Amino Acid Substitutions That Convert the Protein Substrate Specificity of Farnesyltransferase to That of Geranylgeranyltransferase Type I*

(Received for publication, August 7, 1996, and in revised form, October 4, 1996)

Keith Del Villar Dagger , Hiroshi Mitsuzawa Dagger §, Wenli Yang , Isabel Sattler and Fuyuhiko Tamanoi par

From the Department of Microbiology and Molecular Genetics, Molecular Biology Institute, UCLA, Los Angeles, California 90095-1489

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Protein farnesyltransferase (FTase), a heterodimer enzyme consisting of alpha  and beta  subunits, catalyzes the addition of farnesyl groups to the C termini of proteins such as Ras. In this paper, we report that the protein substrate specificity of yeast FTase can be switched to that of a closely related enzyme, geranylgeranyltransferase type I (GGTase I) by a single amino acid change at one of the three residues: Ser-159, Tyr-362, or Tyr-366 of its beta -subunit, Dpr1. All three Dpr1 mutants can function as either FTase or GGTase I beta  subunit in vivo, although some differences in efficiency were observed. These results point to the importance of two distinct regions (one at 159 and the other at 362 and 366) of Dpr1 for the recognition of the protein substrate. Analysis of the protein, after site directed mutagenesis was used to change Ser-159 to all possible amino acids, showed that either asparagine or aspartic acid at this position allowed FTase beta  to function as GGTase I beta . A similar site-directed mutagenesis study on Tyr-362 showed that leucine, methionine, or isoleucine at this position also resulted in the ability of mutant FTase beta  to function as GGTase I beta . Interestingly, in both position 159 and 362 substitutions, amino acids that could change the protein substrate specificity had similar van der Waals volumes. Biochemical characterization of the S159N and Y362L mutant proteins showed that their kcat/Km values for GGTase I substrate are increased about 20-fold compared with that of the wild type protein. These results demonstrate that the conversion of the protein substrate specificity of FTase to that of GGTase I can be accomplished by introducing a distinct size amino acid at either of the two residues, 159 and 362.


INTRODUCTION

Protein farnesyltransferase (FTase)1 catalyzes the transfer of a farnesyl group from farnesyl diphosphate (FPP) to a cysteine residue of a protein substrate such as Ras (Refs. 1 and 2; for a review, see Refs. 3, 4, 5, 6). The enzyme recognizes a tetrapeptide sequence called the CAAX motif (C is cysteine, A is an aliphatic amino acid, and X is the C-terminal amino acid, which is usually methionine, serine, alanine, cysteine, or glutamine) at the C terminus of the protein. A closely related enzyme, geranylgeranyltransferase type I (GGTase I), recognizes the CAAX motif when X is either a leucine or phenylalanine (CAAL motif), but transfers a geranylgeranyl group from geranylgeranyl diphosphate (GGPP) (7, 8). The CAAL motif is found at the C termini of a variety of Rho family G-proteins (3, 4, 5, 6). Although FTase and GGTase I appear to have distinct functions inside the cell, the enzymes are not absolutely specific in vitro; purified FTase could utilize GGTase I protein substrates, and GGTase I could utilize FTase substrates, albeit at a lower rate (9, 10, 11, 12). In addition, overproduction of GGTase I beta -subunit could suppress temperature-sensitive growth due to the mutation of FTase beta  (13, 14).

The property of FTase to recognize the CAAX motif was recently exploited to develop inhibitors of FTase. Farnesylation of Ras is critical for the membrane localization and the transforming activity of Ras. Thus, FTase inhibitors could act to suppress ras-dependent transformation (15, 16). Peptidomimetics were developed by modifying CAAX tetrapeptides, and shown both to inhibit the growth of ras-transformed cells in soft agar and to induce morphological reversion of the transformed cells (17, 18). They also inhibited the growth of ras-induced tumors in mice (reviewed in Ref. 19). Understanding the mechanism by which CAAX and CAAL substrates are recognized by the enzymes responsible for prenyl modification, and the efficiency to which this is achieved provides valuable clues to how the enzyme action can be modulated at the level of protein substrate recognition.

In order to investigate the molecular basis for the recognition of the CAAX motif by FTase, we sought to convert protein substrate specificity of FTase to that of GGTase I. This was accomplished by randomly mutating the beta -subunit of FTase. FTase and GGTase I are heterodimers consisting of a common alpha  subunit and a similar but distinct beta  subunit (3, 20). Cross-linking of FTase with Ras or with peptide substrates implicated the beta  subunits as the subunit specifying protein substrate specificity of these enzymes (21, 22). We have employed the yeast Saccharomyces cerevisiae as a model system, since yeast FTase and GGTase I, like mammalian enzymes, are composed of a common alpha  subunit encoded by the RAM2 gene and related beta  subunits encoded by the DPR1/RAM1 gene and the CAL1/CDC43 gene, respectively (23, 24, 25, 26, 27). Dpr1 protein shares significant sequence identity with the human FTase beta  subunit and with Cal1 protein (14, 28). In addition, yeast FTase shows a divalent cation requirement and a protein substrate specificity similar to those of mammalian enzyme (12, 29). We have developed a screen to identify mutant FTase beta , which could function as a GGTase I beta  (30). The mutant FTases enable farnesylation of GGTase I substrates, which is sufficient to support GGTase I function inside the cell.

Previously, we reported the initial isolation of a mutant FTase beta  subunit, S159N, which replaced the function of GGTase I beta  (30). In the current study, we carried out an extensive screen to identify mutants with altered specificity for protein substrates similar to that reported for S159N. Two mutants in the C-terminal portion of the Dpr1 molecule (Y362H and Y366N) were identified in addition to S159N. Site-directed mutagenesis at the position 159 and 362 residues was carried out to gain further insights into the molecular mechanism by which these residues affect substrate specificity. Our results suggest that introduction of an amino acid of a specific size at residue 159 or at residue 362 results in the conversion of the protein substrate specificity of FTase to that of GGTase I.


EXPERIMENTAL PROCEDURES

Yeast Strains, Media, and Transformation

S. cerevisiae strains used were YOT559-3C (MATa cal1-1 leu2 trp1 ura3 ade2) (14) and YPH250dU (MATa dpr1::URA3 lys2-801 leu2-Delta 1 trp1-Delta 1 ura3-52 ade2-101 his3-Delta 200). YPH250dU was constructed as follows. The 2.4-kb dpr1::URA3 fragment was amplified by PCR from the genomic DNA of strain KMY200-sgp2-12A (31) using the primers DPR1#1 and DPR1#2 (30) and was used to transform strain YPH250 (32) to Ura+. The disruption was confirmed by PCR. Yeast media used were YPD medium (33) and SC-trp medium, synthetic minimal medium (33) supplemented with adenine, uracil, and 0.5% casamino acids. Yeast transformation was carried out by the lithium acetate method (34).

Yeast Plasmids and Construction of Mutated DPR1 Gene Libraries

Yeast high copy (YEp) plasmids containing wild type or mutant DPR1 gene were derivatives of pYO324 (35). Yeast low copy (YCp) plasmids containing wild type or mutant DPR1 gene were constructed by transferring the 2.4-kb XhoI-SacI DPR1 fragment from the high copy plasmids to the XhoI/SacI site of pRS314 (32). The DPR1 gene was randomly mutagenized by PCR with Taq DNA polymerase (Perkin-Elmer) to construct libraries on yeast high copy plasmid. To efficiently identify independent mutations, the procedure described previously (30) was modified as follows. First, the DPR1 gene was divided into two parts and was amplified separately; the 0.9-kb BamHI-NcoI region and the 0.5-kb NcoI-AflII region of pYO324(DPR1)BA were amplified using the primers DPR1#1 and DPR1#4 and the primers DPR1#2 and DPR1#3, respectively. The two regions correspond to amino acids 1-274 and 274-425, and all but the C-terminal six residues were mutagenized during the PCR amplification by inherent infidelity of Taq DNA polymerase. Second, a lower concentration of dNTP (50 µM each instead of 200 µM each) and fewer cycles of reaction (20 cycles instead of 30 cycles) were used to reduce multiple mutations, which we had found in the previous screen. Third, for each region, six libraries were constructed from independent PCR reactions to isolate independent mutations. Each library was derived from 36,000-86,000 Escherichia coli transformants. This approach is largely confined to detect single nucleotide changes in the DPR1 gene.

Isolation of Mutant DPR1 Genes That Suppress cal1

A temperature-sensitive cal1-1 strain, YOT559-3C, was used to screen the mutated DPR1 gene libraries for suppression. This strain cannot grow at the restrictive temperature and grows slower than wild type strain even at the permissive temperature. These phenotypes can be suppressed by the expression of a mutant DPR1, which can function as CAL1. In contrast, the wild type DPR1 gene on a high copy plasmid not only fails to suppress the temperature-sensitive growth, it also impairs the slow growth of the cal1-1 strain even at the permissive temperature (30). This latter property is caused presumably by the competition between Cal1-1 protein and overproduced Dpr1 protein for Ram2, the alpha  subunit shared by FTase and GGTase I. Thus, screening at the restrictive and at the permissive temperatures provides a sensitive assay for CAL1 function, allowing us to select fast growing colonies from a plate containing several thousand colonies.

Mutant DPR1 genes that can suppress the cal1-1 mutation were identified as follows. The mutated DPR1 gene libraries were used to transform the cal1-1 strain. Fast growing transformants were selected from SC-trp plates incubated at 27 °C and then screened for growth at 37 °C. Plasmid dependent suppression was confirmed by retransformation. One clone was chosen from each library to ensure independent origins of mutations and sequenced by dideoxy sequencing with Sequenase (U. S. Biochemical Corp.) using plasmid DNAs as templates.

Protein Prenyltransferase Assays

Protein prenyltransferase assays were carried out as described (30). 5 µM ZnCl2 was included in the reaction mixture as described (30). Yeast crude soluble extracts prepared as described (30) or enzymes purified from E. coli (see below) were used for assays. Glutathione S-transferase (GST) fusion proteins, GST-CIIS and GST-CIIL (26), were used as protein substrates. Prenyl substrates were [3H]FPP (22.3 Ci/mmol) and [3H]GGPP (19.3 Ci/mmol) (DuPont NEN). Incubations were carried out using 76 nM FTase at 30 °C for the periods indicated in figure legends.

Purification of Wild Type and Mutant FTases

FTases were purified by fusing the DPR1 with glutathione S-transferase and using glutathione beads. For this purpose, DPR1 was cloned into pGEX vector to produce pGEX-DPR1. Briefly, DPR1 fragment was PCR-amplified to produce a fragment with a BamHI site at the 5' end and a PstI site at the 3' end of the gene. The fragment was cloned into the BamHI and PstI sites of pBluescript II SK. The DPR1 fragment cut out with BamHI and EcoRI was cloned into the BamHI and EcoRI sites of pGEX-1 (36). S159N and Y362L mutant plasmids were constructed by using PCR to introduce each mutation into pGEX-DPR1. A plasmid, pBC-RAM2, was constructed by placing RAM2 into pBC-KS+ (Stratagene) in two steps (37). First, a XbaI-SphI RAM2 fragment was cloned into XbaI-SphI sites of pGEM-3Z (Promega) to create pGEM-RAM2. The 5' XbaI-BglII portion of RAM2 was replaced with the PCR product amplified from the original RAM2 clone (24) using the primers 5'-GCTCTAGACTCGAGGAAAATGGAGGAG-3' and 5'-CGCGCGGAAGATCTGATAGTTCTTTG3'. The XhoI-HindIII fragment of RAM2 from this construct was cloned into the same sites of pBC-KS+ to yield pBC-RAM2. The constructs utilizing PCR products were sequenced to confirm the mutations and to make sure that no additional mutations were introduced.

E. coli strain DH5alpha carrying pGEX-DPR1 and pBC-RAM2 was grown in LB medium containing chloramphenicol and ampicillin to an A600 of 0.6 and then induced with 1 mM isopropyl-1-thio-beta -D-galactopyranoside. The induction was carried out for 12 h at room temperature. The cell pellets were resuspended in 50 mM Tris-HCl, pH 7.4, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 0.2 mg/ml lysozyme. The cells were broken by sonication, and lysates were centrifuged at 12,000 × g for 30 min. Glutathione beads were added, and the solution was rotated for 2 h at 4 °C. FTase was eluted by the addition of 10 mM glutathione and the eluate was concentrated by Centricon 50 (Amicon). Glycerol was added to 50% and the enzymes were kept at -20 °C. Protein concentrations were determined by Bradford assay. This purification yielded homogeneous preparations that contained two bands, 70 and 40 kDa, corresponding to GST-Dpr1 and Ram2 subunits, respectively, on a SDS-polyacrylamide gel. Scanning of the gel showed that the preparations we used were greater than 90% pure.

Site-directed Mutagenesis of Ser-159 and Tyr-362

Site-directed mutagenesis of Ser-159 and Tyr-362 was carried out by PCR using overlap extension (38, 39) with Pfu DNA polymerase (Stratagene). Mutagenesis of Ser-159 was carried out with the flanking primers DPR1#1 and DPR1#4 and the internal, complementary primers 159S and 159A. The primer 159S contains the degenerate sequence NNN, TWC, GAM, or a sequence TGG at codon 159 (N = A, C, G, or T; W = A or T; M = A or C). Amplified fragment was digested with BamHI plus NcoI and was used to replace the corresponding region of pYO324(DPR1)BA. Plasmids from E. coli transformants were sequenced to determine the residue at 159 and to confirm that no other mutations were introduced. The degenerate primer containing NNN yielded 15 amino acid residues other than Tyr, Phe, Glu, Asp, and Trp. The primer containing TWC, GAM, or TGG yielded Tyr and Phe, Glu and Asp, or Trp, respectively. Mutagenesis of Tyr-362 was carried out with the flanking primers DPR1#2 and DPR1#3 and the internal, complementary primers 362S and 362A. The primer 362S contains the degenerate sequence NNN, RAC, WKG (N = A, C, G, or T; R = A or G; W = A or T; K = G or T) or a sequence ATC. The degenerate primer NNN yielded 15 different amino acid residues other than Asn, Gln, Trp, Met, and Ile. The primer RAC yielded Asn and Gln, whereas WKG yielded Trp and Met. The primer ATC yielded Ile. Amplified fragments were digested with NcoI and AflII and were used to replace the corresponding region of pYO324(DPR1)BA. All mutations were confirmed by dideoxy sequencing (U. S. Biochemical Corp.).

Primer Sequences

Sequences are listed below.
<UP>DPR1#1: </UP><UP>5′-ACAACTGCAGGGATCCTAAAGCTATAGG-3′</UP>(<UP>−40 to −13</UP>)
<UP>DPR1#2: </UP><UP>5′-TATGTGGTTAACTTGGAGAACTTAAGTTGG-3′</UP>(<UP>1274–1303</UP>)
<UP>DPR1#3: </UP><UP>5′-ACAGCTAGTCTAGCAATTTTAAGG-3′</UP>(<UP>796–819</UP>)
<UP>DPR1#4: </UP><UP>5′-CTAAAAGCTTTTCAACATTTATTTGG-3′</UP>(<UP>853–828</UP>)
<UP>159S: </UP><UP>5′-TTATCTCATTTGGCC<UNL>NNN</UNL>ACTTATGCTGCA-3′</UP>(<UP>460–489</UP>)
<UP>362S: </UP><UP>5′-GCCCACTCAGACTTT<UNL>NNN</UNL>CATACAAATTATTGCC-3′</UP>(<UP>1068–1101</UP>)
<SC><UP>Sequences 1–6</UP></SC>

Nucleotides that are not complementary to DPR1 are underlined. 159A and 362A are complementary to 159S and 362S, respectively.

Construction of Double Mutants

DPR1 genes encoding mutations at either residues 159 or 362 were randomly joined to produce the double mutant library. Mutant DNAs at residue 159 except for the strong suppressor, Asn, were mixed in equimolar amounts and digested with NcoI and AflII to produce fragments with N-terminal mutations. Equimolar amounts of mutants at residue 362, except for the strong suppressors, Leu, Met, or Ile, were digested with NcoI and AflII to produce C-terminal fragments. The resulting fragments were ligated, and the library of double mutants was transformed into the cal1-1 strain, YOT559-3C. Transformants were grown on SC-trp at 26 °C for 3 days. Approximately 103 colonies were screened. Fast growing colonies were selected and subsequently tested for growth at restrictive temperatures. The plasmids from these clones were recovered, retransformed into cal1-1 to confirm the suppression, and sequenced.


RESULTS

Isolation of Mutant DPR1 Genes That Can Suppress a cal1 Mutation

We have previously developed a screen to identify mutant yeast FTases with increased ability to utilize GGTase I protein substrates (30). The screen is based on a yeast mutant cal1, which is defective in the beta -subunit of GGTase I. Temperature-sensitive growth of this mutant can be suppressed by introducing mutant DPR1 genes, which could function as GGTase I beta . The mutant DPR1 enables the efficient utilization of GGTase I protein substrates, but attaches a farnesyl instead of a geranylgeranyl group to the substrate proteins. Apparently, farnesylated GGTase I substrates are competent in suppressing cal1 phenotypes. After the initial isolation of a mutant Dpr1, S159N (30), we carried out a more extensive screen in an attempt to identify additional mutants. The PCR mutagenesis procedure employed in the previous screen was modified to isolate independent mutations more efficiently (see "Experimental Procedures"). Briefly, N-terminal and C-terminal portions of the DPR1 gene were separately amplified under conditions optimized so as to generate single point mutations in the region of amplification. Two sets of mutated DPR1 gene libraries, one having the N-terminal portion mutated and the other having the C-terminal portion mutated, were constructed separately on a yeast high copy plasmid. These libraries were then transformed into the cal1-1 strain, and transformants capable of growth at the restrictive temperature were selected. The plasmids from such suppressors were recovered and sequenced. Using the N-terminal mutant libraries, we found six separate G right-arrow A transitions at nucleotide 476 (the same mutation as the previously identified mutation; Ref. 30), which causes a Ser-159 right-arrow Asn substitution (designated S159N). Using the C-terminal mutant libraries, we identified four T right-arrow C transitions at 1084, which cause a Tyr right-arrow His substitution at residue 362 (Y362H); and one T right-arrow A transversion at 1096, which causes a Tyr right-arrow Asn substitution at residue 366 (Y366N).

Suppression of cal1 and Complementation of dpr1 by Mutant DPR1

Fig. 1 shows the suppression of the temperature-sensitive growth of the cal1-1 strain by the three mutant DPR1 genes on a high copy plasmid isolated from the screen described above. The mutant DPR1 genes suppressed the temperature sensitivity of the cal1-1 strain to varying degrees; S159N showed the most efficient suppression, Y366N showed suppression comparable to S159N, and Y362H showed the least efficient suppression. The three mutant DPR1 genes were then transferred to a low copy plasmid and were tested for suppression of cal1-1 and complementation of dpr1::URA3, a disruption allele of DPR1. The results are summarized in Table I. For suppression of cal1-1, wild type DPR1 gene on a low copy plasmid further impaired the slow growth of the cal1-1 strain at permissive temperatures, as did wild type DPR1 gene on high copy plasmid (30), while the mutant DPR1 genes suppressed the slow growth of the strain. Y362H appears to suppress less efficiently than S159N and Y366N, since the doubling time of the Y362H transformant was longer than that of the other two transformants. At restrictive temperatures, S159N showed suppression at both 35 °C and 37.5 °C, whereas Y366N showed suppression at 35 °C but not at 37.5 °C. Y362H showed only weak suppression at the restrictive temperatures. The order of the efficiency of suppression for the three mutants is consistent with the differences observed for the high copy plasmid (Fig. 1).


Fig. 1. Suppression of the cal1-1 mutation by mutant DPR1 genes on high copy plasmid. Transformants of the cal1-1 strain YOT559-3C with the vector pYO324 or mutant plasmid isolated from the libraries were streaked onto a YPD plate and incubated at 37.5 °C for 2 days.
[View Larger Version of this Image (41K GIF file)]


Table I.

Suppression of cal1-1 and complementation of dpr1::URA3 by mutant DPR1 on low copy plasmid

Wild type and mutant DPR1 genes on the low copy plasmid pRS314 were used to transform the cal1-1 strain YOT559-3C and the dpr1::URA3 strain YPH250dU.
DPR1 Growth of cal1 transformants
Growth of dpr1 transformants
27 °Ca 35 °Cb 37.5 °Cb 27 °Ca 37 °Cb

Vector +/-  (3.3)  -  -  -/+ (5.0)  -
Wild type  -/+  (4.3)  -  - +  (2.2) +
S159N +   (2.2) + +/- +  (2.2) +
Y362H +c  (2.6)  -  - +  (2.3) +
Y366N +   (2.3) +  - +  (2.2) +

a  Growth at 27 °C was assessed from colony size of transformants on SC-trp plates incubated for 3 days. The numbers shown in parentheses indicate doubling time, in hours, in liquid SC-trp medium at 25 °C, determined by measuring OD660 of log-phase cultures. Values are average for three (cal1) or two (dpr1) independent transformants. Standard deviation is less than 0.1 h.
b  Growth at 35 °C, 37 °C, or 37.5 °C was scored from streaks of transformants on YPD plates incubated for 2 days.
c  On day 2, the colonies were smaller than those of transformants with S159N or Y366N.

For the complementation of dpr1 disruption mutation, the mutant DPR1 genes, like wild type DPR1 gene, complemented dpr1 mutation for both the slow growth at permissive temperatures and the temperature-sensitive growth. The mutant DPR1 genes also complemented the sterile phenotype of the dpr1::URA3 strain (data not shown). The suppression of cal1 and the complementation of dpr1 suggest that S159N, Y362H, and Y366N can act as the beta -subunit for both GGTase I and FTase in vivo.

Site-directed Mutagenesis of Ser-159

As described above, the substitution of Ser-159 of yeast FTase beta  subunit by Asn conferred cal1 suppression. In order to determine what other amino acid substitutions could affect protein substrate specificity, we substituted Ser-159 of Dpr1 with all 20 amino acids and tested each both for the suppression of cal1-1 and for the complementation of dpr1::URA3. The amino acid substitutions were accomplished by using random oligonucleotides at the serine-159 codon. The ability of the mutant Dpr1 proteins to functionally replace the cal1-1 mutant was assessed from growth of transformants at permissive and restrictive temperatures. The impaired growth of the cal1-1 strain by wild type DPR1 at permissive temperature provided a sensitive assay for CAL1 function (see "Experimental Procedures"). Table II summarizes the results obtained. As can be seen, Asn is the only residue that conferred efficient suppression of both the slow growth at permissive temperature and the temperature-sensitive growth of the cal1-1 strain. Besides Asn, only Asp conferred suppression of the temperature-sensitive growth at restrictive temperatures. However, this latter mutant was not as efficient as the Asn mutant. At the permissive temperature, Cys, Thr, Val, and His conferred the growth of transformants at a rate intermediate between that observed with wild type DPR1 and that observed with vector. In other words, these residues partially relieved the impaired growth by wild type DPR1, suggesting that Cys, Thr, Val, or His confers a weak CAL1 activity that is intermediate between that of DPR1 and that of CAL1. This partial relief from the impaired growth is unlikely to be due to instability of the protein or a defect of the protein in the interaction with the alpha  subunit, because these mutants conferred complementation of dpr1. The other residues showed the same phenotypes as wild type DPR1, i.e. impaired growth at permissive temperature and no suppression at restrictive temperatures.

Table II.

Amino acid substitutions at residue 159 

DPR1 genes with amino acid substitutions at residue 159 on the multi-copy plasmid pYO324 were used to transform the cal1-1 strain YOT559-3C and the dpr1 disruption strain YPH250dU. The wild type residue at 159 is serine, and this is indicated in the table. The values for van der Waals volume for each amino acid are from Creighton (50). For the suppression of cal1, -/+, +, and +/- are the same as in Table I. To indicate the degree of suppression, + to ++++ symbols are used. For the complementation of dpr1, growth (+) or slow growth (+/-) is indicated.
Residue van der Waals volume 26 °C
37 °C
cal1 dpr1 cal1 dpr1

Å3
Gly 48  -/+ +  - +
Ala 67  -/+ +  - +
Ser (wt) 73  -/+ +  - +
Cys 86 + +  - +
Pro 90  -/+ +  - +
Asp 91 ++ + +/- +
Thr 93 + +  - +
Asn 96 ++++ + ++++ +
Val 105 + +  - +
Glu 109  -/+ +  - +
Gln 114  -/+ +  - +
His 118 + +  - +
Ile 124  -/+ +  - +
Leu 124  -/+ +  - +
Met 124  -/+ +  - +
Lys 135  -/+ +  - +
Phe 135  -/+ +  - +/-
Tyr 141  -/+ +  - +/-
Arg 148  -/+ +  - +/-
Trp 163  -/+ +  - +/-

The above results on amino acid residues that confer CAL1 activity can best be explained by the physical size of the amino acids. Table II shows correlation of CAL1 function with the size of the residue. It is interesting that Asn and Asp confer CAL1 function, while Gln and Glu do not. This suggests that the physical size of the residue is important rather than chemical properties. In fact, these two residues, as well as Thr, which exhibit weak CAL1 activity, have similar van der Waals volumes (91-96 Å3; Table II).

The mutant protein could contain any amino acid at 159, except Phe, Tyr, Arg, or Trp, and confer complementation of dpr1 for both the slow growth at permissive temperature and the temperature-sensitive growth. Phe, Tyr, Arg, and Trp conferred complementation for the slow growth at permissive temperature but not for the temperature-sensitive growth. We, therefore, conclude that very large residues at amino acid 159 interfere with DPR1 function.

Site-directed Mutagenesis of Tyr-362

We also substituted Tyr-362, one of the tyrosine residues in the C terminus of Dpr1 with all 20 possible amino acids. Results are summarized in Table III. Three amino acid substitutions, Ile, Leu, and Met, exhibited efficient suppression of the cal1 phenotype. Of the three, Leu and Met mutants suppressed better than the Ile mutant. Interestingly, these amino acid residues have the same van der Waals volume (124 Å3; Table III), suggesting that it is the size of the amino acid that is important for conferring CAL1 activity. It is worth noting that these amino acids are hydrophobic amino acids, suggesting that the apolar character of these residues may also be important for substrate recognition of CAAL motif. Weak suppression was obtained with Thr and Val mutants at restrictive temperatures. It is rather surprising that the amino acid size (93-105 Å3; Table III) of these weak suppressors is close to the size of those identified as suppressors in the Ser-159 mutagenesis described above (see "Discussion"). The original mutant, Y362H, suppressed only weakly at 35 °C. No suppression was noted at 37 °C, even when multi-copy plasmids were used (Table III). In addition to Thr and Val, weak suppression by Gly, Cys, Asp, and Asn was seen at the permissive temperature.

Table III.

Amino acid substitutions at residue 362 

DPR1 genes with amino acid substitutions at residue 362 on the multicopy plasmid pYO324 were used to transform the cal1-1 strain YOT559-3C and the dpr1 disruption strain YPH250dU. The wild type residue at 362 is tyrosine, and this is indicated in the table.
Residue van der Waals volume 26 °C
37 °C
cal1 dpr1 cal1 dpr1

Å3
Gly 48 ++ +  - +
Ala 67  -/+ +  - +
Ser 73  -/+ +  - +
Cys 86 ++ +  - +
Pro 90  -/+ +  - +
Asp 91 ++ +  - +
Thr 93 +++ + + +
Asn 96 ++ +  - +
Val 105 +++ + + +
Glu 109  -/+ +  - +
Gln 114  -/+ +  - +
His 118 +++ +  - +
Ile 124 ++++ + +++ +
Leu 124 ++++ + ++++ +
Met 124 ++++ + ++++ +
Lys 135  -/+ +  -  -/+
Phe 135  -/+ +  - +
Tyr (wt) 141  -/+ +  - +
Arg 148  -/+ +  -  -/+
Trp 163  -/+ +  - +

Fig. 2 compares the effect of some of the Tyr-362 substitution mutants with the S159N mutant on the doubling time of the cal1-1 mutant. As can be seen, the Y362M mutant grew at a rate slightly faster than that seen with the S159N transformant. Y362L and Y362I transformants exhibited growth rates less than that seen with the S159N transformant. A weak suppressor Y362H clearly grew better than the vector control, but its growth rate was approximately 3-fold slower than that observed with the S159N mutant.


Fig. 2. Growth of cal1 mutant cells transformed with a variety of DPR1 mutants. The cal1-1 strain YOT559-3C was transformed with a vector and a variety of mutant DPR1 plasmids indicated in the figure. The transformants were grown at 37 °C in SC-trp media and the growth was followed by OD600. The figure shows the inverse of the doubling time.
[View Larger Version of this Image (15K GIF file)]


We next tested whether these Tyr-362 substitution mutants were capable of complementing dpr1 mutation. As shown in Table III, Arg and Lys mutants were the only ones that did not complement the temperature-sensitive growth of the dpr1 mutant. We conclude that the basic property of these amino acids may interfere with the DPR1 function.

Genetic Evidence for the Cooperation between Residues 159 and 362

To gain insights into possible interaction between residues 159 and 362, we attempted to obtain double mutants having alterations of both serine 159 and tyrosine 362. A library of DPR1 containing mutations at both these residues was constructed by combining the mutant DPR1 genes identified above (see "Experimental Procedures"). Strong suppressors (S159N, Y362L, Y362M, and Y362I) were excluded from the library. The library was transformed into the cal1-1 strain, and transformants capable of suppressing cal1 were identified by their growth at restrictive temperatures. This study led to the identification of a double mutant, which exhibited significant cal1 suppression. As shown in Fig. 2, the double mutant (DM) grew at a rate comparable with that of Y362L. The double mutant contained S159D and Y362N mutations. S159D was observed to be a weak suppressor at restrictive temperatures, and Y362N was shown to exhibit weak suppression only at the permissive temperature. Thus, combining weak suppressors will produce additive effects, suggesting that the two residues 159 and 362 cooperate to recognize the CAAL motif.

Characterization of Mutant FTases

Suppression of the cal1-1 mutation by the mutant DPR1 genes described above suggests that mutant FTases farnesylate or geranylgeranylate GGTase I substrates in vivo. Crude soluble extracts, prepared from cal1-1 transformants carrying wild type or mutant DPR1 genes on a low copy plasmid, were tested for enzymatic activity to farnesylate or geranylgeranylate GST-CIIL protein, a preferred substrate for GGTase I (26). FTase activity from cal1 transformants expressing the mutant beta  subunit S159N, Y362H, or Y366N showed an increased ability to farnesylate the GST-CIIL protein compared with wild type FTase, but showed little activity to geranylgeranylate the GST-CIIL protein (data not shown). These in vitro results suggest that the mutant FTases have altered specificity for protein substrates but not for prenyl substrates and that the suppression is due to the ability of the mutant enzymes to farnesylate GGTase I substrates in vivo.

To further characterize the mutant enzymes, we purified the wild type and two mutant enzymes, S159N and Y362L. To accomplish this, we fused the DPR1 gene with glutathione S-transferase. This fusion construct was transformed into E. coli cells together with a RAM2 construct. Affinity purification with glutathione beads yielded homogeneous preparations (see "Experimental Procedures"). Fig. 3 shows time course of the incorporation of farnesyl or geranylgeranyl radioactivity into FTase substrate, GST-CIIS and into GGTase I substrate, GST-CIIL. As can be seen in Fig. 3 (left panel), the wild type FTase enzyme incorporated farnesyl radioactivity into GST-CIIS approximately 4 times better than that into GST-CIIL. In contrast, both mutant enzymes, S159N and Y362L, incorporated farnesyl radioactivity into GST-CIIS and GST-CIIL with comparable efficiency. Virtually no incorporation of radioactive geranylgeranyl was observed with either GST-CIIS and GST-CIIL in the case of the wild type and S159N enzymes. In the case of Y362L mutant, low but reproducible incorporation of radioactive geranylgeranyl was observed with the GST-CIIS substrate. Thus, both the S159N and Y362L mutants have increased ability to farnesylate the GGTase I substrate. Similar conclusions can be drawn from the results of experiments altering substrate protein concentration (Fig. 4). Kinetic parameters, Km and kcat, are summarized in Table IV. These values were obtained from Lineweaver-Burke plots of the wild type and mutant enzymes carried out in the presence of saturating amounts of farnesyl diphosphate. The wild type enzyme has a Km for GST-CIIS that is approximately 6-fold lower than the Km for GST-CIIL. kcat for CIIS is approximately 10-fold larger than that for GST-CIIL. Because of these differences, kcat/Km value for GST-CIIS is approximately 50-fold higher than the value for GST-CIIL. Thus, the wild type enzyme prefers GST-CIIS over GST-CIIL. In contrast, the mutant S159N shows only a 1.5-fold difference in the kcat/Km values between GST-CIIS and GST-CIIL. In the case of the Y362L mutant, similar kcat/Km values were obtained for the two protein substrates. This suggests that the mutant enzymes farnesylate GST-CIIS and GST-CIIL substrates with an equivalent efficiency.


Fig. 3. Time course of FPP and GGPP incorporation into GST-CIIS and GST-CIIL substrates using wild type, S159N and Y362L mutant enzymes. The wild type, S159N and Y362L mutant enzymes were prepared as described in Experimental Procedures. Prenyltransferase assays were carried out at 30 °C with protein substrate concentrations (GST-CIIS or GST-CIIL) of 4 µM, prenyl diphosphate concentrations (FPP or GGPP) of 1 µM, and enzyme concentrations of 76 nM as described in Experimental Procedures. Aliquots were removed at the time intervals indicated and the incorporation of radioactivity into substrate proteins were examined.
[View Larger Version of this Image (19K GIF file)]



Fig. 4. Incorporation of farnesyl and geranylgeranyl radioactivity as a function of protein substrate concentrations. The wild type, S159N and Y362L enzymes were purified as described in Experimental Procedures and the prenyltransferase assays were carried out at 30 °C for 10 min with varying concentrations of GST-CIIS or GST-CIIL, 1 µM of prenyl diphosphates (FPP or GGPP), and 76 nM of wild type or mutant enzymes as described in Materials and Methods.
[View Larger Version of this Image (19K GIF file)]


Table IV.

Kinetic parameters of the wild type and mutant FTases

Km and kcat values were obtained from Lineweaver-Burk plots of the wild type and mutant enzymes carried out in the presence of 1 µM farnesyl diphosphate and 76 nM wild type or mutant FTase enzymes. These experiments were repeated two to three times with similar results.
FTase Km
kcat
kcat/Km
CIIS CIIL CIIS CIIL CIIS CIIL

nM s-1 M-1 s-1
Wild type 250  ± 30 1440  ± 150 0.112  ± 0.01 0.012  ± 0.001 4.5  × 105 8.0  × 103
S159N 690  ± 60 430  ± 30 0.093  ± 0.008 0.088  ± 0.006 1.4  × 105 2.1  × 105
Y362L 340  ± 70 180  ± 40 0.049  ± 0.01 0.028  ± 0.006 1.4  × 105 1.6  × 105


DISCUSSION

FTase recognizes the CAAX motif, whereas GGTase I recognizes the CAAL motif (3, 4, 5, 6). This suggests that the protein substrate binding pockets of these two enzymes have similar but distinct conformations to allow their specificity differences. In the current report, we show that the conversion of the FTase type recognition to the GGTase I type recognition can be accomplished by a single amino acid change in one of three different residues. These are Ser-159, Tyr-362, and Tyr-366 of yeast FTase beta  subunit, Dpr1 protein. We believe that these residues represent major contributors for the CAAX recognition, since the number of mutations that can be isolated from the screen is likely to be close to saturation because independent libraries yielded the same mutations. As schematized in Fig. 5A, these results suggest that two regions of Dpr1 (one surrounding Ser-159 and the other encompassing Tyr-362 and Tyr-366) are involved in the interaction with the protein substrate. It is possible that these residues are in close proximity to each other in a three-dimensional conformation and form a binding pocket for the protein substrate (see below).


Fig. 5. Amino acid sequence alignment of FTase and GGTase I beta  subunits. A. Positions of residues Ser-159, Tyr-362, and Tyr-366 in Dpr1 protein are shown. B. Sequences aligned are the beta  subunits of S. cerevisiae FTase (44), human FTase (14), rat FTase (28), pea FTase (46), S. cerevisiae GGTase I (14), human GGTase I (48), rat GGTase I (47), Drosophila melanogaster GGTase I (48) and Schizosaccharomyces pombe GGTase I (49). Sequence alignment was carried out with the CLUSTAL program of PC/GENE (IntelliGenetics). Residues corresponding to Ser-159, Tyr-362, and Tyr-366 of the S. cerevisiae FTase beta  subunit are in bold.
[View Larger Version of this Image (49K GIF file)]


Site-directed mutagenesis of Ser-159 and Tyr-362 revealed that size rather than chemical properties of the residue is important for the conversion of FTase substrate binding to GGTase I substrate binding. For suppression of cal1-1, Asn is the only residue that conferred efficient suppression at residue-159. Asp, which is different from Asn in chemical properties but is similar in size, conferred weak suppression. In contrast, Gln, which is chemically similar to but is larger than Asn, showed no suppression. Furthermore, residues that conferred very weak CAL1 function, Thr, Cys, and Val, have similar size relative to Asn despite differences in chemical properties. Thus, size appears more important than chemical properties at residue 159 for function as CAL1. A similar interpretation could be made for the results obtained with the site-directed mutagenesis of Tyr-362. In this case, the three mutants that were capable of suppressing cal1 defect contained amino acids Leu, Met, or Ile, which have identical van der Waals volume. Thus, introducing a distinct size amino acid at either 159 or 362 converts the substrate specificity of FTase to that of GGTase I. Presumably, the introduction of a particular size amino acid alters the structure of the substrate binding site. It is worth noting that tyrosine (wild type residue) has a van der Waals volume considerably larger than that of leucine. This argues against the idea that the conversion of FTase to GGTase I involves the introduction of a larger size amino acid in order to open up a pocket. Rather, a precise change is required to accomplish the conversion. In addition, it is intriguing that the three amino acids (Met, Leu, and Ile) detected at residue 362 are all hydrophobic amino acids. One interpretation is that replacement of Tyr at this residue with a smaller aliphatic residue could introduce a nice apolar binding pocket for the Leu side chain of the CIIL peptide.

We have obtained genetic evidence for the cooperativity between the residues 159 and 362. A single mutant S159D or Y362N only weakly suppresses cal1 mutant. However, the double mutant S159D/Y362N exhibits efficient suppression (Fig. 2). One possibility for this additive effect of the alterations at residues 159 and 362 of FTase is that these residues are proximally located in a three-dimensional structure. This proposal is further supported by the detection of weak suppressors of the cal1 phenotype. Weak suppression was accomplished by the conversion of Tyr-362 to Val or Thr. These residues have van der Waals volumes (91-105 Å3) similar to those amino acids that have been detected at Ser-159 to suppress cal1 mutation. Thus, introduction of a certain amino acids of approximately the same size at either 159 or 362 may result in conversion of an FTase pocket to a GGTase I pocket. A possible interpretation of these results is that the residues 159 and 362 are located in the same substrate pocket.

Biochemical analyses of the mutant proteins S159N and Y362L demonstrate that these proteins have increased affinity for protein substrates with the CAAL motif. The wild type enzyme shows approximately 50-fold higher kcat/Km value for the FTase substrate compared with the GGTase I substrate. In the case of the mutant enzymes, while kcat/Km values for the FTase substrates decrease, kcat/Km values for the GGTase I substrates are now similar to those observed for the FTase substrates. These results could explain the ability of the mutants to suppress cal1 phenotypes, since FTase and GGTase I substrates are modified with equivalent efficiency. In addition, in the case of the Y362L mutant, we observed a low but significant incorporation of radioactive geranylgeranyl into GST-CIIS protein. Thus, this latter mutant can also incorporate radioactive geranylgeranyl. Further experiments with other protein substrates are needed to assess the significance of the geranylgeranylation brought about by this mutant enzyme.

Our results also show that the residues 159 and 362 can be changed to other amino acids without losing the ability to function as a beta -subunit of FTase. Of the 20 amino acids tested for Ser-159, all except Phe, Tyr, Arg, and Trp conferred complementation of dpr1 for temperature sensitivity. These four residues, which did not confer complementation, are chemically different but similar in having large van der Waals volumes. The only amino acids that did not confer complementation in the Tyr-362 replacement studies were Arg and Lys. In this case, the introduction of basic residues appears to interfere with the DPR1 function.

A survey of sequences surrounding the residues 159 and 362 among FTases from a variety of sources shows that many amino acids are conserved (Fig. 5B). Tyrosine 362 is also conserved in FTases. The sequences in the corresponding regions of GGTase I, however, are different from these conserved FTase sequences. In particular, regions around residue 362 are interesting. These regions are conserved among FTase and GGTase I better than the regions around Ser-159. Some residues (e.g. Gly-350, Asp-360, and His-363) are conserved among FTase and GGTase I, and others (e.g. Leu-351, Asp-353, and Cys-367) are not conserved between FTase and GGTase I (Fig. 5B). Given the distinct but closely related specificities for protein substrates of FTase and GGTase I, this region is likely to play a significant role in protein substrate recognition and/or binding.

FTase and GGTase I are zinc metalloenzymes; both require zinc for activity (7, 40) and contain a single zinc atom per molecule (41, 42). The observation that zinc is required for the transfer of a prenyl group to protein substrates suggests a role of zinc in the recognition of protein substrates. Recently, Zhang et al. (43) showed that replacing Zn2+ to Cd2+ in FTase results in the acquisition of the ability to recognize GGTase I protein substrate. This suggests that the protein substrate specificity of FTase can be altered not only by introducing mutations in the enzyme but also by metal ion replacements. However, the exact mechanisms by how these manipulations affect substrate specificity may not be related, since our assays are carried out in the presence of ZnCl2.

In conclusion, our results provide insights into the recognition of protein substrates by FTase. Successful conversion of the FTase recognition to GGTase I recognition by the introduction of a particular size amino acid points to the remarkable versatility of protein prenyltransferases for their substrate recognition. Our data on yeast FTase described here provide valuable information for the understanding of the structure and function of FTase.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant CA41996. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    The first two authors contributed equally to this work.
§   Present address: National Institute of Genetics, Mishima 411, Japan.
   Present address: Hans Knoll Institute, D-07745 Jena, Germany.
par    To whom correspondence should be addressed: Dept. of Microbiology and Molecular Genetics, UCLA, 405 Hilgard Ave., Los Angeles, CA 90095-1489. Tel.: 310-206-7318; Fax: 310-206-5231.
1    The abbreviations used are: FTase, protein farnesyltransferase; FPP, farnesyl diphosphate; GGTase I, geranylgeranyltransferase type I; GGPP, geranylgeranyl diphosphate; GST, glutathione S-transferase; PCR, polymerase chain reaction; kb, kilobase pair(s).

Acknowledgments

We thank Drs. Peter Edwards, David Sigman, and James Bowie for critical reading of the manuscript, and Jun Urano and Dr. Patrick Poullet for valuable comments. We also thank Kenneth Esson for DNA sequencing.


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