THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 259, No. 16, Issue of August 25, pp. 10175-10180,1984 0 1984 by The American Society of Biological Chemists, Inc. Printed in U.S.A.

Evidence for Post-translational Incorporation of a Product of Mevalonic Acid into Swiss 3T3 Cell Proteins*

(Received for publication, March 5, 1984)

Rodney A. Schmidt, Carol J. Schneider, and John A. GlomsetS From the Howard Hughes Medical Institute Laboratory, Departments of Medicine and Biochemistry, the Regional Primate Research Center, University of Washington, Seattle, Washington 98195

Previous studies have identified several cellular re- quirements for mevalonic acid that appear unrelated to cholesterol, dolichol, or ubiquinone. To search for other products of mevalonic acid that might account for these requirements we cultured Swiss 3T3 cells in the presence of mevinolin, an inhibitor of mevalonic acid biosynthesis, then labeled the cells with exogenous radioactive mevalonic acid. Upon analyzing the radio- active material formed, we found that 40-50% of it was not extractable into lipid solvents, and that most of the lipid-insoluble material behaved like protein when treated with sodium dodecyl su1fate:chloro- form:phenol, RNase, or proteinase K. Further analysis by electrophoresis revealed that radioactivity was as- sociated with a few specific proteins that had apparent molecular weights of 13,000-58,000. Control experi- ments indicated that authentic radioactive (R)-meva- lonic acid was the active precursor. Other lines of evidence suggested that mevalonate was first con- verted to an isoprenoid compound, then covalently in- corporated into proteins by way of a cycloheximide- insensitive mechanism. These results suggest that Swiss 3T3 cells possess novel metabolic products of mevalonic acid metabolism that are formed by post- translational incorporation of isoprenoids into specific cell proteins.

Compactin and mevinolin are competitive inhibitors of HMG-CoA' reductase that can effectively block the biosyn- thesis of MVA in cultured cells. Compactin, in particular, has been used to unmask cellular requirements for MVA that appear unrelated to sterol metabolism. Thus, Faust and col- leagues (reviewed in Ref. 1) demonstrated that cellular HMG- CoA reductase activity is regulated not only by a feedback mechanism involving cholesterol, but also by one involving a non-sterol product of MVA. Others (2-6), including ourselves, showed that a product of MVA metabolism other than cho- lesterol is required for cell entry into the S phase of the cell cycle. Finally, we (4) recently demonstrated that MVA is required independently of cholesterol for maintenance of the

* This work was supported by Howard Hughes Medical Institute, National Institutes of Health Grants GM07266 and RR00166, and R. J. Reynolds Industries, Inc. 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 U.S.C. Section 1734 solely to indicate this fact.

$ TO whom all correspondence should be addressed. ' The abbreviations used are: HMG-CoA reductase, 3-hydroxy-3-

methylglutaryl-coenzyme A reductase (EC 1.1.1.34); MVA, mevalonic acid; PBS, phosphate-buffered saline; EGTA, ethylene glycol his(@- aminoethyl ether)-N,N,N',N'-tetraacetic acid; SDS, sodium dodecyl sulfate.

shape of Swiss 3T3 cells. For each of these three requirements the unidentified active products of MVA metabolism are likely to be quantitatively minor cell constituents. When cholesterol is present in the cell medium, none of the requirements become manifest until sufficiently high concentrations of compactin or mevinolin are added to inhibit residual MVA biosynthesis almost completely. Then, uptake of tiny amounts of exogenous MVA suffices to overcome the effects of the inhibitors.

To search for the active products we studied the metabolic fate of radioactive MVA under culture conditions that were chosen to detect relevant, quantitatively minor products. We cultured large quantities of Swiss 3T3 cells in the presence of cholesterol and amounts of mevinolin sufficient to induce MVA-deficient cell rounding. We then added concentrations of radioactive MVA that we previously found were barely sufficient to prevent (50-100 pM) or reverse (600 pM) the shape change. Upon analyzing the intracellular products that were formed, we obtained evidence for what appears to be a hitherto undescribed pathway of MVA metabolism in mam- mals (8).

MATERIALS AND METHODS

Except as noted, all radioisotopes were obtained from New England Nuclear and all reagents and enzymes from Sigma. (R)-[l-"C]Mev- alonolactone (10 mCi/mmol) was a generous gift from Dr. J . Watson (University of California, San Francisco). Mevinolin was the kind gift of Dr. A. Alberts (Merck, Sharp & Dohme Research Laboratories, Rahway, NJ) and was converted to its Na+ salt before use (2). PBS without Ca2+ and M e was prepared by the method of Dulbecco (9). Tissue culture reagents were from Gibco Laboratories (Grand Island, NY).

Cell Culture-Mass cultures of Swiss 3T3 cells (2) were obtained by inoculating approximately 2 X lo6 cells into 850 cm2 roller bottles (Corning Scientific Products, Corning, NY) in 100 ml of Dulbecco's modified Eagle's medium containing 10% calf serum, non-essential amino acids, sodium pyruvate, and penicil1in:streptomycin. The bot- tles were gassed with 5% COs/air, sealed, and maintained a t 37 "C on a roller bottle apparatus (Wheaton Scientific, Millville, NJ) until the cultures reached confluence. The complete culture medium con- tained approximately 60 pg/ml of total cholesterol.

Delipidation and Phenol Extraction of Cells Labeled with Radioac- tive MVA, Uridine, and Leucine-In most experiments we labeled confluent roller bottle cultures with 50 pCi/ml of (RS)-[5-3H]MVA (5.7 Ci/mmol), 5 pCi/ml of [5-3H]uridine (28.5 Ci/mmol), 25 pCi/ml of [4,5-3H]leucine (38.6 Ci/mmol), or 5 pCi/ml of (RS)-[2-I4C]MVA (48.6 mCi/mmol) in 10 ml of fresh complete medium containing 9% calf serum, 30 PM mevinolin, and 67 p M unlabeled MVA. After 24 h the cells were scraped directly into the labeling medium, washed twice with cold PBS, then resuspended in PBS containing 1.6 mM pepsta- tin, 8.7 mM o-phenanthroline, 9.5 mM N-ethylmaleimide, 1.1 mM phenylmethylsulfonyl fluoride, 4.5 mM diisopropyl fluorophosphate, 2.4 mM EDTA, 2.4 mM EGTA at 37 "C for 60 min. Cells from half a roller bottle were then sequentially extracted three times each with 5 ml of cold acetone, 5 ml of cold chloroform:methanol(2:1), and twice with 5 ml of cold 95% EtOH. The delipidated residues were dissolved

10175

10176 Labeling of Swiss 3T3 Cell

in 2.2 ml of 1.4% SDS, 0.1 M NaCl, 10 mM EDTA, 50 mM Tris, pH 7.4, and extracted with phenol and CHC13 according to the method of Penman (10). Macromolecules were precipitated from the final aqueous phase with 2 volumes of 95% EtOH (>95% efficiency). CHC13 was evaporated from the pooled organic phases and macromolecules precipitated by adding 5 volumes of 95% EtOH (>95% efficiency). All solutions were treated with 0.1% diethyl pyrocarbonate and au- toclaved before use, and all glassware was heated to 110 "C for at least 1 h prior to use.

The preceding delipidation procedure appears to be complete with respect to MVA-labeled lipids because further extractions with either the same solvents or with CHCl3:methanol:water (10103) failed to release significant amounts of MVA-derived radioactivity. At least 95% of cellular cholesterol was extracted by this procedure (4). The labeled lipids have not all been identified but approximately 50% of the acetone-soluble material was in a non-saponifiable lipid that co- migrated with cholesterol during thin-layer chromatography on Silica Gel H in heptane:ether:methanol(909:15) (4) while 10-30% behaved as MVA or more polar lipids (not shown).

In other experiments we incubated confluent cultures with 30 p~ mevinolin in fresh medium for 24 h, then scraped the cells directly into the medium. Aliquots of cells were spun down, resuspended in a small volume of the same mevinolin-containing medium, and incu- bated with t3H]MVA (200 pCi/ml) for 3 h at 37 "C in a 5% COn atmosphere with occasional agitation. The labeled cells were washed twice with PBS, incubated with protease inhibitors, extracted three times with 2 ml of acetone, then dissolved in electrophoresis sample buffer.

Partial Purification of f H ] M V A on DEAE-cellulose-For compe- tition experiments we partially purified radioactive and non-radio- active mevalonate on DEAE-cellulose (11). (RS)-[5-3H]MVA was loaded on a column (0.9 X 30 cm) of acid base washed DEAE-cellulose (COZ- form) in 1 mM ammonium carbonate, pH 9. After washes with 1 mM ammonium carbonate and dilute PBS (1;200 in water), f'H] MVA was eluted with full strength PBS containing penicil- 1in:streptomycin and stored at -75 "C. Non-labeled MVA was spiked with trace amounts of [3H]MVA, then DEAE-purified in the same way.

Electrophoresis and Fluorography of Radioactive Cell Material- Samples from equal numbers of cells (approximately 160 pg of pro- tein/gel lane) were dissolved in electrophoresis sample buffer con- taining 5% 2-mercaptoethanol, 2 M urea, and 2% SDS, heated at 100 "C for 10 min, and subjected to electrophoresis in 0.1% SDS, 2 M urea through a 1.5-mm thick 12.5% polyacrylamide slab gel with a 5% stacking gel (12). The gel was stained briefly with Coomassie Brilliant Blue R-250 (13), partially destained, impregnated with EN3HANCE (New England Nuclear), then dried at 60 "C on a gel slab dryer. Preflashed (14) Kodak X-Omat R or AR film was clamped against the gel and exposed at -75 "C. 14C-labeled molecular weight standards (67,000, 43,000, 30,000, 20,000, and 14,000) were chromat- ographed in adjacent lanes. Estimated molecular weights of the MVA- labeled proteins were obtained from densitometric scans of the fluo- rograms.

During the course of this investigation we established that the use of protease inhibitors is not esential for the demonstration of MVA- labeled proteins. The omission of protease inhibitors during the harvesting procedure did not result in the loss of any MVA-labeled bands and resulted in only minor changes in their apparent molecular weights: the 45,000, 29,000, 24,000, and 23,000 bands were altered by less than 5% (not shown). Furthermore, all changes not involving the 45,000-Da band were due to an effect of N-ethylmaleimide which was apparently unrelated to inhibition of proteolysis (not shown). Simi- larly, cells harvested by direct solubilization in hot 0.1 M leupeptin in 1% SDS yielded proteins of the same apparent molecular weights as cells harvested without protease inhibitors (not shown). It might be argued, therefore, that protease inhibitors can be omitted routinely from the cell harvesting procedure.

Selective Enzymatic Digestion 'of Radioactive Macromolecules- Samples from 1/10 of a roller bottle were incubated with or without 20 pg of proteinase K (Beckman) in 220 pl of 1% SDS, 50 mM EDTA, 0.1 M Tris, pH 8 (pretreated with diethyl pyrocarbonate), for 1 h at 45 "C. Remaining macromolecules were precipitated with 2 ml of cold 95% EtOH, then dissolved in electrophoresis sample buffer.

Similar samples were suspended in 200 pl of 50 mM Tris, pH 7.4 (pretreated with diethyl pyrocarbonate), sonicated briefly in a soni- cating water bath, then incubated with or without 20 pg of RNase A (Calbiochem-Behring) in the same buffer (previously boiled for 10

Proteins by Mevalonic Acid

min) at 37 "C for 60 min. Remaining macromolecules were precipi- tated with 2 ml of cold 95% EtOH and redissolved in electrophoresis sample buffer.

Extensive Proteolysis of MVA-labeled Proteins and Subsequent Purification-MVA-labeled proteins from the organic phase of the SDS:CHCb:phenol extraction were suspended in 50 mM Tris acetate, 3 mM calcium acetate, pH 7.8, containing 0.3 mg/ml of Pronase for 3 days at 37 "C (15). After centrifugation the supernatant was dis- carded, and the insoluble material dissolved in 50 mM Tris acetate, 10 mM EDTA, 0.5% SDS, pH 8. Proteinase K (0.1 mg/ml) was added at 0 and 3 h and the digestion continued at 45 "C for 24 h (16). After the pH was adjusted to 5-5.5, carboxypeptidase Y (200 pg/ml) was added at 0 and 3 h, and the incubation continued for 24 h at 37 "C (17).

The resulting hydrolysate was applied directly to a column (1.0 X 10 cm) of Bio-Rad AG 1-X2 (formate) in 1 M ammonium formate, pH 6.6. After washes with 1 M ammonium formate and water the radioactivity was eluted with a nonlinear gradient from water to 88% formic acidEtOH (1:4). Appropriate fractions were pooled, dried in a Speed-Vac evaporator (Savant Instruments, Inc., Hicksville, NY), redissolved in formic acidEtOH (1:4), and chromatographed over Sephadex LH-20 (Pharmacia Fine Chemicals, Piscataway, NJ) in the same solvent (18).

RESULTS

Evidence That MVA or One of Its Products Is Incorporated into Specific 3T3 Cell Proteins-In three separate experiments we incubated roller bottle cultures of Swiss 3T3 cells for 24- 25 h in the presence of 30 p~ mevinolin and 50 PM [3H]MVA (50 pCi/ml), then analyzed the radioactive intracellular prod- ucts that had been formed. We found typically (Table I) that about half of the cell-associated radioactivity could not be extracted with lipid solvents. By three criteria, most of this non-lipid radioactivity seemed to be associated with protein rather than RNA (19). First, when we employed an SDS: CHC13:phenol extraction technique to separate nucleic acids from proteins, we found (Table I) that about 80% of the non- lipid radioactivity was soluble in the protein-containing SDS:CHC13:phenol phase, whereas only 16% of the radioac-

TABLE I Distribution of intracellular radioactivity derived from labeled MVA,

leucine or uridine in mevinolin-treated Swiss 3T3 cells Three confluent roller bottle cultures of Swiss 3T3 cells were

treated with fresh medium containing 9% calf serum, 30 p~ mevi- nolin, 67 p~ unlabeled (RS)-MVA, and either 50 pCi/ml of [3H]MVA, 5 pCi/mI of [3HJuridine, or 25 pCi/ml of [3Hjleucine. After 25 h, the cells were scraped from each roller bottle, washed with PBS, treated with protease inhibitors, delipidated, and partitioned in a phenol extraction as described under "Materials and Methods." Recovery of radioactivity in each fraction was determined and all data were corrected for quenching and background radioactivity. Data shown are from a typical experiment and are derived from duplicate samples for the lipid extracts and multiple aliquots of single samples from the phenol extraction. Of the labeled MVA, uridine, or leucine added to each roller bottle, only 0.16, 0.70, and 0.34% became cell-associated, respectively.

Per cent of initial radioactivity recovered"

MVA Uridine Leucine

Acetone extracts 51.1 0.5 3.3 CHCI3:MeOH (2:l) 5.5 1.3 2.5

extracts Ethanol extracts 0.3 2.2 0.5 Phenol extraction

Aqueous phase 8.5 16.5b 91.2 99.1b 22.7 20.0b Organic phase " 42.6 83.5b 0.9 0.9' 89.9 9b

Initial cell-associated radioactivity for MVA-, uridine-, and leu- cine-labeled cells was 846,930,385,570, and 894,400 cpm, respectively.

Numbers refer to per cent recovery of non-lipid radioactivity in the two phases of the phenol extraction.

108.1 96.0 118.8

Labeling of Swiss 3T3 Cell

tivity was soluble in the RNA-containing aqueous phase. This distribution of radioactivity was similar to that obtained when corresponding non-lipid material from [‘Hlleucine-labeled control cells was fractionated by the same technique, but differed dramatically from that obtained using comparable [“Hluridine-labeled material (Table I) .

Second, SDS-gel electrophoresis revealed that the MVA- derived non-lipid radioactivity was associated with discrete macromolecules (Fig. 1). Most labeled macromolecules were present primarily or exclusively in the organic phase, although prominent bands of less than 14 kDa were found only in the aqueous phase. Eighty per cent of the organic phase label, as demonstrated by gel slicing and counting, was in macromol- ecules that had apparent M,, = 20,000-30,000. Individual leucine-labeled bands from control cells also were found pri- marily in the organic phase, but most of them were macro- molecules larger than 30 kDa.

Third, MVA-labeled macromolecules were degraded under conditions of selective proteolysis. In three experiments, MVA-labeled macromolecules from both the aqueous and organic phases were completely hydrolyzed by proteinase K in 1% SDS, but not by boiled RNase (not shown). The sole exception was the broad MVA-labeled band of approximately 14 kDa that appeared to be completely resistant to proteinase K. Control digestions of leucine- and uridine-labeled material showed that the digestion conditions were completely selec- tive for protein and RNA. Taken together, these results strongly suggest that most of MVA-derived non-lipid radio- activity is tightly linked to protein.

A minor fraction of the MVA-labeled, non-lipid material, however, probably consisted of isopentenylated tRNA. We found previously (20) that some MVA-labeled, aqueous phase material co-migrates with tRNA in an electrophoretic systev

MVA Urldlne Leuclne ‘ A B C D E ’ A B D E ‘ A B C D E ‘ -

- .

67

43

30

20. I 14.4

FIG. 1. Behavior of cell material labeled with MVA, uri- dine, or leucine during delipidation and phenol extraction. Samples of [‘HIMVA-, [3H]uridine-, or [3H]leucine-labeled cell ma- terial derived from equal numbers of cells were obtained a t different stages of the experiment shown in Table I. Electrophoresis and fluorography were performed as described under “Materials and Methods.” For each radioactive precursor, samples in lune A are intact cells dissolved in sample buffer; lune R, acetone-insoluble material; lune C , completely delipidated cell material; lune D, mole- cules recovered from the aqueous phase of the phenol extraction; lane E , material recovered from the organic phase of the phenol extraction. Other lanes contain “C-labeled molecular weight markers. In this experiment, the MVA-labeled non-lipid material contained approxi- mately 40,000 cpm/mg of protein.

Proteins by Mevalonic Acid 10177

(21) for RNA. Furthermore, in the present investigation we hydrolyzed aqueous phase material in 0.3 N KOH for 18 h a t 37 “C and found that 19% of the label was converted to a perchloric acid-soluble form that co-chromatogrhphed with AMP on Sephadex G-25 (not shown). Only 6% of the organic phase label was released in perchloric acid-soluble form and less than 2% of it co-chromatographed with AMP; the re- maining 4% was in unidentified higher molecular weight material.

Evidence That the Radioactivity Incorporated into 3T3 Cell Protein Is Derived from Authentic Labeled (R)-MVA-AI- though the experiments described above provided strong evi- dence that much of the cell-associated radioactivity in our experiments was present in protein, it remained to be dem- onstrated that this radioactivity was actually derived from radioactive MVA. To exclude the possibility that a radioactive contaminant in the preparations of [‘HIMVA might have labeled the proteins, we labeled the proteins with two different radioisomers of MVA that had been synthesized from differ- ent starting materials. Whenever we added either [2-I4C]MVA or [5-3H]MVA to separate cell cultures, we always found labeled proteins of similar molecular weights, and in two experiments where we concomitantly added them to the same cell culture we found the same ratio of ‘H to I4C in delipidated, SDS:CHC13:phenol-extracted cell proteins of 20-30 kDa as in lipids that co-migrated with cholesterol and ubiquinone on thin-layer chromatography (not shown). Moreover, the natu- rally occurring (R)-stereoisomer of MVA appeared to be active because similar proteins were labeled with (R)-[3-I4C]MVA as with either (RS)-[5-3H]- or (RS)-[2-I4C]MVA (not shown). These results strongly suggest that authentic radioactive (R)- MVA is indeed the source of the labeled moiety that becomes incorporated into 3T3 cell proteins.

Using a modified labeling system we obtained evidence suggesting that unlabeled MVA can compete with radioactive MVA for incorporation into cell proteins whether the unla- beled MVA is derived from endogenous or exogenous sources. Thus, when we incubated cells with or without mevinolin for 24 h, then labeled them with radioactive MVA and examined the proteins by SDS gel electrophoresis, we found (Fig. 2) detectable radioactivity in macromolecules only in the cells that had been pretreated with mevinolin. In the same exper- iment we also added increasing concentrations of unlabeled MVA to the culture medium of mevinolin-pretreated cells a t the same time that we added radioactive MVA, and found that concentrations of unlabeled MVA greater than 100 p~ competitively prevented the incorporation of radioactivity into cell protein. Below 100 p~ unlabeled MVA, the amount of protein-bound radioactivity was unaffected, presumably because insufficient MVA was taken up by the cells during the relatively short labeling period to fill all potential protein acceptor sites.

Evidence That Radioactivity from MVA Is Incorporated into Proteins by a Post-translational Mechanism-We tested the abilities of cycloheximide (12 pg/ml) and chloramphenicol (60 or 300 pg/ml) to prevent mevinolin-treated cells from incor- porating radioactive MVA into proteins. Neither inhibitor affected the incorporation of labeled MVA into cell proteins (Fig. 3) even though cycloheximide dramatically reduced the incorporation of labeled leucine into the proteins of control cells (not shown). Similar results were obtained both times the experiment was repeated. Cycloheximide had no effect on the conversion of labeled MVA into lipids which co-migrated with cholesterol, cholesteryl oleate, dolichol, or ubiquinone during thin-layer chromatography in heptane:ether:methanol (90:9:15) (not shown). Thus, it appears that the labeling of

10178 Labeling of Swiss 3T3 Cell Proteins by Mevalonic Acid

1 2 3 4 5 6 A B C D E

FIG. 2. Influence of mevinolin and unlabeled MVA on the incorporation of ['HIMVA into proteins. Swiss 3T3 cells in roller bottles were incubated either without (lane I ) or with (lanes 2-6) 30 PM mevinolin for 24 h then suspended in the same medium and divided into multiple aliquots. 194 pCi/ml (14 p M ) of DEAE-purified ['HIMVA was added to all aliquots and 0,10,100,1000, or 10,000 PM unlabeled, DEAE-purified MVA (lanes 2-6, respectively) was also added to the designated samples. Cells in lane I also received 100 p~ unlabeled MVA. The cells were incubated for 3 h a t 37 "C, then washed with PBS and extracted with acetone. Finally, material from equal numbers of cells was prepared for electrophoresis and fluorog- raphy as described under "Materials and Methods." Molecular weight standards as described in the legend to Fig. 1 are shown in adjacent lanes. The radioactivity per mg of protein was not specifically deter- mined in this experiment.

cell proteins by MVA occurs by a post-translational mecha- nism rather than a pre- or co-translational one.

Partial Purification of Proteolytic Fragments of MVA-la- beled Proteins-We have begun to purify proteolytic frag- ments of MVA-labeled proteins in order to determine the structure of the MVA-derived moiety and its link to protein. In order to monitor our purification we first prepared delipi- dated, phenol-extracted proteins that had been labeled either with [2-14C]MVA (as in Table I) or with 3H-amino acids (by growing cells for 4 days in medium that contained either labeled phenylalanine, tyrosine, tryptophan, cysteine or a labeled algal protein hydrolysate). After separately incubating these proteins with Pronase for 3 days, we found in eight experiments that 96-9996 of the 3H-labeled material became water-soluble whereas 88-92% of I4C-labeled material re- mained insoluble. By centrifuging the hydrolysate and de- canting the supernatant we thus effected an approximately 30-fold enrichment of the MVA-labeled material. We then dissolved the insoluble residues in SDS, pooled them, and further incubated them with proteinase K and carboxypepti- dase Y. Although these digestions had little effect on MVA- labeled material, 3H-labeled material was degraded further (not shown). By passing the final hydrolysate over an AG 1- x 2 (formate) column in 1 M ammonium formate, we separated water-soluble fragments (which washed through the column) from SDS and MVA-labeled material (which bound). We then eluted MVA-labeled products (substantially free of SDS) with formic acid:EtOH (1:4). In seven experiments, this step caused a further 2.2-15-fold purification. Analytic chromatography of the MVA-labeled eluate over Sephadex LH-20 in formic acid:EtOH (1:4) revealed that it contained at least two major components with apparent sizes of 1000 and 500 Da (Fig. 44). By preparative LH-20 chromatography we separated these components from each other (Fig. 4B) and simultaneously

FIG. 3. Effects of cycloheximide and chloramphenicol on the incorporation of 'H from MVA into proteins. Roller bottle cultures of Swiss 3T3 cells were incubated with 30 ~ I U mevinolin for 29 h, then suspended in the same medium in multiple equal aliquots. Either 12 pg/ml of cycloheximide or 60 or 300 pg/ml of chloramphen- icol was added to the designated samples and the cells were incubated at 37 "C for 5 min. 600 PM unlabeled MVA and 172 pCi/ml of [3H] MVA were then added and the incubation continued for another 3 h. Cells in lane A , however, received the [3H]MVA at the end of the incubation (nonspecific binding control). The labeled cells were in- cubated with protease inhibitors for 20 min, washed with PBS, and extracted with acetone prior to electrophoresis and fluorography. Lane B, no inhibitors; lane C, 12 pg/ml of cycloheximide; lane D, 60 pg/ml of chloramphenicol; lane E, 300 pg/ml of chloramphenciol. Molecular weight standards as described in the legend Fig. 1 are shown in adjacent lanes. The acetone-insoluble material in this experiment contained approximately 31,000 cpm/mg of protein.

achieved an additional 3-fold purification. The MVA-labeled material in the resulting pools showed an overall enrichment of 400-750-fold, with recoveries of up to 70%. Nevertheless, it still appeared to contain substantial amounts of other fragments that were prelabeled by the amino acids (not shown).

Both the material of 1000 Da and that of 500 Da were extremely hydrophobic and even insoluble in 6 M guanidine hydrochloride. They also were almost insoluble in hexane but appeared to be very soluble in acidic polar organic solvents. Upon being chromatographed on thin-layer plates of silicic acid in solvent systems for neutral lipids (4,22), they remained at the origin, unlike cholesterol, dolichol, and ubiquinone. On the other hand, they migrated near the solvent front in a phospholipid solvent system (23). These properties, and the results of our other work, are consistent with the possibility that the MVA-labeled, proteolytic fragments are small, hy- drophobic peptides that contain polyisoprenoid side chains.

DISCUSSION

In this investigation we studied the metabolic fate of radio- active MVA in mevinolin-treated cultures of 3T3 cells and

Labeling of Swiss 3T3 Cell Proteins by Mevalonic Acid 10179

‘iA A M INTACT PROTEINS c+a DIGESTED PROTEINS

4 ’ O l M POOL A M POOL B

4.8 c *I a Y 3.0-

L P 3 4 2.5-

STANDARDS

.2 *I0 04 03 05 6 07 *9

e8 2.0 r 1 1 I 1

10 15 20 25 30 35 FRACTION

FIG. 4. Chromatography of MVA-labeled proteins and frag- ments over Sephadex LH-20. MVA-labeled cells were harvested without protease inhibitors, delipidated, and SDS:CHC&:phenol ex- tracted, and the organic phase macromolecules recovered as described. Proteolytic digestion and chromatography over AG 1-X2 and LH-20 were then carried out as described under “Materials and Methods.” Various fractions (from different experiments) were dissolved in formic acid:EtOH (1:4) and rechromatographed on LH-20. A, undi- gested proteins (0) and proteolytic hydrolysis products (0); B, “Pool A (0) and “Pool B (D) rechromatographed after purification through LH-20, C, peak elution positions of bovine serum albumin (1); ubiquinone (2); squalene (3); Met-enkephalin (4); cholesterol (5); oleic acid (6); myristic acid (7); mevalonolactone (8); tryptophan (9); hemin (IO).

obtained results strongly suggesting that a product of authen- tic (R)-MVA was converted to a protein-bound form. Since no protein-bound products of MVA in mammalian cells have been described previously, several important questions arise. Could the labeled proteins be experimental artifacts? In what form is the radioactivity bound to protein? Are the modified proteins likely to be biologically important?

We considered two potential artifacts: 1) spurious labeling of proteins by a radiolabeled contaminant, and 2) protein ‘‘labeling’’ as a result of nonspecific complex formation be- tween proteins and labeled lipids. Our experiments provided strong evidence that authentic (R)-MVA serves as the active precursor (see “Results”). Moreover, they made it seem very unlikely that the MVA-labeled proteins represent noncova- lent complexes of unlabeled proteins with either MVA or labeled isoprenoids (including isopentenylated tRNA). Thus, the radioactive products released by extensive proteolytic digestion clearly differed from MVA during chromatography on Sephadex LH-20, and also behaved differently from cho-

lesterol, dolichol, ubiquinone, and cholesteryl oleate during thin-layer chromatography. Furthermore, we were not able to separate the radioactive moiety from proteins using any of the following methods: extraction with organic solvents, ex- traction with SDS:CHCls:phenol, SDS-gel electrophoresis, or chromatography in formic acid:EtOH (1:4). We were also unable to liberate the radioactivity using extractions for far- nesyl-containing heme a (24-26) or solvents that disrupt tight, noncovalent binding of polyphosphorylated lipids to proteins (27). These results strongly suggest, therefore, that the MVA- derived moiety is covalently linked to protein. Since, to the best of our knowledge, this type of linkage has never been described in mammalian cells, it seems likely that MVA- labeled proteins represent novel products of MVA metabo- lism, either known products linked in novel ways or entirely novel metabolites of MVA.

Radioactivity in MVA-labeled proteins might have reflected the presence of MVA itself, a product of a degradative path- way such as the transmethylglutaconate shunt, or an isopren- oid product of MVA. One type of experiment strongly sug- gests, however, that radiolabeled MVA was not involved. When we incubated mevinolin-treated 3T3 cells with either [1-I4C]- or [2-14C]MVA for 24 h then measured radioactivity in proteins by electrophoresis and fluorography, we found that l-14C was incorporated into proteins at least 91-fold less efficiently than 2-’*C.’ Because C1 is selectively lost from MVA during the formation of isopentenyl pyrophosphate, we postulate the MVA-labeled proteins are actually labeled by either isopentenyl pyrophosphate or one of its metabolic products.

Our results make it seem unlikely that labeled isoprenoid pyrophosphates are first degraded to smaller labeled inter- mediates, then incorporated into proteins. Most importantly, 5-3H-, 3-14C-, and 2-14C-labeled MVA were each incorporated into the same proteins and [2-’4C]- and [5-3H]MVA, when added to cells concomitantly, were both incorporated into proteins in the same ratio as into lipids. This strongly suggests that the bonds between the labeled atoms were not cleaved during the conversion of labeled MVA to labeled proteins. It also specifically argues against a role of the transmethylglu- taconate shunt (28) because the shunt converts [5-3H]- and [2-I4C]MVA to labeled acetyl-coA and acetoacetate, respec- tively. In addition, the conversion of labeled MVA to long chain fatty acids by the shunt is not a quantitatively impor- tant pathway under our culture conditions. Less than 2.5% of the radioactivity in the lipid or protein extracts of MVA- labeled cells was in a saponifiable form that co-migrated with fatty acids in a neutral lipid chromatography ~ y s t e m . ~ Taken together, these results strongly suggest that the fragment of MVA that is incorporated into proteins is not an end product of the shunt pathway or any other degradative pathway. On the other hand, they do not rule out the possibility that labeled proteins are formed from an early intermediate in the shunt (e.g. dimethyl acrylic acid).

A final possibility is that the MVA-labeled proteins contain isoprenoid products of MVA. At least three mechanisms can be envisioned whereby an isoprenoid compound might become covalently bound to protein. In one, a cysteine residue might condense across the double bond in isoprenoids, as may occur in felinine (29). In a second, isoprenoid fatty acids such as have been found in bovine retina (30) might be linked to proteins through amide or ester bonds, although the latter

R. A. Schmidt, C. J. Schneider, and J. Glomset, unpublished

R. A. Schmidt, C. J. Schneider, and J. Glomset, unpublished observations.

results.

10180 Labeling of Swiss 3T3 Cell Proteins by Mevalonic Acid

possibility seems unlikely because the radioactivity is not released by treatment with 0.5 M hydroxylamine, pH 11, or 0.1 N NaOH even though ethyl acetate is hydrolyzed.' In a third mechanism, the isoprenoid precursor might be converted to a reactive intermediate with carbonium ion characteristics which then couples to electron-rich sites in proteins via its C, carbon atom. The latter mechanism is thought to underlie the polymerization of isoprenoids (reviewed in Ref. 31), and may account for the biosynthesis of isopentenyl adenosine (32), dimethylallyl tryptophan (33), heme a (34), and yeast peptidal sex hormones (35). In order to choose among these mecha- nisms, we shall need to determine the structure of the MVA- derived moiety and its linkage to protein from fragments of MVA-labeled proteins. We have made progress toward this goal and have purified labeled proteolytic fragments of 3T3 cell proteins by approximately 400-750-fold. This approach has been limited, however, by the small amount of MVA- labeled protein contained within 3T3 cells (see below) and we are currently investigating alternative starting materials that contain more modified protein.

Three arguments raise the possibility that MVA-modified proteins are related to cellular requirements for MVA such as DNA synthesis, shape control, and HMG-CoA reductase reg- ulation. First, appropriate amounts of MVA-labeled protein are present within 3T3 cells. Thus, when we cultured 3T3 cells for four population doublings in the presence of 32 ptM compactin and 92 PM radiolabeled MVA, we calculated the total protein-bound label to be equivalent to 265 x 10"' mol of MVA per cell (approximately 265 pmol of MVA/mg of cell protein). This estimate is quite similar to our previous finding (4) that the uptake of as little as 250 X 10"' mol of MVA/ cell is sufficient to reverse the cellular shape change due to MVA deficiency. Second, a major fraction (26-46% in five experiments) of the MVA that partially prevents cell rounding in the roller bottle culture system is converted ultimately to protein-bound form. Finally, unpublished results in our PDGF-stimulated cell culture system (2, 4) indicate that MVA-labeled proteins are major metabolic products of MVA that turn over very slowly but nonetheless become deficient at the same time that mevinolin-treated cells change shape and are prevented from synthesizing DNA. Taken together, these results support the existence of MVA-labeled proteins as important novel metabolic products of MVA. Future stud- ies into the identity of the individual labeled proteins, their function within cells, and their relation to cellular require- ments for MVA will be of great interest.

Acknowtedgments-we are pleased to acknowledge Sue Schaefer for her assistance in the preparation of this manuscript, Kate Schmitt for editorial assistance, Dr. John Watson for his gift of l-"C-labeled mevalonic acid, and Maria Calula and Weiling King for lipid analyses.

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