Cell, Vol. 65, 429-434,

May 3, 1991, Copyright

0 1991 by Cell Press

Protein Farnesyltransferase Geranylgeranyltransferase a Common a Subunit Miguel C. Seabra,* Yuval Reiss,” Patrick J. Casey,t Michael S. Brown,* and Joseph L. Goldstein* *Department of Molecular Genetics University of Texas Southwestern Medical Center Dallas, Texas 75235 tsection of Cell Growth, Regulation, and Oncogenesis Duke University Medical Center Durham. North Carolina 27710

Summary Mammalian farnesyltransferase, which attaches a 15 carbon isoprenoid, farnesyl, to a cysteine in ~21”” proteins, contains two subunits, a and p. The p subunit is known to bind ~21”’ proteins. We show here that the a subunit is shared with another prenyltransferase that attaches 20 carbon geranylgeranyl to Ras-related proteins. Farnesyltransferase and geranylgeranyltransferase have similar molecular weights on gel filtration, but are separated by ion exchange chromatography. Both enzymes are precipitated and immunoblotted by multiple antibodies directed against the a subunit of farnesyltransferase. The two transferases have different specificities for the protein acceptor; farnesyltransferase prefers methionine or serine at the COOH-terminus and geranylgeranyltransferase prefers leucine. The current data indicate that both prenyltransferases are heterodimers that share a common a subunit with different p subunits. Introduction A variety of intracellular membrane-bound proteins contain covalently bound prenyl groups that aid in membrane attachment (reviewed in Glomset et al., 1990; Maltese, 1990). Thus far, two types of prenyl groups have been identified in animal cells. Farnesyl, a 15carbon isoprenoid, is found on lamin 6 (Farnsworth et al., 1989), a structural protein of the nuclear envelope, and on the p21ras proteins (Caseyet al., 1989; Reisset al., 199Oa), which are a family of 21 kd GTP-binding proteins that regulate cell growth. Farnesyl is also attached to they subunit of transducin (Lai et al., 1990; Fukada et al., 1990), a GTP-binding protein that transmits the visual signal in the retina. Protein-bound geranylgeranyl (GG), a 20 carbon isoprenoid, is much more abundant than protein-bound farnesyl in animal cells (Rillinget al., 1990; Farnsworth et al., 1990), but sofaronly four proteins have been identified as being geranylgeranylated. These are they subunit of neural G proteins (Mumby et al., 1990; Yamane et al., 1990) and three low molecular weight GTP-binding proteins, including G25K (Yamane et al., 1991), raplB (also called smg ~218) (Kawata et al., 1990), and raplA/krev-1 (Buss et al., 1991). Both types of prenyl groups are attached to cysteine residues in thioether linkage. Thus far, all of the known

and Share farnesylated proteins have a similar COOH-terminal sequence, Cys-aliphatic-aliphatic-X, where X is methionine or serine (Reiss et al., 1991 b). The farnesyl group is transferred to this cysteine from afarnesyl pyrophosphate (FPP) donor, after which the protein is cleaved at the COOHterminal side of the cysteine, and the cysteine COOH group is methylated (Hancock et al., 1989). The four proteins that are known to be geranylgeranylated resemble the farnesylated proteins in that they contain cysteine as the fourth residue from the COOHterminus. However, these proteins terminate in leucine instead of methionine or serine as in the farnesylated proteins. Some of the low molecular weight GTP-binding proteins that are likely to be prenylated do not have a cysteine residue four amino acids from the COOH-terminus. These proteins end in sequences such as C-X-C or GGGCC (Hail, 4 990). Based on the data of Rilling et al. (1990) and Farnsworth et al. (1990), it is believed that many of these proteins are likely to be geranylgeranylated, but this has not been demonstrated. It is also not known whether any of these C-X-C- or GGGCC-terminating proteins are trimmed or methylated after prenylation. A protein farnesyltransferase has been purified to homogeneity from rat brain cytosol (Reiss et al., 1990a, 1990b). This enzyme transfers a farnesyl group from FPP to cysteine residues that are fourth from the COOH-terminus in p21 rasproteins. The enzyme binds with high affinity to peptides as short as four residues in length, provided that cysteine is fourth from the COOH-terminus and provided that the other three residues conform to certain rules (Reiss et al., 1990a, 1991 b). The farnesyltransferase does not recognize peptides of the type that are known to be geranylgeranylated, i.e., those that terminate in leucine. This finding led to the suggestion that at least one other prenyltransferase must exist to transfer GG residues (Reiss et al., 1991b). Indeed, an enzyme with this activity has recently been demonstrated; its characterization will be reported elsewhere (P. J. Casey et al., unpublished data). Gel filtration and SDS-polyacrylamide gel electrophoresis experiments indicate that the rat brain farnesyltransferase is a dimer of nonidentical subunits of approximately 49 and 48 kd, designated a and 8, respectively (Reiss et al., 1990a, 1990b, 1991a). The two subunits can be separated only by denaturation, after which all catalytic and binding functions are lost. Chemical cross-linking studies showed that the 8 subunit binds the Ras protein substrate (Reiss et al., 1991a). The holoenzyme forms a stable complex with FPP that can be isolated by gel filtration. Upon subsequent incubation with a Ras protein, the farnesyl residue is efficiently transferred from the bound FPP to the appropriate cysteine in Ras. Inasmuch as the 8 subunit binds the peptide acceptor, it was hypothesized that the a subunit might be responsible for stable binding of FPP (Reiss et al., 1991a). In this study we report the surprising obr;ervation that the a subunit of the protein farnesyltransferase is also

Cell 430

Coomassie Stain

lmmunoblots 123456 7891

8tA. 1.0 i

I 0.5

--iali

JO Fraction

Number

8. Figure 1. lmmunoblot Antibodies

Analysis

with Anti-Protein

Farnesyltransferase

An 8% SDS-polyacrylamide gel (140 x 120 x 1.5 mm) was loaded with 480 ng of affinity-purified farnesyltransferase in a 5 mm wide lane (lane 1) and with 600 ng of the same preparation on a preparative 50 mm wide lane (lane 2-9). After electrophoresis (25 mA, 4 hr, 22OC), the gel was sliced into two parts: lane 1 was stained with Coomassie blue, and lanes 2-9 were transferred to nitrocellulose. After transfer the nitrocellulose was cut into 3-4 mm strips, each containing approximately 50 ng of protein. The strips were incubated with one of the following IgG antibodies: lane 2, Y533 at 1 uglml; lane 3, Y536 at 3 ngl ml; lane 4, X287 at 10 pglml; lane 5, Y533 at 1 uglml plus X287 at IO uglml. Lanes 6-9 contained corresponding amounts of preimmune IgG from each of the rabbits whose immune IgG was used in lanes 2-5, respectively. After incubation for 1 hr at room temperature, the strips were washed and then incubated for 1 hr with ‘251-labeled goat anti-rabbit IgG (lo6 cpmlml). The strips were exposed to Kodak XAR film for 1 hr at -7OOC. Thegel wascalibrated with theindicated molecular weight marker proteins.

present in an enzyme with GG-transferase activity. The latter enzyme, which can be separated from the farnesyltransferase by ion exchange chromatography, does not have the same 8 subunit. We suggest that both prenyltransferases may be heterodimers that share a common a subunit but have distinct 8 subunits. Results Figure 1 (left) shows the two subunits of the purified rat brain protein farnesyltransferase as visualized by Coomassie staining after SDS gel electrophoresis. Figure 1 (right) shows autoradiograms of immunoblots of this gel performed with antibodies raised against synthetic peptides corresponding to sequences in the a subunit (antibody Y533, lane 2), 8 subunit (X287, lane 4), or a mixture of the two antibodies (lane 5). Lane 3 was immunoblotted with Y.536, an antibody directed against a bacterially expressed fusion protein containing the COOH-terminal326 amino acids of the a subunit. The antibodies clearly identified the two subunits with no cross-reactivity. Figure 2 shows the results of ion exchange chromatography on a Mono Q resin performed with a crude ammonium sulfate fraction of rat brain cytosol. Fractions were assayed for farnesyltransferase activity using [3H]FPP and native p21 H-ras,which contains CVLS as the COOHterminal sequence. The fractions were also assayed for GG-transferase activity using [3H]geranylgeranyl pyrophosphate ([3H]GGPP) and a modified p21HqraSthat contains CVLL as the COOH-terminal sequence. The GGtransferase activity emerged from the column earlier than

Anti-o

w_..,-

Anti-6

.a* 3

8 5

12 11

14 13

16 16

20 19

Fraction

22 21

24 23

26 25

28 27

30 29

t

45 kDa

f

45 kDa

34 32

37

Number

Figure 2. Separation of Protein Farnesyltransferase from GG-Transferase on Mono Q Ion Exchange Chromatography

Protein

(A) A 30%-500/o ammonium sulfate fraction from rat brain (220 mg of protein) (Reiss et al., 199Oa) was applied at 4OC to a Mono Q column (10 x 1 cm) equilibrated in 50 mM Tris-chloride (pH 7.5) 0.05 M NaCI, 1 mM DTT, and 20 pM ZnClp, and 4 ml fractions were collected. The column was washed with 24 ml of the loading buffer (fractions 3-9) followed by a 18 ml gradient of 0.05 to 0.15 M NaCl (fractions 10-13) followed by a second wash with 16 ml of the same buffer containing 0.15 M NaCl (fractions 14-18). The enzymes were eluted with a 112 ml gradient of 0.15 to 1 M NaCl in the same buffer (fractions 19-40) at a flow rate of 1 mllmin. An aliquot of each fraction was assayed for farnesyltransferase activity (0.5 ul) (open circles) and for GG-transferase activity (2 ul) (closed circles) as described below. The protein content of each fraction (dashed line) was estimated from the absorbance at 280 nm. Each farnesyltransferase assay contained in a final volume of 25 ul the following components: 50 mM Tris-chloride (pH 7.5), 100 mM NaCI, 0.2% (w/v) octyl-g-D-glucopyranoside, 1 mM DTT, 5 mM MgCl*, 20 PM ZnC&., 40 nM ~21 H-ia*,0.4 pM 13H]FPP (30,000 dpm/pmol), and the indicated fraction volume. Each GG-transferase assay contained in a final volume of 50 nl the following components: 50 mM Tris-chloride (pH 7.5) 100 mM NaCI, 0.2% octyl+o-glucopyranoside, 2 mM DTT, 5 mM MgCI,, 20 pM ZnClz, 5 nM p21H-‘asCvLL, 2 nM 13H]GGPP (10,000 dpmlpmol), and the indicated fraction volume. All assays were incubated for 30 min at 37OC. (6) An aliquot of each fraction (50 ~1) was subjected to electrophoresis (25 mA, 4 hr, 22OC) on an 8% SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with antibodies as described in Experimental Procedures. IgG-Y533 (1 pglml) was the anti-a farnesyltransferase antibody, and IgG-X287 (10 nglml) was the anti-6 farnesyltransferase antibody. After incubation for 1 hr at room temperature, the filter was washed and then incubated for 1 hr with ‘Wabeled goat anti-rabbit IgG (IO” cpmlml). The filter was then exposed to Kodak XAR film for 3 hr at -7OOC. The arrow denotes the position of migration of the ovalbumin standard (45 kd).

the farnesyltransferase. The peak fraction of GG-transferase (fraction 22) had no detectable farnesyltransferase activity, and the opposite was true of the peak farnesyltransferase fraction (fraction 24). Figure 28 shows immunoblots of the column fractions probed with the anti-a and anti-6 subunit antibodies. The a subunit immunoreactivity showed two peaks in fractions 22 and 24. In contrast, the 6 subunit showed only a single peak in fraction 24, and none was detectable in fraction 22. Further immunoblot analyses were performed with fractions 22 and 24 (Figure 3). The fraction 22 material (containing GG-transferase activity) reacted with the anti-a

Common 431

a Subunit

for Prenyltransferases

Fraction 22

Fraction24 I I ABCD

IA

-95

Anti- a -w*

r-w

-55 -43 ,x

EFGH 16

18 17

20 19

22 21

16 23

18 17.

20 19

22 21

23

Fraction Figure 3. lmmunoblot Mono Cl Column

Analysis

of Prenyltransferase

Fractions

from

Mono Q fraction 22 (750 ug) and Mono Q fraction 24 (500 ug) were subjected to electrophoresis (25 mA, 1 hr, 22’C) on a preparative 7% SDS-polyacrylamide gel (80 x 70 x 0.75 mm) as indicated and transferred to nitrocellutose paper. Replicate strips (3-4 mm wide) containing 50 ug (fraction 22) or 33 sg of protein (fraction 24) were cut and probed with one of the following: lane A, preimmune anti-a IgG-Y533 at 1 uglml; lane B, immune IgG-Y533 at 1 uglml; lane C, immune IgG-Y533 at 1 pg/ml plus 25 uM of the synthetic peptide against which the antibody was raised (Reiss et al., 1991a); lane D, immune IgG-Y533 at 1 uglml plus 25 uM of an irrelevant synthetic peptide; lane E, preimmune anti-8 IgG-X287 at IO uglml; lane F, immune IgG-X287 at 10 sglml; lane G, immune IgG-X287 at 10 uglml plus 25 uM of the synthetic peptide against which the antibody was raised; lane H, immune IgG-X287 at 10 uglml plus 25 pM of an irrelevant synthetic peptide. After incubation for 1 hr at room temperature, the strips were washed and then incubated for 1 hr with ‘*Wabeled goat anti-rabbit IgG (10’ cpmlml). The strips were exposed to Kodak XAR film for 2 hr at -7OOC. The gels were calibrated with the indicated molecular weight marker proteins. Arrows denote the position of migration of the a (top) and 8 (bottom) subunits of farnesyltransferase.

antipeptide antibody (lane B), but not with preimmune IgG from the same animal (lane A). Binding was prevented by inclusion of the synthetic peptide against which the antibody was made (lane C), but not by an irrelevant peptide (lane D). This pattern of immunoreactivity with the anti-a antibody was the same when fraction 24 (containing farnesyltransferase) was probed. The anti-p antibody gave a different pattern, however. Fraction 22 showed no evidence of a 46 kd protein that reacted with this antibody (fraction 22, lanes E-H). The only binding that was observed was nonspecific and was not prevented by an inclusion of an excess of the relevant peptide. In contrast, fraction 24 showed a dense band of reactivity at 46 kd (Figure 3, lane F) that was competed with the relevant peptide (lane G), but not with an irrelevant peptide (lane H). These data confirm that the GG-transferase-containing fraction contains the a subunit, but not the 6 subunit of the protein farnesyltransferase. Similar results were obtained with another anti-a farnesyltransferase antibody (IgG-X284) directed against a different synthetic peptide derived from the a subunit amino acid sequence (data not shown). The distinction in subunit composition was also evident when the separated GG-transferase and farnesyltransferase activities were subjected to gel filtration on the same

Figure

4. Gel Filtration

of Farnesyltransferase

and GG-Transferase

Mono Q-purified farnesyltransferase (0.4 mg of fraction 24) (A)or Mono Q-purified GG-transferase (0.6 mg of fraction 22) (B) were subjected to gel filtration at 4OC on Superdex 75 FPLC columns (Pharmacia-LKB Biotechnology) in 50 mM sodium HEPES (pH 7.5) 0.2% OCtyl-P-D100 mM NaCI, and 1 mM DTT at a flow rate of 0.25 & copyranoside, mllmin. Fractions of 0.5 ml were collected. A 3 ul aliquot of each fraction was assayed for farnesyltransferase activity, and a 5 ul aliquot was assayed for GG-transferase activity for 30 min at 37OC as described in the legend to Figure 2. The column was calibrated with thyroglobulin (670 kd), j3-globulin (158 kd), ovalbumin (44 kd), myoglobin (17 kd), and vitamin 812 (1.35 kd). Arrows denote the elution position of the 158 kd and the 44 kd markers. A 75 ul aliquot of each fraction was subjected to electrophoresis on an 8% SDS-polyacrylamide gel, blotted onto nitrocellulose paper, and then subjected to immunoblot analysis with a mixture of anti-a (IgG-Y533 at 1.2 uglml) and anti-6 (IgG-X287 at 10 uglml) anti-farnesyltransferase antibodies as described in Experimental Procedures. The nitrocellulose filter was exposed to XAR Kodak film for 16 hr at room temperature. The position of migration of the a and 6 subunits is denoted by arrows on both sides.

column, but at different times (Figure 4). The peak activities of both enzymes emerged in the same fractions and at the same positions relative to molecular size standards. Both were calculated to have apparent molecular sizes of about 100 kd. The fractions from both columns were subjected to SDS gel electrophoresis and blotted with a mixture of antibodies directed against both subunits. The farnesyltransferase reacted with antibodies against both the a and p subunits (Figure 4A), whereas the fractions containing GG-transferase activity reacted only against the anti-a subunit (Figure 48). To demonstrate that the a subunit is present in the GGtransferase enzyme itself and not in some copurifying protein, we performed an immunoprecipitation experiment (Figure 5). Fractions containing eitherthe farnesyltransferase or the GG-transferase were incubated either with preimmune serum or with an anti-a subunit antibody, after which protein A-agarose was added and the supernatant was assayed for prenyltransferase activities. The activities of both prenyltransferases were decreased by incubation with the same concentrations of anti-a subunit IgG. There was no loss of activity in the supernatant solution when the anti-a antibody was added in the absence of protein A-agarose (data not shown). Thus, the antibody-mediated decrease in activity was attributable to precipitation rather than to direct inhibition.

Cdl 432

2

/ A. Farnesyl Transferase

0.2

0.4

1 B. Geranylgeranyl Transferase

1

0.6

that the transferred radioactivity was [3H]GG because the i3H]GGPP had no detectable [3H]FPP as assessed by thin-layer chromatography. However, further chemical studies will be necessary to confirm this finding. GG-transferase did not transfer a significant amount of [3H]farnesyl to either of the two substrates. As expected, the enzyme transferred [3H]GG to p21H-rasCVLL,but virtually none was transferred to p21 H-ra5.

IgG (mg / ml)

Discussion Figure

5. lmmunoprecipitation

of Prenyltransferase

Activities

(A) Mono Q fraction 24 (70 pg) was mixed with the indicated concentration of IgG-Y536 anti-a farnesyltransferase immune (closed circles) or preimmune (open circles) IgG in 50 ~1 of buffer containing 50*mM Trischloride (pH 7.5), 100 mM NaCI, 0.2% octyl-P-D-glucopyranoside, 1 mM DTT, and 20 yM ZnCL After incubation for 3 hr at 4OC with shaking, 100 PI of a 1:l suspension of protein A-agarose in the same buffer was added to each tube, and the mixture was incubated for 1 hr at 4°C with shaking. The protein A-agarose beads were precipitated by spinning in a microfuge for 15 s, and the supernatant was collected. An aliquot of each supernatant (5 ~1) was assayed for farnesyltransferase activity at 37OC for 30 min as described in the legend to Figure 2. The “100% of control” value was 44 pmol per supernatant. The triangle represents a sample incubated without IgG and protein A-agarose. Each value is the average of duplicate incubations. (6) Mono Q fraction 22 (90 pg) was incubated with the indicated concentrations of IgG-Y536 anti-a farnesyltransferase immune (closed circles) or preimmune (open circles) antibody in 50 pl of buffer containing 50 mM Tris-chloride (pH 7.5), 100 mM NaCI, I mM DTT, 0.1% octyl-p-D-glucopyranoside, and 10 PM &Cl, for 3 hr at 4OC and processed exactly as described in (A). An aliquot of the supernatant (25 pl) was assayed for GG-transferase activity at 37OC for 30 min as described in the legend to Figure 2. The “100% of control” value was 21 pmol per supernatant. The triangle represents a sample incubated without IgG and protein A-agarose. Each value is the average of duplicate incubations.

Table 1 shows an experiment designed to compare the substrate specificities of the farnesyltransferase and GGtransferase. As expected, the farnesyltransferase transferred [3H]farnesyl to p21 H-ras,but very little was transferred to p21 H-ra*CVLL. The enzyme also transferred a small amount of 3H radioactivity from [3H]GGPP to ~21 H-ras,but virtually none was transferred to the CVLL derivative. We believe

Table

1. Substrate

Specificities

of Farnesyltransferase

The current data reveal that a protein GG-transferase from rat brain contains a subunit that appears to be similar, if not identical, to the a subunit of the protein farnesyltransferase from the same tissue. The two subunits have virtually identical apparent molecular weights on SDS-PAGE. Moreover, both react with all available antibodies directed against the a subunit of the farnesyltransferase. One of these is an anti-peptide antibody and another was prepared against a fusion protein that contains the COOHterminal 326 amino acids of the a subunit. We have also observed cross-reactivity with a third antibody directed against a different synthetic peptide from the a subunit of farnesyltransferase (unpublished data). This latter antipeptide antibody corresponds to a 19 amino acid epitope in the a subunit that is separated by 269 amino acids from the epitope recognized by IgG-Y533, which is directed against the COOH-terminal 15 amino acids (Reiss et al., 1991 a; unpublished data). This extensive immunologic cross-reactivity suggests that the two a subunits are not merely closely related, but that they are identical. Although the data seem compelling, it remains possible that the two a subunits are not identical gene products. The GG-transferase did not react with an antibody directed against the p subunit of the farnesyltransferase. Nevertheless, the farnesyltransferase and the GG-transferase both show the same apparent molecular weight of ~100,000 on gel filtration. These data suggest that both enzymes are heterodimers that share acommon a subunit in association with different p subunits.

and GG-Transferase

Substrate Enzyme

Prenyl

Farnesyltransferase

13H]FPP

Pyrophosphate

13H]GGPP

GG-transferase

[“HJFPP [3H]GGPP

Each assay either 1 pg as indicated, the amount of triplicate

Ras Protein

13H]Prenyl Group (pmol/lO minlmg

P21 n~rarCYLL

562 6.0 21 6.0

P21 n.rar P21 H-rasCvLL P21 n.raa P2f H-rasCvu

3.5 5.3 0.2 136

P2 1 Was P21n-rarcvLL p21 H-faS

Transferred Protein)

(25 pl) contained 50 mM Tris-chloride (pH 7.5), 20 mM KCI, 20 PM ZnClz, 5 mM MgCI,, 1 mM DTT, 0.2% octyl+-D-glucopyranoside, of Mono Q-purified farnesyltransferase or 6 pg of Mono Q-purified GG-transferase as indicated, either 40 NM p21n‘BB or 10 pM p21”‘BscyLL and either 0.6 KM [3H]FPP (15,251 dpmlpmol) or 0.6 pM 13H]GGPP (9423 dpm/pmol) as indicated. After incubation for 10 min at 37OC, of [3H]farnesyl or 13H]GG transferred to Ras protein was measured as described in Experimental Procedures. Each value is the mean incubations.

Common 433

a Subunit

for Prenyltransferases

The mechanism of the farnesyl transfer reaction is complex and not yet understood., It is known that the p subunit bindsthe p21raSsubstrate(Reissetal., 1991a). In addition, the holoenzyme forms a stable noncovalent complex with FPP. Such a complex is unusual for a transferase enzyme and suggests that the enzyme has an FPP carrier function that is distinct from its catalytic activity. The FPP that is bound to this carrier site is preferentially transferred to p2 1r=, suggesting that the FPP is transferred directly from the carrier site to the catalytic site without dissociation from the enzjrme (Reiss et al., 1991 a). We do not yet know the function of the a subunit that is shared by the two prenyltransferase activities. It might be the prenyl pyrophosphate carrier or it might be a regulatory subunit. If it is the carrier of prenyl pyrophosphates, one must explain how it can assist in different transfer reactions when it is present on the two enzymes. A detailed kinetic study of the transferase reactions with purified enzymes will be necessary to answer these questions. The GG-transferase activity that was measured in this study transfers [3H]GG to a p21H-raSvariant in which the COOH-terminal amino acid has been changed from serine to leucine. As discussed in the Introduction, it seems likely that cells contain other GG-transferases that use other protein substrates whose COOH-terminal sequences end in C-X-C or GGCCC. If such enzymes exist and if they share the common a subunit, it should be possible to isolatetheseenzymes byimmunoaffinitychromatographyusing antibodies prepared against the a subunit of the purified rat brain farnesyltransferase. Experimental

Procedures

Materials 13H]FPP (20 Cilmmol) was custom synthesized by New England Nuclear. [3H]GGPP (8 Cilmmol) was purchased from Dr. Ft. Kennedy Keller (University of South Florida, Tampa, FL). Recombinantwild-type p21H’BS protein was produced in a bacterial expression system and purified as previously described (Reiss etal., 199Oa). Unlabeled GGPP was generously provided by Dr. Dale Poulter (University of Utah). Unlabeled FPP was synthesized by Dr. J. R. Falck (University of Texas Southwestern Medical Center at Dallas). Synthetic peptides were either purchased from Peninsula Laboratories or synthesized by Sarah Stradley and Lila Gierasch (University of Texas Southwestern Medical vector was kindly proCenter at Dallas). The p21n”CvLL expression vided by Channing Der (La Jolla Cancer Research Foundation). Protein A-agarose was purchased from Schleicher & Schuell. Other materials were obtained from previously reported sources (Reiss et al., 1990a, 1990b). Expression and Purification of ~21”-“~“~~ The p2, H-rWC”LLconstruct (H-ras with a leucine-for-serine

substitution at the COOH-terminus) in a plasmid expression vector based on pXVR (Feig et al., 1986) was transformed into Escherichia coli strain PRISQ. After induction with isopropyl-a-D-thioglucopyranoside, the cells were disrupted by passage through a French pressure cell and centrifuged at 30,000 x g. The pellet fraction, which contained the bulk of the ras-CVLL, was extracted with 3.5 M guanidine-HCI, diluted 50-fold in buffer containing 50 mM Tris-chloride (pH 8.0), 1 mM EDTA, 1 mM DlT, 3 mM MgCI,, and 10 PM GDP, and loaded onto a DEAE-Sephacel column that was eluted at 200 mM NaCI. The protein peak was pooled, concentrated to 3 mg/ml in a Centriprep IO concentrator in the above buffer, and stored in multiple aliquots at -70%. Purification of Protein Protein farnesyltransferase

Farnesyltransferase was purified from rat brain according

to a

previously published cations as described

procedure (Reiss et al., 199Oa) with minor modifi(Reiss et al., 1990b).

Assay for Protein Farnesyltransferase Activity The amount of [3H]farnesyl transferred from IBH]FPP to p21 H-‘88protein was measured by precipitation of the [3H]farnesylated p21 H-raEwith trichloroacetic acid followed by filtration on a glass fiber filter as previously described (Reiss et al., 199Oa). The components in each reaction mixture are listed in the legends. Assay for Protein GG-Transferase Activity Protein GG-transferase activity was determined by measuring the amount of 13H]GG transferred from [3H]GGPP to a mutant recombinant p21H-” protein in which the COOH-terminal amino acid had been changed from serine to leucine, which changed the COOH-terminal four amino acids to CVLL instead of CVLS (p21H-r@‘LL). The components in each reaction mixture are listed in the legends. After incubation at 37’-‘C, the reactions were processed exactly as described above for measurement of protein farnesyltransferase activity. Antibodies and lmmunoblotting Antibody Y536 is a polyclonal rabbit antibody directed against a fusion protein containing Schistosoma japonicum glutathione S-transferase (Smith and Johnson, 1988) fused to the COOH-terminal 326 amino acids of the Q subunit of rat farnesyltransferase (Reiss et al., 1991a). Antibodies X284 and X287 are polyclonal rabbit antibodies directed against synthetic peptides corresponding to the amino acid sequences derived from tryptic digests of the a and b subunits, respectively, of purified rat farnesyltransferase (Reiss et al., 199la). Antibody Y533 is apolyclonal rabbit antibody directed against asynthetic peptidewhose amino acid sequence was derived from a partial cDNA clone of the a subunit. The amino acid sequence of the three peptides used for immunization is reported elsewhere (Reiss et al., 199la). The details of the cloning of the a and B subunits will be published elsewhere (W. J. Chen et al., unpublished data; D. Andres et al., unpublished data). IgG fractions were prepared from rabbit serum (preimmune and immune) by protein A-agarose chromatography (Beisiegel et al., 1961). lmmunoblot analysis was performed as previously described (van Driel et al., 1987) with minor modifications (Reiss et al., 1991a). Other Methods SDS-polyacrylamide gel electrophoresis was carried out as described by Laemmli (1970). Gels were calibrated with low range SDS-PAGE standards (Bio-Rad) or with prestained molecular weight markers (Diversified Biotech) for immunoblot analysis. The protein content of samples was measured by the method of Lowry et.al. (1951) unless otherwise stated.

We thank Richard Gibson, Debra Noble, Natalie Poppito, Nora Poppito, and John Moomaw for excellent technical assistance. This research was supported by grants from the National Institutes of Health (HL 20948), the Lucille P. Markey Charitable Trust, and the Perot Family Foundation. M. C. S. is the recipient of a graduate fellowship from Fundacao Calouste Gulbenkian of Portugal and a Fullbright Scholarship. Y. R. is the recipient of a Chaim Weizmann postdoctoral fellowship award. P. J. C. is supported by a grant from the American Cancer Society (IN-158C) to the Duke Comprehensive Cancer Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

March

8, 1991.

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Cell 434

Buss, J. E., Quilliam, L. A., Kato, K., Casey, P. J., Solski, P. A., Wong, G., Clark, R., McCormick, F., Bokoch, G. M., and Der, C. J. (1991). The COOH-terminal domain of the RaplA (Krev-1) protein is isoprenylated and supports transformation by an H-ras:raplA chimeric protein. Mol. Cell. Biol. 71, 1523-1530. Casey, P. J., Solski, P. A., Der, C. J., and Buss, J. E. (1989). ~21”~ is modified by a farnesyl isoprenoid. Proc. Natl. Acad. Sci. USA. 86, 6323-8327. Farnsworth, C. C., Wolda, S. L., Gelb, M. H., andGlomset, J. A. (1989). Human lamin 6 contains a farnesylated cysteine residue. J. Biol. Chem. 264,20422-20429. Farnsworth,C. C.,Gelb, of geranylgeranyl-modified 322.

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