THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 6, Issue of February 25, I+ 3362-3368, 1990 Printed in U.S.A.

RASZ Protein of Succharomyces cereuisiae Undergoes Removal of Methionine at N Terminus and Removal of Three Amino Acids at C Terminus*

(Received for publication, August 25, 1989)

Asao FujiyamaS and Fuyuhiko Tamanoigll From the $National Institute of Genetics, Mishima, Shizuoka, 411, Japan, and the §Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637

RAS2 protein of Saccharomyces cerevisiae under- goes post-translational modifications involving methyl esterification and palmitic acid addition, resulting in their association with the plasma membrane. In this paper, we provide evidence that two kinds of proteo- lytic events accompany the biosynthesis. This is shown by separating and characterizing three intracellular forms of RASP protein: precursor, intermediate, and mature (fatty acid-acylated) forms. N-Terminal se- quencing has revealed that all three forms start with proline, which is the second amino acid expected from the RAS2 gene sequence. Thus, the first methionine is removed very early during the biosynthesis. Isolation and sequencing of C-terminal peptides indicate that three C-terminal amino acids present in the precursor form are removed in the intermediate and in the fatty acid acylated forms. C-Terminal proteolysis appears to accompany methyl esterification, since the methyla- tion occurs with the intermediate and the fatty acid- acylated forms, but not with the precursor. Palmitic acid is identified as the major fatty acid attached to the fatty acid-acylated form.

Products of ras genes are guanine nucleotide binding pro- teins which play a major role in transduction of growth signal across the plasma membrane (reviewed in Barbacid, 1987). Expression of mutated forms of mammalian ras proteins in NIH 3T3 cells results in the transformation of these cells. In yeast, RASl and RAS2 proteins are required for the stimu- lation of adenylate cyclase. This results in an increase of CAMP which is needed for the transition from Gl to S phase during the cell cycle (reviewed in Tamanoi, 1988).

The ras proteins are predominantly localized in the inner surface of the plasma membrane, and this localization is essential for the function of these proteins (Shih et al., 1982; Willingham et al., 1980; Fujiyama and Tamanoi, 1986). This intracellular targeting of the ras proteins appears to be facil- itated by a conserved C-terminal sequence, CysAAX (A is an aliphatic amino acid and X is the C-terminal amino acid). This sequence is termed CAAX. box (Magee and Hanley, 1988). Viral ras proteins without the CAAX box or having the

* This work was supported by National Institutes of Health Grant CA41996 and also by a grant from the Cancer Research Foundation. 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.

V Established Investigator of the American Heart Association. To whom correspondence and reprint requests should be addressed.

cysteine in the CAAX box changed to serine remain in the cytosol and are deficient in transforming NIH 3T3 cells (Willumsen et al., 1984; Weeks et al., 1985). Analogous mu- tants of yeast RAS2 protein remain in the cytosol and do not support the growth of cells lacking the RAS genes (Deschenes and Broach, 1987). The requirement for the membrane local- ization of yeast RAS2 protein can be bypassed by overpro- ducing the mutant RAS2 proteiri (Deschenes and Broach, 1987).

In order for the ras proteins to be localized in the mem- brane, they have to undergo a series of post-translational modification events. The modification involves palmitic acid attachment (Sefton et al., 1982; Buss and Sefton, 1986; Fuji- yama and Tamanoi, 1986; Magee et al., 1987) as well as a novel methyl esterification (Clarke et al., 1988). Using yeast RASl and RAS2 proteins as well as H-ras expressed in yeast, we have previously shown that the modification occurs in two distinct steps: 1) conversion of a precursor form to an inter- mediate form and 2) palmitic acid addition to the intermediate form producing a fatty acid-acylated form (Fujiyama and Tamanoi, 1986; Tamanoi et al., 1988). The intermediate form exhibits a slightly increased mobility on a SDS’-polyacryl- amide gel. Conversion of the precursor form to the interme- diate form occurs in the cytosol. A similar conclusion has been obtained by Gutierrez et al., (1989) from the analyses of mammalian ras protein.

Further insights into the biosynthesis of ras protein were obtained by a comparison to the processing of a yeast mating factor, a-factor. Yeast mutants, dprl and ram, defective in the biosynthesis of ras protein were isolated (Fujiyama et al., 1987; Powers et al., 1986). These mutants were also found to be defective in the production of u-factor, and the mutation was allelic to ste16 (Wilson and Herskowitz, 1987), a mutation known to affect post-transcriptional steps in the biosynthesis of the u-factor. Like ras proteins, the u-factor is synthesized as a precursor which ends with the CAAX box (Brake et al., 1985). The mature protein ends with cysteine, and this C- terminal cysteine is methyl-esterified (Betz et al., 1987). In addition, farnesyl moiety is attached to the cysteine (Anderegg et al., 1988). Availability of the mutants enabled the cloning of a ras biosynthesis gene. This gene, DPRl/RAM encodes a hydrophilic protein of 431 amino acids which can be identified as 43-kDa protein on a SDS-polyacrylamide gel (Goodman et al., 1988).

Recent realization that a large number of proteins contain

1 The abbreviations used are: SDS, sodium dodecyl sulfate; HPLC, high performance liquid chromatography; BSA, bovine serum albu- min.

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Biosynthesis of rus Proteins 3363

the CAAX box at their C termini supports the idea that the ras biosynthesis mechanism is utilized by a wide range of proteins. These proteins include y-subunits of transducin (Hurley et al., 1984; Yatsunami et al., 1985) and mammalian G protein (Gautum et al., 1989), as well as STEM protein, a putative y-subunit of yeast G-protein (Whiteway et al., 1989). Furthermore, the CAAX box is found at the C termini of various fungal mating factors (Akada et al., 1987; Brake et al., 1985).

To define events during the biosynthesis of ras proteins, we have purified and characterized intracellular forms of yeast RASP protein. Their N-terminal sequences have been deter- mined by sequencing the proteins after labeling with radio- active amino acids. The C-terminal structures of these forms have been compared by isolating C-terminal peptides. In addition, we have characterized methylation and palmitic acid attachment.

EXPERIMENTAL PROCEDURES

Preparation of Labeled RAS2 Protein-To label RAS2 protein, yeast cells carrying plasmid YEp51-RAS2 which overproduce RASP protein in the presence of galactose (Fujiyama and Tamanoi, 1986) were used. The cells were grown at 30 “C in synthetic medium containing 5% galactose, lacking appropriate amino acids, and labeled with [?S]methionine (1150 Ci/mmol), [?S]cysteine (1308 Ci/mmol), [3H]serine (37 Ci/mmol), [3H]isoleucine (106 Ci/mmol), [3H]palmitic acid (54 Ci/mmol), or [3H]myristic acid (48 Ci/mmol) at a final concentration of 13 - 25 pCi/ml. The radioactive compounds were purchased from Amersham. For methyl esterification experiments, [methyl-3H]methionine (80 Ci/mmol, Du Pont-New England Nu- clear) was used at a concentration of 5 - 12.5 ,.&i/ml. Labeled RAS2 protein was purified by the immunoaffinity purification procedure (Cobitz et al., 1989) utilizing an anti-ras monoclonal antibody Y13- 259 covalently attached to Sepharose beads (259-Sepharose). Briefly, cells were resuspended in 50 mM KPO, (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 1 mM phenylmethylsulfonyl fluo- ride, 20 nM p-nitrophenylguanidine benzoate and broken with glass beads (diameter 0.5 mm), followed by centrifugation for 10 min at 10,000 rpm in a Sorval HB4 rotor. The supernatant was adjusted to a final NaCl concentration of 0.5 M, mixed with 259-Sepharose, and rotated overnight at 4 “C. The 259Sepharose beads were collected by centrifugation and washed extensively with 50 mM KPO, (pH 7.4), 500 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 1 mM phenyl- methylsulfonyl fluoride, 20 nM p-nitrophenylguanidine benzoate and finally with 50 mM KPOa (DH 7.4). 15 mM NaCl. Bound nrotein was eluted with 0.1 M acetic acid, 0.02% Triton X-100 (259Sepharose fraction).

HPLC Analyses of Proteins and Peptides-Separation of three intracellular forms of RASP protein was carried out by using a C4 HPLC column (Pierce, Aquapore butyl). The 259-Sepharose fraction was mixed with trifluoroacetic acid (final concentration 1%) and injected into the column. Elution was carried out by increasing acetonitrile concentration.

To carry out proteolytic digestion, the peak fractions were pooled and lvophilized after addinn 100 ua of BSA. The dried material was first dissolved in 8 M urea,-20 rn&-Tris-HCl (pH 7.4), 5 mM dithio- threitol and then diluted with 0.1 M 2-amino-2-methyl-1,3-propane- diol (pH 9.5) to 2 M urea. Digestion was carried out with 5 pg of lysyl endopeptidase (Wako Pure Chemicals, Achromobacter protease I (EC 3.4.21.50), Masaki et al., 1978) for 4 to 8 h at 37 “C. In some earlier experiments, the lysyl endopeptidase digestion was followed by over- night incubation with 5 pg of trypsin at 37 ‘C. The extent of the digestion was assessed by running a small aliquot on a Cl8 HPLC column and examining the digestion of BSA. After adding methanol and trifluoroacetic acid to 25% and l%, respectively, peptides were separated on a Cl8 or C4 HPLC column. In some cases, the sample was treated with alkali to remove fatty acid and passed through gel filtration column prior to reverse phase HPLC.

HPLC Analyses of Fatty Acids-Removal of palmitic acid from RAS2 protein was carried out in the following manner. Fatty acid- acylated RAS2 protein isolated as above on a HPLC column was lyophilized, suspended in 8 M urea, and then adjusted to 2 M urea in 5 mM Tris-HCI (pH 7.4), 1 mM dithiothreitol. Treatment with KOH was carried out by adding KOH to a final concentration of 0.1 M and

incubating at 23 “C for 30 to 60 min. Treatment with hydroxylamine was carried out by adding equal volumes of freshly prepared 2 M NHYOH (DH 7.0) and incubating: at 23 “C for 30 to 60 min. From the reaction mixture, fatty acids were extracted twice with 4 volumes of chloroform/methanol mixture (2:l). The extracted sample was dried in uacuo, dissolved in methanol containing authentic fatty acid mark- ers, and then injected into a C4 HPLC column. The fatty acids were eluted from the column with increasing concentrations of acetonitrile.

Sequence Determination of Proteins and Peptides-Amino acid sequence was determined using Applied Biosystems model 470A gas phase Sequencer. Recovery of the protein on membrane in each cycle is estimated to be about 98% for protein and 90% for peptides.

Chemical Synthesis of Peptides-Decamer SGSGGCCIIS corre- sponding to the C-terminal peptide of lysyl endopeptidase digest of RAS2 was prepared by Dr. Satoe Nakagawa (University of Chicago) using Applied Biosystems 430A peptide synthesizer and purified by HPLC.

Analysis of Carboxymethylation of RAS2 Protein-A test for the methyl esteriflcation of RAS2 protein was performed by the vapor- phase method essentially as described (Stock et al., 1984) except concentration of NaOH was lowered to 0.1 M to avoid breakdown of S-CH3 groups. RAS2 protein labeled with [ methyl-3H]methionine was purified by 259-Sepharose followed by HPLC as described above. The fractions containing precursor form, intermediate form, and fatty acid-acylated form RASP were pooled separately and lyophilized together with 100 pg of BSA as a carrier. Labeled precursor RAS2 protein was purified from dprl strain. Labeled proteins were dissolved in 100 1~1 of 0.1 M NaOH in a 1.5-ml Microfuge tube. transferred into a 20-mi scintillation vial containing 8 ml of scintillation fluid (Beck- man, Ready-Gel), tightly sealed, and incubated at 37 “C for 20 to 24 h. Radioactive methanol from the methyl group of carboxyl methyl ester was assayed using liquid scintillation counter.

RESULTS

Three Distinct Species of RAS2 Protein Can Be Identified during Its Biosynthesis-Based on pulse-chase with [35S]me- thionine and subcellular fractionation, we have previously proposed that RAS2 protein is synthesized first as a precursor which is converted to an intermediate form that migrates slightly faster than the precursor form on a SDS-polyacryl- amide gel. The palmitic acid addition occurs on the interme- diate form to produce a fatty acid-acylated form which is predominantly localized in the plasma membrane (Fujiyama and Tamanoi, 1986). We have purified these three different forms on a C4 HPLC column. Yeast cells overproducing RAS2 protein were labeled for a brief period with [35S]cysteine. The labeling period was designed so that biosynthesis intermedi- ates could be isolated. The cells were broken, and RAS2 protein was purified by using anti-ras monoclonal antibody Y13-259 covalently coupled to Sepharose beads. When the purified RAS2 protein was loaded onto a C4 HPLC column and eluted with an increasing concentration of acetonitrile, we observed the appearance of two peaks as shown in Fig. 1A. The position of the second peak (peak II in Fig. 1A) matches the RAS2 protein labeled with [3H]palmitic acid (Fig. 1B). Thus, fatty acid-acylated RAS2 protein (mature RAS2 pro- tein) is separated from other forms of RAS2 protein presum- ably due to its increased hydrophobicity. When peak ZZ of Fig. L4 was treated briefly with 0.1 M KOH to remove the fatty acid and was reapplied to the HPLC column, the peak of the radioactivity moved to the position of peak Z (Fig. 1C). Thus, the different elution positions of the fatty acid-acylated form and the other forms appear to be due to the presence of palmitic acid.

When a shallower gradient was applied, peak Z was split into two peaks, and a total of three peaks was detected (Fig. 2). Each peak can be identified by SDS-polyacrylamide gel electrophoresis (Fig. 2, insets). The first peak (peak 1) rep- resents the precursor RAS2 protein as shown by its slower mobility on a SDS-polyacrylamide gel (Fig. 2, inset, lane I). Some degradation products of RAS2 protein also elute in the

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3364 Biosynthesis of r-as Proteins

Fraction Number

FIG. 1. Separation of immunoaffinity-purified RAS2 pro- tein by HPLC. 259Sepharose fraction of [““Slcysteine-labeled (panel A) or [3H]palmitic acid-labeled protein (panel B) was sepa- rated by C4 column with the gradient of acetonitrile from 0 to 100% for 100 min at 1 ml/min. The peak II of cysteine-labeled sample was treated with alkali and was separated under the same condition (panel 0.

cm 201

1Of

C

1234

69- 43- ---

30-

10 20 30 40 50 60

Fraction Number

FIG. 2. HPLC separation of [35S]methionine-labeled RAS2 protein. 259.Sepharose fraction was injected into a C4 HPLC col- umn and eluted with the gradient from 40 to 60% acetonitrile for 60 min at 1 ml/min. Inset, peak fractions designated I to 3 were pooled separately and subjected to SDS-polyacrylamide gel electrophoresis. Lanes I to 3 correspond to the peaks 1 to 3, respectively. Lane 4 shows alkali-treated sample from peak 3. Molecular weight markers are albumin (M, = 69,000), ovalbumin (M, = 43,000), and carbonic anhydrase (M, = 30,000).

same peak. The second peak (peak 2) represents the inter- mediate form as judged by its slightly increased mobility (Fig. 2, inset, lane 2). The third peak (peak 3, corresponding to peak II of Fig. 1) which was eluted with an even higher acetonitrile concentration represents the mature, fatty acid- acylated form. As described before, a brief alkali treatment of

peak 3 changes its elution position to that of the earlier peaks. In this shallow gradient system, the peak after the alkali treatment corresponds to that of the intermediate form (data not shown). This treatment does not affect its electrophoretic mobility on a SDS-polyacrylamide gel (Fig. 2, inset, lanes 3 and 4), confirming our earlier observation that fatty acid addition does not affect the mobility of ras proteins on a SDS- polyacrylamide gel (Tamanoi et al., 1988).

Palmitic Acid Is the Major Fatty Acid Attached to RASB- The combination of immunoaffinity purification and HPLC method enables purification of RAS2 protein to near homo- geneity in two steps. The availability of highly purified RAS2 protein allowed us to further characterize the fatty acid acy- lation event. Yeast cells overproducing RAS2 protein were labeled with [3H]palmitic acid, and the RAS2 protein was purified by 259-Sepharose. When the sample was applied onto HPLC, the radioactivity appeared as a single peak with ap- proximately 90% recovery. To examine the stability of the fatty acid attachment, the “H-labeled RAS2 proteins were subjected to two different treatments. As shown in Table I, the fatty acid radioactivity, which was stably bound to the protein in 0.5 M Tris-HCl (pH 7.4) at 23 “C, was released almost quantitatively by the treatment with either 1 M

NH,OH (pH 7.0) at 23 “C for 30 min or with 0.1 M KOH at 23 “C for 30 min. This suggests that the fatty acid attachment involves a thioester linkage. HPLC analyses of the fatty acid released after the alkaline treatment revealed that a majority of the fatty acid is palmitic acid (Fig. 3A).

We have found that [3H]myristic acid can also be used to label RAS2 protein. A significant level of radioactivity can be incorporated into the RAS2 protein. However, when the 3H radioactivity was released and analyzed, we found that it consisted predominantly of palmitic acid (Fig. 3B), and only minor amounts of myristic acid were detected. It is likely that the radioactive myristic acid is intracellularly converted to palmitic acid before attaching to the protein. A strong pref- erence for palmitic acid may indicate that a putative fatty acyltransferase for ras protein has a specificity for palmitic acid.

The First Methionine Is Removed at an Early Stage during Biosynthesis-To further define biosynthetic events for RAS2 protein, we carried out characterization of their N-terminal structure. To determine their N-terminal sequence, RAS2 protein was labeled with [“Hlleucine or [3H]isoleucine and was purified using 259Sepharose followed by C4 HPLC as described. For the isolation of labeled precursor form, dprl mutant cells were used, RAS2 protein remains as a precursor in the mutant (Fujiyama et al., 1987). Intermediate and fatty acid-acylated forms were isolated from wild type cells. The purified proteins were mixed with BSA which was added as a carrier and then subjected to automated gas-phase Edman degradation. As shown in Fig. 4A, when the precursor RAS2

TABLE I Stability of bound fatty acids

Fatty acid-acylated form of RAS2 protein labeled with [lH]palmitic acid was isolated by 259.Sepharose followed by C4 HPLC column chromatography. The sample was incubated with 0.1 M KOH or with 1 M NH,OH as described under “Experimental Procedures,” and the radioactivity released was determined by extracting with chloroform/ methanol.

No treatment 0.1 M KOH 1 M NHJOH, pH 7.0

Percentage of radmactwity in organic phase

% 0

92.9 95.6

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Biosynthesis of ras Proteins 3365

[MJPLNKSN I REYKLVVVGGGGVG--- A

0 I 0

x

E e

3-

2-

l-

Fin-

-

0 D L MPS

I I I III

1

, 0

,

AU J I I

0 D LMPS

I I I I I I .

6-

0 10 20 30 40 50 60 70

Fraction Number

FIG. 3. Analysis of fatty acids isolated from mature RAS2 protein. Panel k, fatty acids were recovered from [3H]palmitic acid- labeled RASZ. HPLC separation was performed using C4 column and linear gradient of acetonitrile from 0 to 100% for 100 min at 1 ml/ min with authentic fatty acid markers, octanoic acid (0), decanoic acid (D). lauric acid (~5). mvristic acid (M). nalmitic acid (P). and _, _ . . stearic acid (S). Arrows indicate the elution position of each marker. Panel B, fatty acids were recovered from [3H]myristic acid-labeled RAS2 protein and analyzed as described above.

protein labeled with [3H]leucine was subjected to Edman degradation, the radioactivity was recovered in cycles 2 and 12. This is the result expected if the RAS2 protein starts with proline which is the second amino acid expected from its nucleotide sequence (shown at the top of Fig. 4). The mature, fatty acid-acylated form also starts with proline, since the same result was obtained when a [3H]leucine-labeled, fatty acid-acylated RAS2 protein was subjected to Edman degra- dation (the radioactivity appeared in cycle 2) (Fig. 4C). The intermediate RASP protein also starts with proline, since isoleucine radioactivity appeared in cycle 7 when the inter- mediate RAS2 protein labeled with [3H]isoleucine was sub- jected to Edman degradation (Fig. 4B). Therefore, all three forms of RASP protein start with proline. The first methio- nine predicted from its nucleotide sequence is removed. The removal appears to occur very early during the biosynthesis.

Three C-Terminal Amino Acids Are Removed during Bio- synthesis-we next investigated C-terminal structure of the three forms. RAS2 protein was labeled with [35S]cysteine and subjected to digestion with lysyl endopeptidase. Because there are only two cysteines in RASP protein and both of them are located very close to the C terminus (318th and 319th residues in the 322 residue RAS2 protein), it is possible to selectively label the C-terminal portion and isolate C-terminal peptides after the protease digestion. When the lysyl endopeptidase digests of [35S]cysteine-labeled precursor RAS2 protein were separated on a Cl8 HPLC column, the C-terminal peptide was eluted with 37% acetonitrile (Fig. 5A). This elution profile was similar to that of a synthetic decamer peptide, SGSGGCCIIS (Fig. 5A). This peptide would be expected to

A cpm 400

‘*” Precursor

B ” 400. IlO

Intermediate

eye les

FIG. 4. Gas-phase sequence analyses of precursor, inter- mediate, and fatty acid-acylated form RAS2 protein. Amino acid sequence predicted from the nucleotide sequence is shown at the top. Panel A, analysis of [3H]leucine-labeled precursor form. Panel B, analysis of [3H]isoleucine-labeled intermediate form. Panel C, analy- sis of fatty acid-acylated form labeled with [3H]leucine. Phenylthio- hydantoin amino acids were recovered from the Sequencer, and the radioactivity in each fraction was assayed by scintillation counting.

be generated from the precursor (amino acid sequence deduced from the nucleotide sequence). C-Terminal peptidefrom the fatty acid-acylated protein, on the other hand, eluted at a higher acetonitrile concentration (46% acetonitrile) even after the removal of palmitic acid (Fig. 5B). C-Terminal peptide from the intermediate form also eluted at this position (data not shown). Thus, C-terminal structures of the intermediate and fatty acid-acylated forms appear to be different from that of the precursor form.

To further define the structure of the C-terminal peptides, they were subjected to Edman degradation. We first charac- terized the C-terminal peptide of the precursor form. When the peptide from the [3H]serine-labeled precursor form was subjected to Edman degradation, the radioactivity was re- covered in cycles 1 and 3. A small but significant radioactivity was also recovered in cycle 10 (Fig. 6A). Low levels of radio- activity detected in cycles 6 and 7 are most likely due to conversion of serine to cysteine within the cell. Presence of 2 isoleucine residues in the C-terminal peptide was demon- strated by sequencing the peptide prepared from [3H]Ile- labeled RAS2 protein. As shown in Fig. 6A, the radioactivity was recovered in cycles 8 and 9. These results are in agreement with the sequence of the C-terminal residues predicted from the RAS2 gene sequence, SGSGGCCIIS.

Similar sequencing analyses were carried out with the C- terminal peptide of the mature, fatty acid-acylated form. The C-terminal peptides were pretreated briefly with alkali to remove palmitic acid. When the C-terminal peptide from the [3H]serine-labeled mature form was subjected to Edman deg- radation, the radioactivity was recovered in cycles 1 and 3 but not in cycle 10 (Fig. 6B2). When the C-terminal peptide prepared from [3H]isoleucine-labeled mature form was ana- lyzed, very little radioactivity was recovered, and this was distributed in every cycle (Fig. 6B2). In fact, very little [3H]- isoleucine radioactivity was recovered in the C-terminal pep-

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3366 Biosynthesis of ras Proteins

CT b

X

E n 0

A

6

4 :

Precursor

B Elution Position (min)

“b IO

X

E Ef 5

Fatty Acylated (Alkali treated)

I A

lpLLL 0 IO 20 30 40 50

Elution Position (min)

FIG. 5. Identification of peptides from the C-termini of RAS2 proteins. Precursor and fatty acid-acyiated form RAS2 pro- teins were purified as described under “Experimental Procedures.” Panel A, precursor RAS2 protein was digested with lysyl endopepti- dase and separated by a Cl8 column with a gradient of acetonitrile from 0 to 100% over 100 min at 1 ml/min. The arrows indicate the position of synthetic SGSGGCCIIS peptide. Panel B, lysyl endopep- tidase digest of alkali-treated fatty acid-acylated form RAS2 was analyzed as above.

tide of the fatty acid-acylated form, which contrasts with a quantitative recovery of [3H]isoleucine radioactivity in the C- terminal peptide of the precursor form (Table II). These results suggest that the three C-terminal amino acids Ile/Ile/ Ser are missing in the mature form. The 2 cysteines adjacent to the isoleucine, however, are present in the C-terminal peptide. This is demonstrated by a similar analysis carried out with a C-terminal peptide prepared from [35S]cysteine- labeled mature form. As shown in Fig. 6B1, the radioactivity was recovered in cycles 6 and 7. Thus, we conclude that the C-terminal sequence of the mature form is SGSGGCC.

Intermediate and Fatty Acid-acylated RAS2 Proteins Are Methyl-esterified-Another event that takes place at the C- terminus of yeast RAS2 protein is methyl esterification (Deschenes et al., 1989). To investigate which intracellular form of RAS2 protein is methyl-esterified, cells were labeled with [ methyL3H]methionine, and RAS2 protein was purified by 259-Sepharose column followed by a C4 HPLC column. Each intracellular form of RAS2 protein was pooled and treated with alkali, and the radioactivity released as radioac- tive methanol was determined by the vapor phase method (Stock et al., 1984). As shown in Table III, 21.6% and 25.9% of the radioactivity incorporated into the intermediate form and the fatty acid-acylated form, respectively, was released as

A

cm 0 Ser . IlO

500

rsYi!Li * O 1 5 IO

511&J!J 20:u 1 5 10 1 5 10

Cycles Cycles

FIG. 6. Sequence analyses of C-terminal peptides from lysyl endopeptidase digests. Panel A, [3H]serine-labeled (0) precursor RAS2 protein and [3H]isoleucine-labeled (0) precursor RAS2 were subjected to gas-phase Edman degradation separately. Radioactivity recovered from each cycle was analyzed by liquid scintillation counter. Panel B, C-terminal peptides from [35S]cysteine-labeled, fatty acid- acylated RAS2 (B,), [3H]serine-labeled (0), and [3H]isoleucine-la- beled (0) RAS2 were sequenced as above (B,).

TABLE II

Recovery of r3H]Ile radioactivity in C-terminal peptides after protease digestion

Precursor and fatty acid-acylated forms of RAS2 protein labeled with [3H]isoieucine were separated by 259~Sepharose followed by C4 HPLC column chromatography. After mixing corresponding forms of [35S]cysteine-labeled RAS2 protein, they were digested with lysyl endopeptidase, and the samples were loaded onto a Cl8 column. The positions of C-terminal peptides were identified by the appearance of [35S]cysteine radioactivity. Percentage of [3H]isoleucine radioactivity in the C-terminal peptides was determined and is shown in the “Actual” column. Percentage of [3H]isoleucine radioactivity expected to be recovered in the C-terminal peptide based on RAS2 gene sequence is 14.3%. The expected percentage is 0% if three C-terminal amino acids are removed in the fatty acid-acylated form.

Actual Expected

Precursor Fatty acid-acylated

7% 13.7 14.3

1.5 0

TABLE III

Methyl&ion of RAS2 protein Three intracellular forms of RAS2 protein labeled with [methyl-

3H]methionine were isolated as described under “Experimental Pro- cedures.” Precursor form is isolated by overproducing RAS2 protein in dprl mutant, and the intermediate and fatty acid-acylated forms were isolated from wild type cells. These samples were subjected to alkali treatment, and the radioactivity recovered as methanol was determined as described under “Experimental Procedures.”

Total Alkali-labile (MeOH) Released

Precursor Intermediate Fatty acid-acylated

cpm cm % 27,783 78 0.3

1,650 356 21.6 1,928 499 25.9

radioactive methanol after the alkali treatment. In contrast to this, virtually no radioactivity was released from the pre- cursor form after the alkali treatment. Thus, the intermediate

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Biosynthesis of ras Proteins

and fatty acid-acylated forms of RAS2 protein are modified by methyl esterification, but the precursor form is not.

DISCUSSION

Membrane association of ras proteins involves palmitic acid addition and methyl esterification. It is believed that these modifications contribute to the increase in the hydrophobicity of the protein, thus enhancing its affinity for membrane lipids. This strategy represents an alternative to the well studied secretory pathway. In this paper, we have demonstrated sep- aration of three intracellular forms of RAS2 protein (precur- sor, intermediate, and fatty acid-acylated forms) which rep- resent proteins at different stages of biosynthesis. An increase of hydrophobicity during its biosynthesis is observed as judged by their order of elution from a C4 HPLC column. The fatty acid-acylated form is the most hydrophobic, whereas the intermediate form is only slightly more hydrophobic than the precursor form. Thus, palmitic acid appears to play a major role in the increase of the overall hydrophobicity of RAS2 protein. Treatment of the fatty acid-acylated form with mild alkali converts back to the intermediate form and quantita- tively releases fatty acids attached to RASP protein. We have identified palmitic acid as the major fatty acid attached to the protein. Since palmitic acid can be released by treatment with hydroxylamine, it appears that palmitic acid attachment in- volves a thioester linkage. The site of palmitic acid attach- ment appears to be very close to the C terminus, since there are only 2 cysteines in the RAS2 molecule, and they are located at the 4th and the 5th positions from the C-terminus.

Structural characterization of the intracellular forms of RAS2 protein has led to the finding that two proteolytic events, removal of methionine at the N terminus, and removal of three amino acids at the C terminus, take place during the biosynthesis. The removal of N-terminal methionine was discovered when N-terminal sequencing was carried out, which demonstrated that the RAS2 proteins start with pro- line. The removal of methionine appears to take place very early in biosynthesis, since even the precursor form is missing methionine. Our finding that N-terminal methionine is re- moved from RAS2 protein is consistent with the previously reported and widely accepted suggestion that many eukaryotic as well as prokaryotic proteins lose their N-terminal methio- nine during biosynthesis (reviewed in Tsunasawa et al., 1985). By compiling N-terminal sequences of a number of proteins, Tsunasawa et al. (1985) concluded that N-terminal cleavage occurs when the methionine precedes residues of alanine, glycine, proline, serine, threonine, and valine but not when it precedes residues of arginine, asparagine, aspartic acid, glu- tamine, glutamic acid, isoleucine, leucine, lysine, or methio- nine. RAS2 protein seems to conform to this rule, since the methionine precedes proline.

In this paper, we have provided evidence that 3 C-terminal amino acid residues are removed during the biosynthesis. We have previously suggested this possibility from the analogy with the biosynthesis of a yeast mating factor, a-factor (Ta- manoi et al., 1988). Processing of a-factor is catalyzed by DPRl gene which is known to play a major role in the processing of RAS proteins. The a-factor is synthesized as a precursor ending with the CAAX box present at the C ter- minus of ras proteins. Structural characterization of the a- factor established that it ends with cysteine, demonstrating that three amino acids after the cysteine are removed (Betz et al., 1987). We have shown that RAS2 protein loses three C-terminal amino acids during its biosynthesis by following isoleucine radioactivity in the C-terminal peptide of RAS2 protein. The three-amino acid removal occurs during the

conversion of precursor to intermediate form, since the elution profile of the C-terminal peptide of the intermediate form from a Cl8 column is similar to that of the C-terminal peptide of alkali-treated fatty acid-acylated form. Removal of three C-terminal amino acids from mammalian ras protein was recently reported (Guttierez et al., 1989). In this case, tryp- tophan was introduced into one of the three C-terminal amino acids of H-ras protein, and the loss of this tryptophan during the processing was shown. A different line of inquiry also led to a similar conclusion. Deschenes et al. (1989) have shown that the C terminus of RAS2 protein is modified by methyl esterification and that this methylation takes place on cys- teine. The implication of this finding is that the cysteine must be at the C terminus. Thus, all these results point to the idea that the three C-terminal amino acids are removed placing cysteine at the C terminus.

Our finding indicates that precisely three amino acids at the C terminus of RASP protein are removed. This may be catalyzed by a novel protease, presumably some type of car- boxypeptidase. If it is an exopeptidase, the activity has to stop at the cysteine. Alternatively, an endopeptidase cleaving after the cysteine might be involved. The presence of an activity to remove three C-terminal amino acids was implied by an experiment reported by Molenaar et al. (1988). They analyzed ras-related yeast YPTl protein which ends with cysteine. The presence of cysteine at its C-terminus appears to be essential for its function. Addition of three extra amino acids (two aliphatic amino acids and the terminal amino acid) to the C terminus of YPTl protein, generating ras-typical C-terminal end, did not destroy its function and resulted in the appear- ance of precursor protein which could subsequently be con- verted into mature protein.

Another event that happens at the C terminus of RAS2 protein is methyl esterification. We have shown that this modification occurs on the intermediate and fatty acid-acyl- ated forms but not on the precursor form (see Table III). The methy ester group has been Iocalized to the C-terminal SGSGGCC peptide of the intermediate and fatty acid-acyl- ated forms.2 This is consistent with the results obtained by Deschenes et al. (1989) who showed that cysteine is the site of methylation. Although both the precursor and the inter- mediate forms are present in the cytosol along with a putative methyltransferase, it is highly likely that methylation occurs only on the protein that lacks C-terminal extension of three amino acids.

Recently, another type of C-terminal modification of ras proteins has been reported. Hancock et al. (1989) have shown that mammalian ras proteins can be labeled with [3H]meva- ionic acid, suggesting that polyisoprenylation occurs on ras proteins. The modification appears to take place on the cys- teine in the CAAX box. A possibility that yeast RAS2 protein is also modified by polyisoprenylation has been raised by Schaffer et al. (1989) from their analyses of the effect of blocking mevalonic acid synthesis by using mutants in hy- droxymethylglutaryl-CoA reductase. We have recently suc- ceeded in labeling RAS2 protein with [3H]mevalonic acid. The incorporated radioactivity can be localized in the C- terminal peptide after lysyl endopeptidase digestion2 The polyisoprenylation may explain the hydrophobic nature of the C-terminal peptide of mature RAS2 proteins as reported in this paper. The C-terminal peptides of the intermediate and fatty acid-acylated forms, even after the removal of methyl ester and palmitic acid, are considerably more hydrophobic than a synthetic heptamer having the expected sequence. Thus, it is likely that the yeast RAS2 protein is also modified

ZXA. Fujiyama, unpublished results.

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3368 Biosynthesis of ras Proteins

by polyisoprenylation. However, further work is needed to understand the nature of this modification and to determine exact structure of the C terminus of RAS2 protein. The C- terminal peptide obtained from mature RAS2 protein should provide valuable substrates for further investigation.

Acknowledgments-We thank Dr. Theodore Steck, Dr. Kan Agar- wal, and Laurie Goodman for critical reading of this manuscript and Drs. Fumio Sakiyama and Susmu Tsunasawa for discussions. We also thank Dr. Satoe Nakagawa for the synthesis of oligopeptides and Mary Gramhofer for secretarial assistance.

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A Fujiyama and F Tamanoiterminus and removal of three amino acids at C terminus.

RAS2 protein of Saccharomyces cerevisiae undergoes removal of methionine at N

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