Grant HL20948 and by a research grant from the Perot Family Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
The first two authors contributed equally to this work.

Supported by Medical Scientists Training Grant GM08014.

(^1)
The abbreviations used are: FTase, farnesyltransferase; FPP, farnesyl pyrophosphate; GG, geranylgeranyl; GGPP, geranylgeranyl pyrophosphate; GGTase-1, geranylgeranyl transferase-1; His-tagged, histidine-tagged; PCR, polymerase chain reaction.

(^2)
James, G. L., Goldstein, J. L., and Brown, M. S. (1995) J. Biol. Chem.270, 6221-6226


ACKNOWLEDGEMENTS

We thank Kara Robinson for excellent technical assistance, Jeff Cormier and Michelle Laremore for DNA sequencing; Pat Casey for providing the cDNA for the beta-subunit of CAAX GGTase-1, Doug Andres for providing the Ras-CVLL plasmid, Guy James for helpful suggestions and critical review of the manuscript, and Michael Gelb for helpful discussions and for providing purified brain GGTase-1, which was useful during the preliminary stages of this work.


REFERENCES

  1. Casey, P. J. (1992) J. Lipid Res. 33, 1731-1740 [Medline] [Order article via Infotrieve]
  2. Schafer, W. R., and Rine, J. (1992) Annu. Rev. Genet. 30, 209-237
  3. Brown, M. S., and Goldstein, J. L. (1993) Nature 366, 14-15 [CrossRef][Medline] [Order article via Infotrieve]
  4. Reiss, Y., Stradley, S. J., Gierasch, L. M., Brown, M. S., and Goldstein, J. L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 732-736 [Abstract]
  5. Yokoyama, K., and Gelb, M. H. (1993) J. Biol. Chem. 268, 4055-4060 [Abstract/Free Full Text]
  6. Yokoyama, K., Goodwin, G. W., Ghomashchi, F., Glomset, J. A., and Gelb, M. H. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5302-5306 [Abstract]
  7. Moores, S. L., Schaber, M. D., Mosser, S. D., Rands, E., O'Hara, M. B., Garsky, V. M., Marshall, M. S., Pompliano, D. L., and Gibbs, J. B. (1991) J. Biol. Chem. 266, 14603-14610 [Abstract/Free Full Text]
  8. Seabra, M. C., Reiss, Y., Casey, P. J., Brown, M. S., and Goldstein, J. L. (1991) Cell 65, 429-434 [Medline] [Order article via Infotrieve]
  9. Chen, W.-J., Andres, D. A., Goldstein, J. L., Russell, D. W., and Brown, M. S. (1991) Cell 66, 327-334 [Medline] [Order article via Infotrieve]
  10. Moomaw, J. F., and Casey, P. J. (1992) J. Biol. Chem. 267, 17438-17443 [Abstract/Free Full Text]
  11. Reiss, Y., Seabra, M. C., Armstrong, S. A., Slaughter, C. A., Goldstein, J. L., and Brown, M. S. (1991) J. Biol. Chem. 266, 10672-10677 [Abstract/Free Full Text]
  12. Reiss, Y., Brown, M. S., and Goldstein, J. L. (1992) J. Biol. Chem. 267, 6403-6408 [Abstract/Free Full Text]
  13. Trueblood, C. E., Ohya, Y., and Rine, J. (1993) Mol. Cell. Biol. 13, 4260-4275 [Abstract]
  14. Cremers, F. M., Armstrong, S. A., Seabra, M. C., Brown, M. S., and Goldstein, J. L. (1994) J. Biol. Chem. 269, 2111-2117 [Abstract/Free Full Text]
  15. Seabra, M. C., Goldstein, J. L., Südhof, T. C., and Brown, M. S. (1992) J. Biol. Chem. 267, 14497-14503 [Abstract/Free Full Text]
  16. Armstrong, S. A., Seabra, M. C., Südhof, T. C., Goldstein, J. L., and Brown, M. S. (1993) J. Biol. Chem. 268, 12221-12229 [Abstract/Free Full Text]
  17. Seabra, M. C., Brown, M. S., Slaughter, C. A., Südhof, T. C., and Goldstein, J. L. (1992) Cell 70, 1049-1057 [Medline] [Order article via Infotrieve]
  18. Andres, D. A., Seabra, M. C., Brown, M. S., Armstrong, S. A., Smeland, T. E., Cremers, F. P. M., and Goldstein, J. L. (1993) Cell 73, 1091-1099 [Medline] [Order article via Infotrieve]
  19. Adamson, P., Marshall, C. J., Hall, A., and Tilbrook, P. A. (1992) J. Biol. Chem. 267, 20033-20038 [Abstract/Free Full Text]
  20. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  21. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  22. James, G. L., Goldstein, J. L., Brown, M. S., Rawson, T. E., Somers, T. C., McDowell, R. S., Crowley, C. W., Lucas, B. K., Levinson, A. D., and Marsters, J. C., Jr. (1993) Science 260, 1937-1942 [Medline] [Order article via Infotrieve]
  23. Zhang, F. L., Diehl, R. E., Kohl, N. E., Gibbs, J. B., Giros, B., Casey, P. J., and Omer, C. A. (1994) J. Biol. Chem. 269, 3175-3180 [Abstract/Free Full Text]
  24. O'Reilly, D. R., Miller, L. K., and Luckow, V. A. (1992) Baculovirus Expression Vectors: A Laboratory Manual , W. H. Freeman & Co., New York
  25. Jahner, D., and Hunter, T. (1991) Mol. Cell. Biol. 11, 3682-3690 [Medline] [Order article via Infotrieve]
  26. James, G. L., Brown, M. S., Cobb, M. H., and Goldstein, J. L. (1994) J. Biol. Chem. 269, 27705-27714 [Abstract/Free Full Text]
  27. Seabra, M. C., Brown, M. S., and Goldstein, J. L. (1993) Science 259, 377-381 [Medline] [Order article via Infotrieve]

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