Protein lsoprenylation in Suspension-Cultured Tobacco Cells · The Plant Cell, Vol. 5, 433-442, April 1993 O 1993 American Society of Plant Physiologists Protein lsoprenylation in

The Plant Cell, Vol. 5, 433-442, April 1993 O 1993 American Society of Plant Physiologists

Protein lsoprenylation in Suspension-Cultured Tobacco Cells

Stephen K. Randall,' Mark S. Marshall,b and Dring N. Crow@ll 'I'

a Department of Biology, Indiana University-Purdue University at Indianapolis, Indianapolis, Indiana 46202-5132

University at Indianapolis, Indianapolis, Indiana 46202-5121 Department of Medicine, Hematology/Oncology Section and Walther Oncology Center, Indiana University-Purdue

Many mammalian and yeast proteins, including small ras-like GTP binding proteins, heterotrimeric G protein y subunits, and nuclear lamins, have been shown to be covalently linked to isoprenoid derivatives of mevalonic acid. lsoprenylation of these proteins is required for their assembly into membranes and, hence, for their biological activity. In this report, it is shown that cultured tobacco cells, when pretreated with an inhibitor of endogenous mevalonic acid synthesis (lovastatin), incorporate radioactivity from 14C-mevalonic acid into proteins. Most of these proteins are membrane associated, and many are similar in mass to mammalian ras-like GTP binding proteins and nuclear lamins. Furthermore, it is shown that tobacco cell extracts catalyze the transfer of radioactivity from 3H-farnesyl pyrophosphate and 3H-geranylgeranyl pyrophosphate to protein substrates in vitro. These studies indicate the presence of at least two distinct prenykprotein transferases in tobacco extracts: one that utilizes farnesyl pyrophosphate and preferentially modifies a substrate protein with a CAlM carboxy terminus (farnesykprotein transferase) and one that utilizes geranylgeranyl pyrophosphate and pref- erentially modifies a substrate protein with a CAlL carboxy terminus (geranylgeranykprotein transferase type I). This work provides a basis for future work on the role of protein isoprenylation in plant cell growth, signal transduction, and membrane biogenesis.

INTRODUCTION

The mevalonate pathway participates in the biosynthesis of sterols, dolichols, ubiquinones, heme A, and tRNAs (Brown and Goldstein, 1980; Goldstein and Brown, 1990). As shown in Figure 1, it also leads to the formation of cytokinins, abscisic acid, gibberellins, plastoquinones, carotenoids, chlorophylls, and numerous other products in plants. The importance of this pathway in control of cell growth has been recognized in re- cent years due to reports of an additional requirement of a product of mevalonic acid for cell growth. This conclusion is based in part on the observation that mammalian cells treated with lovastatin (mevinolin), a potent inhibitor of 3-hydroxy-3- methylglutaryl coenzyme A reductase, cease growth and do not resume normal growth when supplemented with exoge- nous cholesterol, ubiquinone, or dolichol (Brown and Goldstein, 1980; Maltese and Sheridan, 1985,1987; Goldstein and Brown, 1990; Maltese, 1990). In addition, it was shown by Schmidt et al. (1984) that a product of mevalonate is post-translationally incorporated into mammalian cell proteins.

Recent work on the modification of cell proteins by isopre- noid products of mevalonate has revealed the chemical nature of the isoprenoid adduct as well as the identity of many isoprenylated proteins. All known isoprenylated proteins are linked through a cysteinyl thioether bond to either a farnesyl or a geranylgeranyl moiety (most are geranylgeranylated)

To whom correspondence should be addressed.

(Maltese and Erdman, 1989; Farnsworth et al., 1990; Reiss et al., 1990; Rilling et al., 1990). The isoprenylated cysteine residue typically occurs at the carboxy terminus of the protein (Maltese, 1990). Among the proteins that have been identified as having an isoprenoid adduct are various small GTP bind- ing proteins of the ras superfamily (p21ras, raplB [smg ~2161, racl, rac2, G25K), heterotrimeric G protein y subunits, and nu- clear lamins (prelamin A and lamin 9) (Wolda and Glomset, 1988; Casey et al., 1989; Farnsworth et al., 1989; Hancock et al., 1989; Vorburger et al., 1989; Didsbury et al., 1990; Fukada et al., 1990; Kawata et al., 1990; Maltese and Robishaw, 1990; Maltese and Sheridan, 1990; Maltese et al., 1990; Mumby et al., 1990; Yamane et al., 1990; Lutz et al., 1992). The sequence of events leading to a mature, isoprenylated protein is best un- derstood for ras, a protein involved in regulation of cell growth (Schafer et al., 1989,1990). Farnesylation of ras occurs at Cys- 186 and is dependent on the isoprenoid donor, farnesyl pyrophosphate (Manne et al., 1990; Reiss et al., 1990; Schaber et al., 1990). Following this reaction, the three carboxy-terminal amino acids are cleaved, and the new carboxy terminus at Cys- 186 is methylated (Clarke et al., 1988; Gutierrez et al., 1989). For some ras proteins (H-ras and N-ras), upstream cysteine residues are then palmitoylated (Hancock et al., 1989).

Protein isoprenylation is involved in signal transduction and cell growth control. Farnesylation is required for the function of p21ras, since a mutant p21 ras that contains serine at position

434 The Plant Cell

Acetyl COA + Aceioacetyl COA

i HMG COA Reduciase

t + Mevalonaie-PP

t Isopentenyl-PP

Dlmeihylallyl-PP

t Geranyl-PP

/ i \ sesqulterpenes

Ublqulnone

Dollchols plaStogulnOne ’qualene Geranylgeranyl-PP

i l~arne~y la ted ~ r ~ t e l n s I Phytosterols Dlterpenes

Glbberelllns

CarOtenOldB Chlorophylls

AbSCISIC Acld

Geranylgeranylaied Protelns

Figure 1. Biosynthesis of lsoprenoids in Plant Cells.

This diagram is based on information contained in the following pub- lished reports: Brown and Goldstein (1980), Bach and Lichtenthaler (1983), Letham and Palni (1983), Akiyoshi (1984), Bach (1986), Goldstein and Brown (1990), Parry and Horgan (1991), Crowell and Salaz (1992). HMG COA, 3hydroxy4”thylglutaryl coenzyme A; PP, pyrophosphate.

186 in place of cysteine is not farnesylated, does not associate with the plasma membrane, and does not transform mam- malian cells (Jackson et al., 1990). The isoprenylation of heterotrimeric G proteins (Fukada et al., 1990; Maltese and Robishaw, 1990; Mumby et al., 1990; Yamane et al., 1990) is consistent with a role for isoprenylation in signal transduction and cell growth control, since many of these proteins trans- duce signals that are initiated by the interaction of growth factors with cell surface receptors. Receptor kinases and cGMP phos- phodiesterases have also been shown to be isoprenylated (Anant et al., 1992; lnglese et al., 1992a, 1992b).

Protein isoprenylation is involved in the organization of the cellular cytoskeleton. Nuclear lamins, which form a protein- aceous layer on the inner surface of the nuclear envelope, are

isoprenylated (i.e., prelamin A and mature lamin B are isoprenylated) (Wolda and Glomset, 1988; Farnsworth et al., 1989; Vorburger et al., 1989; Lutz et al., 1992). These proteins mediate interactions between chromatin and the nuclear mem- brane, and regulate the assembly and disassembly of the nuclear envelope throughout the cell cycle. Furthermore, it has been shown that at least one isoprenylated protein regulates intracellular actin polymerization (Fenton et al., 1992) and that rac and rho (two small GTP binding proteins) are involved in this process (Ridley and Hall, 1992; Ridley et al., 1992).

Protein isoprenylation is involved in intracellular vesicle trans- port. A number of small, isoprenylated GTP binding proteins are involved in the movement of vesicles between intracellu- lar organelles (Balch, 1990; Khosravi-Far et al., 1991; Kinsella and Maltese, 1992; Rothman and Orci, 1992; Seabra et al., 1992). Proteins encoded by rab genes, for example, are isoprenylated and have been localized to various membrane- ous structures, including endoplasmic reticulum, Golgi complex, secretory vesicles, and endosomes (Salminen and Novick, 1987; Goud et al., 1988; Rothman and Orci, 1992). These small GTPases, which contain carboxy-terminal se- quences that are essential for proper membrane localization and function, are believed to catalyze interactions between pro- teins on various intracellular membranes (Bourne et al., 1990; Chavrier et al., 1991; Rothman and Orci, 1992).

The functional significance of protein isoprenylation differs among the various isoprenylated proteins. Most isoprenylated proteins are membrane associated (Glomset et al., 1990), and current data suggest that the isoprenoid group anchors the proteins through hydrophobic interactions with membrane lipids and/or proteins (Hancock et al., 1989; Jackson et al., 1990). However, a second hydrophobic modification is often required for efficient membrane association. This second modification usually involves fatty acylation (Hancock et al., 1989). Thus, it appears that the isoprenoid modification is necessary but, in some cases, insufficient for membrane as- sociation. Examination of the assembly of lamin A into the nuclear lamina suggests an alternative role for isoprenylation (Vorburger et al., 1989; Lutz et al., 1992). In this case, the car- boxy terminus of prelamin A bearing the isoprenoid moiety must be proteolytically removed prior to assembly of lamin A into the nuclear lamina. These observations suggest that iso- prenylation has a role in the regulation of assembly of lamin A.

To date, one farnesy1:protein transferase (FTase) and two dis- tinct geranylgerany1:protein transferases (GGTases) have been identified (Reiss et al., 1990; Kohl et al., 1991; Moores et al., 1991; Seabra et al., 1992). These have been distinguished on the basis of isoprenoid substrate specificity and isoprenyla- tion site specificity. The FTase and the GGTase type I recognize a CaaX consensus sequence at the carboxy terminus of the target protein, where “ C is cysteine, “a” is any aliphatic amino acid, and “ X is one of severa1 different amino acids. For the FTase, “ X is often serine, methionine, alanine, cysteine, or glutamine, and for the type I GGTase, “ X is usually leucine (Reiss et al., 1990; Moores et al., 1991). In contrast, the type II GGTase recognizes a CC or CXC consensus sequence at

Protein lsoprenylation in Tobacco 435

the carboxy terminus of the target protein and also recognizes interna1 sequence information (Moores et al., 1991; Seabra et al., 1992).

Rase and GGTase type I are heterodimeric complexes (Reiss et al., 1990; Kohl et al., 1991; Seabraet al., 1991; Mayer et al., 1992), whereas GGTase type II is more complex (Seabra et al., 1992). The FTase from rat (or bovine) brain, for example, is a heterodimer consisting of a 47-kD a subunit and a 45-kD I3 subunit (Reiss et al., 1990, 1991; Kohl et al., 1991; Seabra et ai., 1991). Current evidence indicates that the three prenyl: protein transferases (PTases) have related p subunits and that the FTase and type I GGTase have identical a subunits (Kohl et al., 1991; Seabra et al., 1991; Mayer et al., 1992). In fact, co-expression of the yeast RAMP (a subunit) gene with RAM7 (FTase (3 subunit gene) or CDC43 (type I GGTase p subunit gene) results in measurable FTase or type I GGTase activity in Escherichia coli, respectively (Mayer et al., 1992).

Bovine and yeast PTases exhibit considerable sequence con- servation (Kohl et al., 1991). A comparison of the nucleotide sequences of a bovine FTase a subunit cDNA and the yeast RAMP gene revealed 58% deduced amino acid similarity. In addition, antibodies raised against the yeast RAM7 gene prod- uct cross-reacted with the bovine FTase p subunit (Kohl et al., 1991). The relatedness of these enzymes has been exploited by Yang et al. (1993), who recently isolated a pea homolog of the FTase p subunit gene by polymerase chain reaction.

Post-translational isoprenylation of small GTP binding pro- teins, heterotrimeric G protein y subunits, and nuclear lamins is believed to be required for the proper membrane associa- tion and function of these proteins. Since small GTP binding proteins and G proteins have now been found in higher plants (Dillenschneider et ai., 1986; Zabulionis et al., 1988; Matsui et al., 1989; Bossen et al., 1990; Anuntalabhochai et al., 1991; Fairley-Grenot and Assmann, 1991; Hagege et al., 1992), pro- tein isoprenylation may be involved in the assembly and biological activity of regulatory proteins in plants as well. Con- sistent with this hypothesis, we show that cultured tobacco cells incorporate mevalonate-derived material into proteins and that many of these proteins are similar in molecular mass to mammalian small GTP binding proteins and nuclear lamins. Furthermore, we show that tobacco cell extracts contain both FTase and type I GGTase activity.

RESULTS

Labeling of Tobacco Proteins with 14C-Mevalonate-Derived Material

As shown in Figure 2, treatment of suspensioncultured tobacco cells with lovastatin arrested cell growth (Crowell and Salaz, 1992). This inhibition was completely relieved by adding ex- ogenous mevalonic acid to the growth medium, indicating that growth inhibition in the presence of lovastatin was caused by depletion of endogenous mevalonic acid. Therefore, cells

treated with lovastatin and then incubated in the presence of 14CC-mevalonic acid were expected to efficiently incorporate ra- dioactivity into mevalonic acid-derived material.

To test the hypothesis that tobacco cells would incorporate radioactivity from i4C-mevalonic acid into proteins, cells were treated with 10 WM lovastatin for 16 hr and then incubated in the presence of 7 WCilmL (R,S)-2-14C-mevalonolactone (50 mCilmmol) for 4 hr. Labeling of cellular proteins with carbon- 14 was then detected by SDS-PAGE and fluorography. As shown in Figure 3, most of the radiolabeled proteins had esti- mated molecular masses between 55 and 66 kD and between 14 and 31 kD and were, thus, similar in mass to mammalian isoprenylated proteins (nuclear lamins and small GTP bind- ing proteins, respectively). As expected for isoprenylated proteins, the majority of these proteins were membrane as- sociated (Figure 3C). Two polypeptides of m66 and 36 kD (Figure 3B), however, were primarily represented in the solu- ble fraction (the 66-kD polypeptide was also present to a significant extent in membrane fractions). Interestingly, sev- era1 isoprenylated proteins were detected in whole cell extracts (Figure 3A) that were not found in the membrane or soluble fractions, indicating that these proteins were proteolyzed dur- ing fractionation or were associated with cell debris (and discarded).

As shown in Figure 3, incorporation of 14C-mevalonate- derived material into membrane-associated and soluble proteins was dependent on pretreatment of the cells with

. Control 10 -

10 pM Lov. s . E a, 8 - ~10pMLov.+6mMMev.

1 KM LOV.

1 pM Lov. + 6 mM Mev. v

- E,

J ’ - 6 - -

3 4 c c o) 0

2

O O 2 4

DaY

6 8

Figure 2. Mevalonic Acid Restores Growth to LovastatinTreated Tobacco Cells.

All values, which were recorded on days O, 2,4, 6, and 8 of the experi- ment, represent cell volumes after 10 min of settling (measured in milliliters). Microscopic examination of the cells revealed no changes in average cell size, indicating that changes in cell volume reflect changes in cell number. Error bars represent standard deviations. Lov., lovastatin; Mev., mevalonic acid.

436 The Plant Cell

14c Incorporation Protein

Lovaslalin [jiM I 0 1 5 10 20 50 MW 0 1 5 10 20 50

lovastatin. Optimal incorporation, and maximal growth inhibi-tion, was observed at 1 u.M lovastatin (Figures 2 and 3; Crowelland Salaz, 1992). Lovastatin treatment (up to 50 \iM) had noapparent effect on the relative abundance of proteins detectedby Coomassie Brilliant Blue R 250 staining (Figure 3, right side),but the amount of protein recovered per milliliter of settled cellsdecreased significantly at higher concentrations of lovastatin(at 50 u.M lovastatin, protein yields were ~50% of control).

Mevalonate-Derived Products Are Linked to Protein

To confirm the proteinaceous nature of the mevalonate-modifiedmaterial shown in Figure 3,14C-labeled products were testedfor trichloroacetic acid (TCA) precipitability, acetone insolubility,RNase resistance, and protease sensitivity. As shown in Figure4A, high molecular mass, 14C-labeled products were recov-ered by TCA precipitation of membrane-associated proteins.

TCAAcetoneRNaseTrypsin

Figure 3. Labeling of Tobacco Cells with 14C-Mevalonic Acid RevealsHigh Molecular Mass Isoprenoid Products.

Cultured tobacco cells were pretreated for 16 hr with various concen-trations of lovastatin (as indicated) and then incubated with"C-mevalonic acid as described in Methods. Cells were washed twicein homogenization buffer and either lysed directly by incubation at 90°Cfor 4 min in SDS-sample buffer or were homogenized, fractionatedby sequential centrifugation at 2,000g and 70,000g, and then heatedin SDS-sample buffer. Coomassie blue-stained gels (right side) andthe corresponding fluorograms (left side) are shown. Protein concen-trations (Bradford, 1976), determined on membrane fractions (70,000gpellets), were used to normalize both pellets and supernatants for SDS-PAGE. Twice as much membrane-associated protein as soluble pro-tein (relative to settled cell volume) was loaded on the gel. Molecularmass markers (MW) are shown and correspond (in descending order)to rabbit muscle phosphorylase b (97.4 kD), BSA (66.2 kD), chickenegg white ovalbumin (45.0 kD), bovine carbonic anhydrase (31.0 kD),soybean trypsin inhibitor (21.5 kD), and chicken egg white lysozyme(14.4 kD).(A) Whole cells.(B) 70,000g supernatants.(C) 70,000g pellets.

s t.«*t

•a*

Figure 4. 14C-Mevalonic Acid-Derived Material Is Incorporated intoProtein.

Cultured tobacco cells grown in the presence of lovastatin (10 nM) werelabeled with "C-mevalonic acid as described in Methods. Sampleswere fractionated by centrifugation at 70,000g and treated as followsprior to SDS-PAGE and fluorography: lane 1, TCA (12.5% w/w) precipi-tation; lanes 2 to 7, TCA precipitation followed by acetone extraction;lanes 3 and 7, 125 ng/mL RNase A for 60 min at 4°C (done prior toTCA precipitation and acetone extraction); and lanes 4 and 6,500 ng/mLtrypsin in the presence of 0.1% Triton X-100 for 60 min at 4°C (doneprior to TCA precipitation and acetone extraction). Acetone extractionswere performed twice by resuspending TCA pellets in 500 nL of 100%acetone, followed by centrifugation at 10,000g for 10 min. Molecularmass markers (MW) are indicated and correspond to those describedin Figure 3.(A) 70,000g pellets.(B) 70,000g supernatants.

Protein Isoprenylation in Tobacco 437

200 ,

O 1 0 20 30 40 50

Time (minutes)

Figure 5. lncorporation of 3H-Farnesyl and 3H-Geranylgeranyl into Protein 1s Linear with Time.

Assays contained 1 pCi of 3H-FPP or 3H-GGPP and 0.030 mg of tobacco cell protein in 50 pL of assay mix (described in Methods). Results are expressed as picomoles of product formed per milligram of tobacco cell protein versus time. The FTase assay utilized aras pro- tein substrate with a CAlM carboxy terminus. The GGTase type l assay utilized a ras protein substrate with a CAlL carboxy terminus.

Furthermore, acetone extraction of the TCA precipitates removed most of the low molecular m a s products from the samples (<14 kD), suggesting that these products corresponded to unin- corporated mevalonic acid and/or hydrophobic derivatives of mevalonic acid, but had no effect on the recovery of high mo- lecular mass products. Ribonuclease A did not affect the recovery of high molecular mass products, but trypsin com- pletely degraded the radiolabeled products in the 55- to 66-kD range and partially degraded the products in the 14- to 31-kD range. Thus, 14C-mevalonate-derived material was incorpo- rated into tobacco proteins. The relative protease resistance of the 14- to 31-kD proteins is due to their association with mem- branes, since the same proteins are completely degraded (Figure 46) when trypsin digests are performed on soluble frac- tions (70,OOOg supernatants) of labeled cells (although the 14- to

31-kD proteins are primarily membrane associated, a small amount is recovered in the soluble fraction).

Development of in Vitro Assays for Tobacco FTase and GGTasè Type I To begin the molecular characterization of the enzymes that catalyze protein isoprenylation in tobacco cells, in vitro assays for the tobacco Rase and type I GGTase were developed. These assays were performed on crude extracts of cultured tobacco cells (4 days post-subculture) and utilized either 3H-farnesyl pyrophosphate (FPP) or 3H-geranylgeranyl pyrophosphate (GGPP) and a yeast rasl substrate (only the amino-terminal 26 kD) containing either a CAlM or a CAlL carboxy terrninus (Reisset al., 1990; Moores et al., 1991), respectively. Asshown below, these proteins were optimal substrates for the tobacco FTase and type I GGTase.

To establish the linearity of the FTase and type I GGTase assays with respect to time and tobacco cell protein, quantita- tive measurements of activity were made. The results shown in Figure 5 demonstrate that incorporation of radioactivity (TCA- precipitable, ethanol insoluble counts per minute) into ras was time dependent (for both FPP and GGPP, incorporation was linear for at least 40 min). Furthermore, as shown in Figure 6, modification of ras substrates was dependent on the con- centration of the tobacco extract in the assay. The type I GGTase assay was prone to higher experimental error because the spe- cific activity of this enzyme in tobacco extracts was lower than that of the FTase (GGTase type I activity was 9% of FTase activity), and the nonspecific binding of 3H-GGPP to glass fi- ber filters was higher than that of 3H-FPl? The data shown in Figures 5 and 6 demonstrate the utility of these assays in the purification and in vitro characterization of plant PTases.

Characterization of Tobacco FTase and GGTase Type I

To begin the characterization of tobacco FTases, the ability of tobacco cell extracts to catalyze the isoprenylation of four differ- ent ras substrates was examined. These substrates contained either an SVLS, CVLS, CAIM, or CAlL carboxy-terminal amino acid sequence. SVLS-ras was included as a negative control (it lacks a carboxy-terminal cysteine residue, which is required for covalent linkage of isoprenoid moieties) (Hancock et al., 1989; Reiss et al., 1990; Moores et al., 1991). As shown in Fig- ure 7, no radioactivity derived frorn 3H-FPP or 3H-GGPP was incorporated into protein in the absence of tobacco extract or in the presence of SVLS-ras. However, tobacco extracts effi- ciently and specifically incorporated radioactivity from 3H-FPP into a ras protein containing a CAlM carboxy terminus (upon long exposure, incorporation of radioactivity into CVLS-ras and CAIL-ras was also detectable; data not shown). Quantitative estimates of activity, shown in Table 1, indicated that the rate of incorporation of radioactivity from 3H-FPP into CAIM-ras was at least 80- to 100-fold greater than the rate of incorpora- tion into CVLS-ras or CAIL-ras (these assays were performed

438 The Plant Cell

2000

1800

1600

1400

1200

1000

800

600

„ 4[>0O

.0 'Z 200

II—

FTase

B Boiled Extract• Extract

GGTase I

Boiled ExtractExtract

Protein (mg) x 103

Figure 6. Incorporation of 3H-Farnesyl and 3H-Geranylgeranyl intoProtein Is Linear with Tobacco Cell Protein.

Assays contained 1 nCi of 3H-FPP or 3H-GGPP in 50 nL of assay mix(described in Methods) and were terminated after 40 min. Results areexpressed as picomoles of product formed per minute versus milli-gram of tobacco cell protein. The FTase assay utilized a ras proteinsubstrate with a CAIM carboxy terminus. The GGTase type I assayutilized a ras protein substrate with a CAIL carboxy terminus.

DISCUSSION

This paper presents direct evidence of protein isoprenylationin plants. Lovastatin-treated tobacco cells are shown to incor-porate product(s) of 14C-mevalonic acid into high molecularmass components. These components, most of which areassociated with membranes, are TCA precipitable, acetoneinsoluble, RNase resistant, and trypsin sensitive, suggestingthat they correspond to isoprenylated proteins. Furthermore,tobacco cell extracts are shown to contain both FTase activityand type I GGTase activity in vitro. These data do not provethat the proteins detected by 14C-mevalonic acid labeling orthose detected in vitro by 3H-FPP or 3H-GGPP labeling aremodified by farnesyl or geranylgeranyl groups. A rigorousdemonstration of the chemical nature of these adducts will re-quire cleavage of the isoprenoid groups from labeled proteins(by reaction with methyl iodide), followed by chemical analy-sis of the released material.

In mammalian and yeast cells, various proteins have beenshown to be isoprenylated, including small, ras-like GTP bind-ing proteins, heterotrimeric G protein y subunits, and nuclearlamins (prelamin A and lamin B). Two isoprenylated proteins

minusenzyme

Isoprenoid 3H-FPP 3H-GGPP

ras «Substrate >

2 _i ">< < >U U en

5 j< <

CL.OO

u u MW

ras

under conditions where incorporation was linear with time andtobacco protein concentration; furthermore, the CVLS-ras andCAIL-ras proteins used in this experiment have been shownby Mayer et al. [1992] to be good substrates for the yeast FTaseand GGTase type I, respectively). In the presence of 3H-GGPP,tobacco extracts preferentially incorporated radioactivity intoa ras protein containing a CAIL carboxy terminus. Incorpora-tion of 3H-geranylgeranyl-derived material into CAIM-ras wasalso detectable (as was incorporation of radioactivity into CVLS-ras after long exposure to film), perhaps due to recognitionof CAIM-ras by the type I GGTase, utilization of 3H-GGPP bythe FTase, or prenylation of CAIM-ras by a third PTase. Thesedata demonstrate that tobacco cells contain FTase and type IGGTase activities with distinct substrate specificities.

Figure 7. Substrate Specificity of the Tobacco FTase and Type I GGTase.

All PTase assays contained 1 nCi of 3H-FPP or 3H-GGPP and 0.030mg of tobacco cell protein in 50 nL of assay mix (described in Methods)and were terminated after 40 min. The carboxy-terminal sequencesof the ras substrates and the radiolabeled isoprenoids used in thisexperiment are indicated. The location of the modified (i.e.,isoprenylated) ras protein is also indicated. Molecular mass markers(MW) are indicated and correspond to those described in Figure 3.

Protein Isoprenylation in Tobacco 439

. .

Table 1. FTase and Type I GGTase Activities in Extracts of Cultured Tobacco Cells

Carboxy Terminus of ras Su bstrate

SVLS CVLS CAlM CAlL

lncorporation of 3H-Farnesyl (CPW

2,691 f 13 3,077 f 359

248,950 f 20,619 2,809 f 35

lncorporation of 3H-Geranylgeranyl (com)

2,135 f 52 2,402 f 70 8,548 f 387

24,462 f 235

Assays were performed as described in Methods. Data (determined in the same experiment as depicted in Figure 7) represent the aver- age of two determinations from a single extract and agree with data from severa1 independent experiments (no corrections for background have been made). Specific activity estimates were 8.2 f 0.6 pmoll min/mg protein for the tobacco FTase and 0.74 f 0.01 pmol/min/mg protein for the tobacco type I GGTase. Control values obtained in the absence of tobacco extract were 1198 f 51 and 3604 f 159 for farnesyl incorporation (CAIM-ras as protein substrate) and geranyl- geranyl incorporation (CAIL-ras as protein substrate), respectively. Values under 4000 cpm do not differ sianificantlv from backaround.

were observed in tobacco cells with molecular masses of m66 and 63 kD, suggesting that they may correspond to prelamin A and lamin 6, respectively (although, to our knowledge, no reports of plant lamins have appeared in the literature). Con- sistent with this hypothesis, the 66-kD protein was found in the soluble fraction (prelamin A is larger than lamin B and does not associate with the nuclear lamina until the isoprenylated peptide at the carboxy terminus is removed). In addition, numerous isoprenylated tobacco proteins were observed with estimated molecular masses between 14 and 31 kD, suggest- ing that they may correspond to small ras-like GTP binding proteins. The identification of these proteins and their puta- tive role(s) in cell cycle regulation and signal transduction is a focus of study in our laboratories. We are using the in vitro assays described in this paper to select cDNA clones that en- code proteins recognized by plant PTases (i.e., by in situ enzymatic screening of a cDNA expression library). These cDNAs, some of which may encode GTP binding proteins or nuclear lamins, will provide tools necessary for studying the role of isoprenylation in targeting of proteins to membranes.

Similar carboxy termini are recognized and prenylated by mammalian, yeast, and higher plant PTases (Figure 7; Reiss et al., 1990; Mooreset al., 1991; Mayer et al., 1992). Thetobacco GGTase type I clearly discriminates between substrates with CVLS and CAlL carboxy termini (it preferentially prenylates the substrate with a CAlL carboxy terminus), similar to other eukaryotic type I GGTases. However, unlike the mammalian FTase, the tobacco FTase does not appear to discriminate be- tween substrates with CVLS and CAlL carboxy termini (both are prenylated at low levels). Rather, the tobacco FTase, like the yeast FTase (Mark S. Marshall, unpublished data), effi- ciently prenylates protein substrates with a CAlM carboxy

terminus, suggesting differences in substrate specificity be- tween the plant FTase and the mammalian FTase. Interestingly, although most isoprenylated proteins have been shown to be geranylgeranylated in other systems, we find that FTase activ- ity is 40-fold greater than GGTase type I activity in extracts of cultured tobacco cells (this has been observed in yeast as well; Mark S. Marshall, unpublished data). We are currently exploring the possibility that the relative activities of these en- zymes vary in a cell cycle-dependent manner.

Proteins involved in cell growth control, signal transduction, organization of the cytoskeleton, and intracellular trafficking of membrane vesicles have been shown to be isoprenylated. It is, therefore, reasonable to speculate that isoprenylated pro- teins have a multitude of functions in plants as well. Since mevalonic acid restores growth to lovastatin-treated tobacco cells, products of mevalonic acid, including, perhaps, iso- prenylated proteins, are required for cell growth. Furthermore, it is tempting to speculate that plant-specific processes, includ- ing photomorphogenesis and phytohormonal responses, are regulated by isoprenylated proteins (i.e., G proteins). Given the recent discovery of isoprenoid and peptide inhibitors of PTases (Reiss et al., 1990; Crowell et al., 1991; Fenton et al., 1992), the stage is now set for testing the hypothesis that protein isoprenylation is required for plant-specific processes. These studies will contribute to a better understanding of the physio- logical significance of protein isoprenylation in plants.

METHODS

Tissue Culture

All experiments were performed on suspension cultures of Nicotiana tabacum cell line BY-2 (derived from N. tabacum cv Bright Yellow-2 callus), which were grown in Murashige-Skoog medium (Murashige and Skoog, 1962) containing 0.9 pM (0.2 mg/L) 2,4-D at 26 f IoC in continuous fluorescent light (Crowell and Salaz, 1992). Additions to the cultures (e.g., lovastatin and mevalonic acid) were made 24 hr af- ter subculture (day O is defined as the day that additions were made). Growth was monitored every 2 days by measuring cell volumes after 10 min of settling (clumping precluded accurate measurement of cell number) and by microscopic examination of the cells (to monitor cell size) (Crowell and Salaz, 1992). All experiments were performed in duplicate.

Labeling of Tobacco Cells with 14C-Mevalonic Acid

Early-log-phase tobacco cultures were incubated for 16 hr (starting 1 day after subculture) in MurashigsSkoog medium containing 0.9 pM 2,4-D and lovastatin (O to 50 pM) and then labeled in the same media for 4 hr with 7 pCi/mL (R,S)-2-14C-mevalonolactone (50 mcilmmol). Cells (35 pL of settled cells) were washed with 1.0 mL of homogenization buffer (Randall and Sze, 1986) and then homogenized in 2 volumes of homogenization buffer containing 5 mM EGTA, 0.1% BSA, 1 mglmL leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 0.1 mg/mL aprotinin (to reduce proteolysis). In Figure 3, unbroken cells

440 The Plant Cell

were sedimented at 2,0009 for 2 min and discarded. The cell extract was then centrifuged at 70,OOOg for 45 min to sediment membranes. The high-speed pellet (derived from 25 pL of settled cells) and high- speed supernatant (derived from 32.5 pL settled cells) were then resolved on 14% polyacrylamide gels and subjected to fluorography for 2 weeks at -8OOC. Whole cell pellets were also suspended in SDS-sample buffer, boiled, and analyzed.

In Vitro Assay for Tobacco PTases

PTase assays (Moores et al., 1991; Mayer et al., 1992) contained ras protein (5.0 pM), protein from tobacco cells grown for 4 days without lovastatin, 20 mM MgClp, 5 mM DTT, and 50 mM Hepes, pH 7.5. Af- ter pre-equilibration at 3OoC for 2 min, 20 pCi/mL of either W-FPP (15 Cilmmol) or 3H-GGPP (15 Cilmmol) was added, and the incubation was continued for 40 min at 3OoC (125 pL total volume). Two 50-pL portionsof the reaction were then terminated with 1 M HCI in ethanol, and the precipitates were collected and washed with ethanol on Whatman GF/C glass fiber filters. The filters were counted by liquid scintillation. lsoprenylation of protein was confirmed by SDS-PAGE and fluorography of a 10-pL portion of the assay mix (more unincor- porated 3H-FPP and 3H-GGPP runs at the gel front in the presence of tobacco extract, perhaps due to nonspecific binding of label to tobacco proteins).

The ras proteins used in these assays were encoded by plasmids containing a truncated yeast rasl gene (truncated at the 3’end) under the control of the lac promoter (Moores et al., 1991). The plasmids (ob- tained with permission from Merck, Sharpe and Dohme Research Laboratories, West Point, PA) were modified by site-directed mutagen- esis such that different constructs encoded 26-kD ras proteins with different carboxy-terminal tetrapeptide sequences. The constructs were introduced into Escherichia coli cells by the method of Hanahan (1985) and then induced to express the ras gene by the addition of 0.1 mM isopropyl-P-o-thiogalactopyranoside to cultures of transformed cells. Expressed proteins were purified from E. coli cell extracts to greater than 90% purity by medium pressure liquid chromatography on an HR10/10 monoQ column (Pharmacia).

ACKNOWLEDGMENTS

We thank Brian Caplin for expert technical assistance. This work was supported by National Science Foundation Grant No. DMB-9220099 to D.N.C. and S.K.R., a National lnstitutes of Health Biomedical Re- search Support Grant and an Indiana University-Purdue University at lndianapolis (IUPUI) School of Science Faculty Award to S.K.R., an IUPUl Faculty Development Grant to D.N.C., and a donation from the Ballve family to M.S.M.

Received December 15, 1992; accepted February 8, 1993.

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DOI 10.1105/tpc.5.4.433 1993;5;433-442Plant Cell

S K Randall, M S Marshall and D N CrowellProtein isoprenylation in suspension-cultured tobacco cells.

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