Amino Acid Residues That Define Both the Isoprenoid and CAAX Preferences of the Saccharomyces cerevisiae Protein Farnesyltransferase
CREATING THE PERFECT FARNESYLTRANSFERASE*

Brian Erich CaplinDagger §, Yoshikazu Ohya, and Mark S. MarshallDagger §par **

From the Dagger  Walther Oncology Institute, Indianapolis, the Departments of § Biochemistry and Molecular Biology and par  Medicine, Division of Hematology and Oncology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5121, and the  Department of Biology, Faculty of Science, University of Tokyo, Hongo, Tokyo 113, Japan

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Studies of the yeast protein farnesyltransferase (FTase) have shown that the enzyme preferentially farnesylates proteins ending in CAAX (C = cysteine, A = aliphatic residue, X = cysteine, serine, methionine, alanine) and to a lesser degree CAAL. Furthermore, like the type I protein geranylgeranyltransferase (GGTase-I), FTase can also geranylgeranylate methionine- and leucine-ending substrates both in vitro and in vivo. Substrate overlap of FTase and GGTase I has not been determined to be biologically significant. In this study, specific residues that influence the substrate preferences of FTase have been identified using site-directed mutagenesis. Three of the mutations altered the substrate preferences of the wild type enzyme significantly. The ram1p-74D FTase farnesylated only Ras-CIIS and not Ras-CII(M,L), and it geranylgeranylated all three substrates as well or better than wild type. The ram1p-206DDLF FTase farnesylated Ras-CII(S,M,L) at wild type levels but could no longer geranylgeranylate the Ras-CII(M,L) substrates. The ram1p-351FSKN FTase farnesylated Ras-CIIS and Ras-CIIM but not Ras-CIIL. The ram1p-351FSKN FTase was not capable of geranylgeranylating the Ras-CII(M,L) substrates, giving this mutant the attributes of the dogmatic FTase that only farnesylates non-leucine-ending CAAX substrates and does not geranylgeranylate any substrate. These results suggest that the isoprenoid and protein substrate specificities of FTase are interrelated. The availability of a mutant FTase that lacked substrate overlap with the protein GGTase-I made possible an analysis of the role of substrate overlap in normal cellular processes of yeast, such as mating and growth at elevated temperatures. Our findings suggest that neither farnesylation of leucine-ending CAAX substrates nor geranylgeranylation by the FTase is necessary for these cellular processes.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

One recently discovered protein post-translational modification in eukaryotic organisms is protein isoprenylation (1, 2). Protein isoprenylation is the first in a series of post-translational modifications which serve to increase the hydrophobicity of the modified proteins, producing membrane association for proteins that otherwise lack membrane binding structures (3, 4). Prenylated proteins are modified by either a 15-carbon isoprenoid, farnesyl, or a 20-carbon isoprenoid, geranylgeranyl. The carboxyl-terminal amino acid sequence of the modified proteins serves to direct the addition of either of these isoprenoids.

Three general classes of COOH-terminal sequences serve as substrates for the protein prenyltransferases. The first class comprises those that conform to the consensus sequence -CAAX, where C is cysteine, a is an aliphatic amino acid, and X is Ala, Cys, Glu, Met, Ser, or Val (5). Proteins ending in -CAAX sequences which are modified with farnesyl include the mammalian H-Ras (CVLS), K-Ras (CVIM), and lamin B (CAIM) well as the yeast a-factor (CVIA), Ras1p (CIIC), Ras2p (CIIS), and Rho4p (CIIM) proteins. The second class is that of proteins that terminate in the consensus sequence CAAL, which includes such proteins as the yeast Rho2p (CIIL), Rsr1p (CTIL), and Rho1p (CVLL) proteins. The third class of COOH-terminal motifs includes the sequence -CC or -CXC found in the Rab family GTPases, which are double geranylgeranylated. Two additional modifications follow the isoprenylation of -CAAX and -CAAL proteins. The COOH-terminal tripeptide is endoproteolyzed, and the exposed isoprenyl-cysteine is carboxylmethylated. Isoprenylation and the subsequent carboxylmethylation serve to increase the hydrophobicity of the modified protein, allowing it to associate physically with intracellular membranes (4). Protein prenylation is catalyzed by at least three distinct protein prenyltransferases: protein farnesyltransferase (FTase),1 the type I protein geranylgeranyltransferase (GGTase-I), and the type II protein geranylgeranyltransferase (GGTase-II) (5-8, 37). GGTase-II transfers geranylgeranyl pyrophosphate to protein substrates that have carboxyl-terminal sequences of -CXC or -CC. FTase and GGTase-I transfer either farnesyl diphosphate (Fpp) or geranylgeranyl diphosphate (GGpp) to protein substrates with carboxyl-terminal -CAAX and -CAAL sequences, respectively. FTase and GGTase-I have been identified in organisms as diverse as yeast (9-12), rat (6, 8, 13, 14, 37), and man (15). FTase and GGTase-I are heterodimeric enzymes containing a common alpha  subunit and highly related beta  subunits (11). The conservation of enzyme structure and substrate preferences of these enzymes suggests that they share a common catalytic mechanism. Kinetic studies indicate that both FTase and GGTase-I preferentially bind isoprenoid before binding the -CAAX or -CAAL protein substrate (16, 17). Furthermore, both enzymes catalyze product formation from ternary complex (isoprenoid substrate, protein -CAAX or -CAAL substrate, and enzyme) faster than the release of isoprenoid substrate (17, 18), suggesting the conservation of catalytic mechanism of the two enzymes.

On the basis of their similar subunit composition, the tertiary structures of FTase and GGTase-I are likely to be quite high. In addition to their identical alpha  subunits, the beta  subunits of FTase and GGTase-I share approximately 35% amino acid sequence identity. Perhaps not surprisingly, these enzymes have measurable isoprenoid and protein substrate overlap in vitro and in vivo (19-21). Substrate overlap between FTase and GGTase-I is particularly evident with substrate proteins that terminate in CIIM; these substrates are geranylgeranylated by both FTase and GGTase-I (20). FTase can farnesylate protein substrates ending in the sequence CIIL, and GGTase-I farnesylates the yeast RhoB, which has a carboxyl-terminal CKVL sequence (21). Yeast FTase and GGTase-I enzymes also show substrate overlap in vivo, where Ras proteins in yeast that lacks FTase are farnesylated by GGTase-I, and yeast that lacks GGTase-I has a faster growth rate when FTase is overexpressed (19).

Mutational analysis of both the alpha  and beta  subunits of FTase indicates that each subunit plays a role in enzymatic catalysis (22-24). The alpha  subunit is important for stabilization of the heterodimer and may also directly affect the catalytic reaction (25). Mutagenesis of a Zn2+-coordinating Cys residue in the beta  subunit of FTase indicates a role for this subunit in isoprenoid binding and a direct role in enzymatic catalysis (26). Photoaffinity cross-linking of peptides to FTase indicates the presence of an intersubunit location for the catalytic active site (27, 28). Further studies of the FTase beta  subunit indicate that distinct NH2-terminal and COOH-terminal regions of this subunit are involved in determining the -CAAX specificity of this enzyme and that single site mutations are sufficient to produce a mutant FTase that has an increased affinity for -CAAL substrates (29).

The results from these and other studies suggest that the FTase beta  subunit determines both isoprenoid and protein substrate specificities. This is supported logically by the fact that their beta  subunits are the only difference between the FTase and GGTase-I enzymes. For this study it was hypothesized that regions highly conserved among eukaryotic FTase beta  subunits that differed from the known GGTase-I beta  subunits contained the residues that determined a preference for farnesyl pyrophosphate and CAA(A,S,C,M) over CAAL. Seven such regions and individual residues were identified in the yeast FTase beta  subunit which met these criteria. Each site was changed from the yeast FTase beta  subunit sequence to the corresponding yeast GGTase-I beta  subunit amino acid sequence. Mutations affecting residues 74, 206-212, and 351-354 in the beta  subunit altered -CAAX and isoprenoid specificity upon reconstitution with the alpha  subunit. The ram1p-351FSKN FTase was surprising in that rather than behaving more like the GGTase-I, it was found to behave as a dogmatic FTase: able to farnesylate only non-leucine-ending -CAAX proteins and unable to geranylgeranylate any substrate tested. Expression and analysis of the ram1p-351FSKN mutant enzyme in ram1 Saccharomyces cerevisiae yeast lacking the wild type beta  subunit of the FTase strongly suggested that the loose substrate specificity observed for FTase in vitro and in vivo does not play a critical physiological function in S. cerevisiae.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

[3H]Fpp (15 Ci/mmol) and [3H]GGpp (15 Ci/mmol) were purchased from American Radiolabeled Chemicals. All other chemical reagents were from U. S. Biochemical Corp. and Sigma. Restriction endonucleases were from New England Biolabs. GF/F filters were direct from Whatman. BioSafe II scintillation mixture was from Research Products International. Protein assay reagents for the Bradford and BCA assays were purchased from Bio-Rad and Pierce, respectively. HyperfilmMP was purchased from Amersham. DNA sequencing was performed with Sequenase version 2.0 from U. S. Biochemical Corp. YL1/2 antibody was purified from hybridoma cells (30). Construction of Ras1-CAAX mutants and Ras protein expression and purification are described elsewhere (5). Ras1-CAAX will be referred to as Ras followed by the specific CAAX sequence, for example Ras-CIIS. The S. cerevisiae strains used were YO2A (MATa RAM1 ade2 lys2 leu2 trp1 his3 ura3), YO4D(MATalpha RAM1 ade2 lys2 leu2 trp1 his3 ura3), YO3C (MATa ram1:LEU2 ade2 lys2 leu2 trp1 his3 ura3), and YO5C (MATa ram1:LEU2 ade2 lys2 leu2 trp1 his3 ura3), PT1 (MATalpha ).

Site-directed Mutagenesis of the RAM1 Gene-- Specific amino acid changes in the RAM1 protein were made in pMM101 (pRAM1-EEF) as described (30). The mutations were confirmed by DNA sequencing. The oligonucleotides used are as follows, named according to the amino acid changes produced in the RAM1 protein: R57HV60FL61FS63RV64H (5'-ATCATCGTATATCTCTAAGTGACGTTGGAAAAATTTGTAATGTGCTTCTGTAGTGTCGGT-3'); I74D (5'-GAGAGCAGGTTCATCATTCTTTTCATCATC-3'); G206DV208DT210L G212F (5'-GCTCAAGGCACAGTATATAAATCTTAGATGTCTTCATCGACTTCTAAACAGGTCTT-3'); G308T (5'-AAAACTATAGCAAGTGTCAACAAGTTTGTT-3'); L351FR352 SD353KK354N (5'-TGAGTGGGCCCCTGGATTCTTACTAAAACCAGGTTGCTCTTTTTC-3'); C367G (5'-CAGTCCTAATAGGCCATAATTTGTATGGTA-3'); S385K (5'-CTTAATATTATGTGGTTTATCATTAGGAGT-3'). Mutations will be referred to by the amino acid changes produced in the Ram1 protein, for example the R57HV60FL61FS63RV64H changes will be referred to as Ram1p-57HFFRH. All RAM1 mutagenic oligonucleotides were synthesized by an Oligo 1000 DNA Synthesizer from Beckman Instruments Inc.

Construction of RAM1 Yeast Expression Vectors-- The yeast shuttle vector YCP50-ADH containing a 5'-EcoRI cloning site and a 3'-XbaI cloning site was designed to express the RAM1, RAM1-351FSKN, and RAM1-74D genes with a constitutive alcohol dehydrogenase promoter. Plasmids YCP50-ADH-RAM1, YCP50-ADH-RAM1-351FSKN, and YCP50-ADH-RAM1-74D were produced by subcloning EcoRI/XbaI fragments of the appropriate vectors according to the method of Maniatis et al. (38). The identity of the vectors was confirmed by restriction endonuclease digestion with EcoRI and XbaI enzymes and dideoxy sequencing (U. S. Biochemical Corp., Sequenase version 2.0).

Expression of Prenyltransferases in Escherichia coli-- Coexpression of each protein prenyltransferase was accomplished by the cotransformation of E. coli strain DH5alpha with 0.1 µg each of pBH57 (pRAM2) and pMLM4 (pCDC43), pMM101-3 (pRAM1), pBC27-3 (pRAM1-57HFFRH), pBC22-81 (pRAM1-74D), pBC26-13 (pRAM1-206DDLF), pBC25-7 (pRAM1-308T), pBC30-5 (pRAM1-351FSKN), pBC21-41 (pRAM1-367G), or pBC24-8 (pRAM1-385K)(32) and selection on Luria Broth plates containing 100 mg/ml ampicillin and 10 mg/ml chloramphenicol. Recombinant FTase and GGTase-I enzyme expression in E. coli and cell lysate preparation was performed as described previously (20, 31, 33).

Isoprenylation of Ras-CAAX Substrates with Crude Bacterial Lysates and Purified Enzymes-- Prenyltransferase assays were carried out and analyzed as described previously (20, 31, 33). Kinetic studies were performed with purified wild type and ram1p-351FSKN FTases using 0.5-20 µM Ras-CII(S,M) or 1.0-38 µM Ras-CIIL for protein substrates and 0.021-20 mM [3H]-Fpp or [3H]-GGpp. In a standard FTase assay, the reaction was terminated after 10 min by the addition of 0.9 ml of 1 M HCl in ethanol. After protein precipitation for 15 min at room temperature, samples were slow filtered over pre-wet Whatman GF/F filters and washed four times with 2-ml volumes of 100% ethanol. Filters were air dried and scintillation counted in 5 ml of BioSafe II liquid scintillation mixture.

Time course assays for both the wild type and ram1p-351FSKN FTases with each substrate combination were performed to determine the appropriate incubation times necessary to acquire initial rate values. Kinetic data were collected from at least four separate experiments for each substrate. Steady-state kinetic parameters were determined by fit to an ordered Bi-Bi steady-state reaction mechanism using GraFit version 3.02 (Erithacus Software, Inc., 1996). Equation 1 is for such a model (34).
   <FR><NU>&ngr;</NU><DE>V<SUB><UP>max</UP></SUB></DE></FR>=<FR><NU>[<UP>Isoprenoid</UP>] · [<UP>C</UP><IT>AA</IT><UP>X</UP>]</NU><DE><AR><R><C>K<SUB>d(<UP>isoprenoid</UP>)</SUB> · K<SUB>m(<UP>C</UP><IT>AA</IT>X)</SUB>+K<SUB>m(<UP>C</UP><IT>AA</IT>X)</SUB> · [<UP>isoprenoid</UP>]</C></R><R><C> <UP>+</UP> K<SUB>m(<UP>isoprenoid</UP>)</SUB> · [<UP>C</UP><IT>AA</IT>X]+[<UP>isoprenoid</UP>] · [<UP>C</UP><IT>AA</IT>X]</C></R></AR></DE></FR> (Eq. 1)

Purification of Wild Type FTase and ram1p-351FSKN FTase-- DH5alpha cells cotransformed with the RAM2 and either RAM1 or RAM1-351FSKN genes were used to inoculate 1-liter volumes of Luria Broth with 100 mg/ml ampicillin and 10 mg/ml chloramphenicol. Protein expression and cell pellets were obtained according to the protocol outlined above. Cell pellets were resuspended into 10 ml of purification lysis buffer (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 4 mM dithiothreitol, 20 µM ZnCl2, 0.2 mM phenylmethylsulfonyl fluoride, 0.1 mM leupeptin, 0.1 µM pepstatin A) and lysed by sonication using five 30-s pulses with a Fisher model 300 sonicator at 35% power. Lysates were cleared by centrifugation at 14,000 × g at 4 °C. Cleared lysates were loaded onto three sequential 5-ml Econo Q columns (Bio-Rad) at a flow rate of 0.5 ml/min. The columns were washed with TDZ buffer (20 mM Tris-HCl, pH 7.5, 4 mM dithiothreitol, and 20 µM ZnCl2). The column was eluted with a 10% step gradient from 0 to 100% TDZN buffer (TDZ buffer plus 1 M NaCl). Prenyltransferase activity eluted in the 200-500 mM NaCl fractions. The active fractions were pooled and concentrated in Amicon Centriprep 10 concentrators. Wild type FTase and ram1p-351FSKN FTase enzyme purification was confirmed by SDS-polyacrylamide gel electrophoresis, Coomassie Brilliant Blue (U. S. Biochemical Corp.) staining, and Bio-Rad Bradford protein analysis.

The wild type FTase and the ram1p-351FSKN FTase enzymes prepared by Econo Q purification were prepared for Glu-Glu-Phe immunoaffinity purification adapted from Refs. 12 and 35. Concentrated active fractions from Econo Q columns were diluted with TDZE (TDZ + 0.1 mM EDTA) to bring the final salt concentration to 100 mM NaCl. Protease inhibitors were added (0.2 mM phenylmethylsulfonyl fluoride, 0.1 mM leupeptin, 0.1 µM pepstatin A) (YL1/2 loading buffer). The active fraction was loaded onto a Gamma Bind Plus/YL1/2 antibody column at a flow rate of 0.05 ml/min in a continuous loop overnight at 4 °C. The preparation of this column has been described elsewhere (12). The column was washed with 10 volumes of TDZE at a flow rate of 0.1 ml/min. Column elution was achieved with YL1/2 elution buffer (TDZ + 5 mM Asp-Phe dipeptide; Sigma) at 0.1 ml/min; fractions were 0.5-ml volumes. The active fractions were assayed by the method of Bradford (Bio-Rad) for protein content and by filter binding prenyltransferase assays for activity. Active fractions were dialyzed at 4 °C, in 250 ml of 50 mM Na-HEPES, pH 7.5, 20 mM MgCl2, 20 µM ZnCl2, 2 mM dithiothreitol, and 15% glycerol. The dialysate was separated into 0.05-ml aliquots and flash frozen in N2 (liquid) and stored at -80 °C until ready for use. The presence of prenyltransferase was confirmed by 10% SDS-polyacrylamide gel electrophoresis and a combination of Coomassie Brilliant Blue staining and Western blot analysis with YL1/2 (anti-tubulin (EEF)) primary antibody and Amersham horseradish peroxidase (anti-rat) secondary antibody. The presence of immunoreactive bands was determined by ECL detection (Amersham). Purified enzymes were used for Michaelis-Menten kinetic analysis as described above.

Yeast Cell Growth Curves and Mating Assays-- Yeast cells YO2A, YO4D, YO3C, and YO5C were transformed by the lithium acetate method (CLONTECH). YO3C and YO5C cells are each transformed with pMJ13 (YCP50-ADH), pBC31 (YCP50-ADH-RAM1-351FSKN), pBC32 (YCP50-ADH-RAM1), or pBC33 (YCP50-ADH-RAM1-74D). Transformants were selected on SD-Ura plates at 30 °C to produce strains BC3CY (YCP50-ADH, MATa), BC3CY31 (YCP50-ADH-RAM1-351FSKN, MATa), BC3CY32 (YCP50-ADH-RAM1, MATa), BC3CY33 (YCP50-ADH-RAM1-74D, MATa), BC5CY (YCP50-ADH, MATalpha ), BC5CY31 (YCP50-ADH-RAM1-351FSKN, MATalpha ), BC5CY32 (YCP50-ADH-RAM1, MATalpha ), BC5CY33 (YCP50-ADH-RAM1-74D, MATalpha ). Transformants were grown in 5 ml of SD-Ura selective medium overnight at 30 °C, 200 rpm. The cell number was determined by counting with a hemacytometer. These overnight cultures were used to inoculate cultures for growth curves. 1 × 106 cells of each transformant were seeded in 25 ml of SD-Ura broth. Cultures were grown at either 28 or 35 °C, 300 rpm, and 0.8-ml samples were collected. Cell growth was determined by measuring A600 at indicated time points. The cell number was determined by hemacytometry and was found to be linear with respect to optical density.

Mating assays consisted of BC3CY, BC3CY31, BC3CY32, and BC3CY33 cells (MATa) each mated to PT1 cells (MATalpha ). Briefly, an equal number of MATa cells, which had been sonicated to separate individual cells, was mixed with a 10-fold excess of MATalpha cells and filtered onto individual nitrocellulose filters. MATa and MATalpha cells on filters were incubated on YPD plates for 4 h at 30 °C. After incubation cells were diluted in minimal media and sonicated. Mated cells were plated onto minimal agar plates. Revertant controls were subjected to the same conditions as the mated cells, and the cell number control experiments were plated onto minimal agar plates with nutrients adenine (20 mg/ml), lysine (30 mg/ml), tryptophan (20 mg/ml), and histidine (20 mg/ml). A detailed description of yeast mating assays can be found in Ref. 39.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Identification of Amino Acid Residues Conserved among Eukaryotic FTase beta  Subunits but Not Conserved with the beta  Subunits of the GGTase-I-- The beta  subunits of FTase and GGTase-I sequences were aligned using the CLUSTAL version 1.6 multiple sequence alignment program (Baylor College of Medicine, Houston). A visual search strategy was employed to identify amino acid residues potentially important for determining the unique substrate preferences of the two enzymes. Briefly, amino acid residues conserved between both enzymes were presumed to be necessary for the tertiary structure of the enzymes. Amino acid residues specifically conserved in only the FTase or the GGTase-I beta  subunits, but not conserved for both enzymes, were hypothesized to determine the distinct substrate specificities of the two enzymes. Seven such regions or individual residues were identified and selected for mutagenesis. The amino acid residues of the yeast FTase Ram1p beta  subunit were substituted with the corresponding residues of the yeast Cdc43p GGTase-I beta  subunit. The following RAM1 mutants were prepared: RAM1-57HFFRH, RAM1-74D, RAM1-206DDLF, RAM1-308T, RAM1-351FSKN, RAM1-367G, and RAM1-385K. The result of this search strategy and the substitutions made in the FTase enzyme are depicted in Fig. 1. The conserved sequences that met the criteria of the search strategy generally were found to be embedded within the context of short stretches of conservation for both the FTase and GGTase-I enzymes.


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Fig. 1.   Sequence alignment of protein isoprenyltransferases with amino acid substitutions. beta  subunits of yeast and mammalian FTases and GGTase-I were aligned using CLUSTAL version 1.6. Regions of sequence conservation in the FTase beta  subunit were replaced by the corresponding residues of the GGTase beta  subunit. Amino acid substitutions to the yeast Ram1p are indicated in bold.

Substrate Preferences of Mutant FTase Enzymes-- Recombinant wild type FTase was expressed in E. coli, and the bacterial lysate was assayed for its ability to farnesylate or geranylgeranylate the Ras-CIIS, Ras-CIIM, and Ras-CIIL proteins (Fig. 2). The wild type FTase enzyme efficiently farnesylated Ras-CIIS, Ras-CIIM, and Ras-CIIL and also geranylgeranylated Ras-CIIM (31). The quantity of [3H]farnesyl and [3H]geranylgeranyl incorporated into each Ras substrate by the wild type FTase under standardized conditions was determined by time course assays to represent the initial rate of reaction and was considered the 100% control value for comparison with the mutant FTase enzymes. Extracts were prepared from E. coli expressing each mutant ram1p FTase: ram1p-57HFFRH, ram1p-74D, ram1p-206DDLF, ram1p-308T, ram1p-351FSKN, ram1p-367G, and ram1p-385K. Each was assayed for the ability to isoprenylate Ras-CIIS, Ras-CIIM, and Ras-CIIL with [3H]farnesyl pyrophosphate (Fig. 3A) and [3H]geranylgeranyl pyrophosphate (Fig. 3B).


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Fig. 2.   Substrate specificity of recombinant wild type FTase. Recombinant wild type FTase was expressed in E. coli, and 50 µg of bacterial lysate was assayed in the presence of either 67 pmol of [3H]Fpp or 67 pmol of [3H]GGpp and 5 µM Ras-CIIS (black bars), Ras-CIIM (shaded bars), or Ras-CIIL (stripped bars) proteins.


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Fig. 3.   Relative substrate preferences of mutant beta  subunit FTases. Recombinant mutant FTase enzymes were expressed in E. coli, and 50 g of bacterial lysate was assayed in the presence of 67 pmol of [3H]Fpp (panel A) or 67 pmol of [3H]GGpp (panel B) and 5 µM Ras-CIIS (black bars), Ras-CIIM (shaded bars), or Ras-CIIL (stripped bars) proteins. Data are expressed as percent of wild type FTase activity.

The effect of the mutations could be divided into three categories: 1) wild type activity and substrate specificity, 2) impaired activity, and 3) altered substrate specificity. In the first category were the ram1p-367G and ram1p-385K mutants that showed nearly wild type farnesylation and geranylgeranylation activity for each of the substrates tested. The second class of mutants included the ram1p-57HFFRH and ram1p-308T mutants that exhibited a general impairment of isoprenyl transferase activity. The third class of mutations included ram1p-74D, ram1p-206DDLF, and ram1p-351FSKN, each of which showed a unique set of substrate specificities. The ram1p-74D mutant was restricted to farnesylating only the Ras-CIIS protein while retaining the ability to geranylgeranylate each Ras-CAAX protein tested. The ram1p-74D mutant demonstrated a nearly 3-fold increase in ability to geranylgeranylate Ras-CIIM. The ram1p-206DDLF mutant enzyme was no longer able to use geranylgeranyl as a substrate, yet it still farnesylated each of the Ras-CAAX proteins including the Ras-CIIL protein. The third mutant enzyme of this class, ram1p-351FSKN, was unique in that it was still able to farnesylate Ras-CIIS and Ras-CIIM but not Ras-CIIL. Furthermore, the ram1p-351FSKN enzyme had no capacity to geranylgeranylate any of the Ras-CAAX proteins. By the criteria established before these studies, the ram1p-351FSKN FTase mutant was an ideal FTase in that it was unable to farnesylate Ras-CIIL and had no geranylgeranyltransferase activity.

To confirm that these unusual results were not artifacts based upon assaying the enzymes in bacterial lysates, the wild type, and ram1p-308T, ram1p-351FSKN mutants were expressed in E. coli and purified by a combination of ion exchange and immunoaffinity chromatography. SDS-PAGE analysis of purified enzymes indicates that these proteins purify equally well by this approach. Purified enzymes displayed substrate specificities and activity comparable to those observed in the cell lysates (data not shown).

Steady-state Kinetic Analysis of Wild Type Ram1p and ram1p-351FSKN FTase-- The altered substrate specificity of the mutant ram1p-351FSKN FTase suggested that there were fundamental changes in substrate recognition by the mutant enzyme. To determine the nature of these changes, steady-state kinetic analysis was performed using purified wild type Ram1p and ram1p-351FSKN FTases. Both enzymes were analyzed using a combination of Ras-CIIS, Ras-CIIM, Ras-CIIL, and either Fpp or GGpp as substrate. The data were fit to several bisubstrate models using GraFit version 3.02 (Robin Leatherbarrow, Erithacus Software, 1996) and were found to fit best (F test and reduced chi 2) to an ordered Bi-Bi kinetic model and as such are consistent with the findings with the mammalian FTase (16, 18).

Table I depicts the kinetic relationships between the affinity for isoprenoid (Fpp or GGpp) and the affinity for the Ras-CAAX substrates. A clear preference for Fpp is observed for both the wild type enzyme and the ram1p-351FSKN enzyme, where Km(farnesyl) is <1 µM, and the Km(geranylgeranyl) is >3.5 µM. The ram1p-351FSKN mutant enzyme has at least a 4-fold reduction in affinity for GGpp over the wild type enzyme. The Km and Kd of GGpp for the ram1p-351FSKN mutant enzyme are 17.5 and 43.5 µM, and 3.59 and 7.61 µM for the wild type Ram1p FTase. Additionally, the ram1p-351FSKN mutant enzyme is reduced in its affinity for Ras-CIIL, with a Km(CIIL) of 22.8 µM versus 10.2 µM for wild type FTase. Analysis of the Kcat data indicate that although the Kcat values of the wild type Ram1p and ram1p-351FSKN enzymes are similar for the combination of farnesyl and either Ras-CIIS or Ras-CIIM substrates, the ram1p-351FSKN FTase is clearly impaired relative to the wild type enzyme when utilizing either GGpp and Ras-CIIM or Fpp and Ras-CIIL. These results are consistent with those seen in the standard isoprenyltransferase assays (Fig. 3, A and B).

                              
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Table I
Kinetic constants for the wild type Ram1p and mutant Ram1p-351FSKN FTase enzymes
Kinetic constants were determined by best fit to an ordered Bi-Bi steady-state reaction (Equation 1). Substrate concentrations for [3H]farnesyl diphosphate and [3H]geranylgeranyl diphosphate were 0.021-20 µM, and Ras-CAAX concentrations were 0.5-38 µM. Kinetic constants were determined by GraFit version 3.02 (Erithacus Software, Inc., 1996). Abbreviations: W.T., wild type; Km(iso), Km for isoprenoid; Km(CaaX) Km for Ras-CAAX substrate; Kd(iso), Kd for isoprenoid; Kcat, intrinsic catalytic rate at saturating substrate concentrations; ND, not able to be determined.

RAM1-351FSKN Restores Yeast Mating and Growth at Elevated Temperatures-- The overlapping substrate preferences of the yeast FTase and GGTase-I suggest the possibility that the FTase may farnesylate CAAL-ending proteins or geranylgeranylate other CAAX substrates in vivo. With a restricted ability to only farnesylate non-leucine-ending CAAX substrates, the ram1p-351FSKN FTase provided a tool to address this question genetically. Using this mutant, we determined the ability of a "perfect" FTase to complement the growth and mating defect of ram1-1 S. cerevisiae cells. S. cerevisiae strains BC3CY[ram1], BC3CY31[ram1p-351FSKN], BC3CY32 [Ram1p], and BC3CY33[ram1p-74D] were grown at either a permissive (28 °C) or restrictive (35 °C) temperature in minimal medium (Fig. 4, A and B, respectively). Each strain grown at the permissive temperature showed similar growth kinetics, with doubling times of approximately 6 h in mid-log phase, reaching stationary phase approximately 28 h postinoculation. Strains grown at the restrictive temperature showed dramatically different growth characteristics. The BC3CY32[Ram1] and BC3CY31[ram1p-351FSKN] both grew at the nonpermissive temperature with similar doubling times in mid-log phase of approximately 5 h demonstrating full functional complementation of the growth defect by the "perfect" FTase. The YO3C[ram1-1] yeast strain grew only poorly at the restrictive temperature (36), with a doubling times of 12 h but never showing logarithmic growth. The substrate specificity mutant, ram1p-74D FTase, was tested for complementation of the growth defect of BC3CY[ram1-1]. We observed that this mutant was incapable of restoring growth at the restrictive temperature and in fact grew more poorly than the parental ram1:LEU2 strain (Fig. 4B).


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Fig. 4.   Complementation of the temperature-sensitive growth defect of in ram1-1 S. cerevisiae. Yeast was transformed with constitutive expression vectors YCP50-ADH (open squares), YCP50-ADH-RAM1 (open circles), YCP50-ADH-RAM1-351FSKN (open triangles), or YCP50-ADH-RAM1-74D (asterisks) and grown at either 28 °C (panel A) or 35 °C (panel B). The number of cells was found to be linear at A600 nm, and cell growth was determined by optical density measurements.

The same mutant beta  subunit FTase enzymes were evaluated for restoration of mating in the ram1:LEU2 strain (Table II). Multiple steps in mating responsiveness require isoprenylated proteins. Mating assays of S. cerevisiae (ram1-1 MATa) expressing RAM1, RAM1-74D, or RAM1-351FSKN beta  subunit indicate that both mutant FTases are capable of prenylating the protein substrates necessary for efficient mating.

                              
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Table II
Mating assays to test for complementation of the mating defect in ram1-1 yeast
MATa S. cerevisiae with the ram1-1::LEU2 allele were transformed with constitutively expressed vectors YCP50-ADH, YCP50-ADH-RAM1, YCP50-ADH-RAM1-351FSKN, or YCP50-ADH-RAM1-74D. Each was mated with PT1 MATalpha yeast. The percent of mated cells versus the total number of viable MATa cells in the mating is depicted in the table. The data are representative of four separate matings.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The alignment of primary amino acid sequences of enzymes with similar catalytic properties is a time-tested approach to identifying those amino acid residues that distinguish one enzyme from another. Using a simple strategy for identifying amino acid residues that may be important for the substrate specificity differences observed between the FTase and GGTase-I enzymes, we constructed seven Ram1p beta  subunit mutants containing one to five residue substitutions each. The recombinant expression mutant ram1p and wild type Ram2p proteins to form FTase heterodimers in E. coli proved to be a rapid method for characterizing both isoprenoid and protein substrate preferences. In terms of relative activity and substrate preferences, the mutant FTases fell into three classes: wild type enzymes, catalytically dead enzymes, and altered substrate preference enzymes. The recent publication of the crystal structure of the rat FTase provides insight into the results observed for each of the these three classes of mutations (25) (Fig. 5).


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Fig. 5.   Calpha tracing of the wild type rat FTase with ram1p-351FSKN mutations. The Calpha trace of the rat FTase (25) was redrawn. The location of the 351FSKN mutation is depicted in bold. An asterisk represents the position of the putative isoprenoid binding pocket.

The first class of mutant enzymes displayed substrate preferences that were similar to those of the wild type FTase enzyme. These include the ram1p-367G and the ram1p-385K mutant enzymes. In the beta  subunit, cysteine 367 is adjacent to tyrosine 366, believed to be involved in forming the isoprenoid binding groove of the enzyme. This substitution does not alter the positioning of this crucial tyrosine residue. The conserved tyrosine residue at position 362 has been suggested to be important for substrate binding (29). Despite the close proximity of the ram1p-367G mutation to tyrosine 362, the FTase had the same substrate specificity of the wild type FTase. The location of the ram1p-385K substitution in the three-dimensional structure of the FTase appears to have substrate preferences and catalytic properties similar to those of the wild type enzyme, as only the farnesylation of the Ras-CIIL substrate is impaired. This is likely caused by the positioning of this residue on a tail structure that is located on a face of the enzyme which is opposite that of the substrate binding pocket (25). The rat FTase x-ray structure reveals this region of the enzyme to be involved in intersubunit contacts, and it may be important for maintaining proper positioning of the alpha  and beta  subunits. Clearly, this region has only a modicum of influence on the substrate preference of the enzyme.

Those mutant enzymes that were affected catalytically can be explained structurally using the rat FTase crystal structure as a model. The ram1p-57HFFRH substitution is located in the first alpha  helix of the enzyme. The affected portion of this alpha  helix is located on the surface of the protein adjacent to the Ras-CAAX binding groove. Although these same residues are found in the wild type GGTase-I enzyme they may not provide the proper amino acid context to form the first alpha  helix of the beta  subunit, which may be required for proper protein substrate binding (25). The ram1p-308T mutant is more easily understood in terms of the possible cause of its catalytic impairment. Residue 308 of the wild type Ram1p is a glycine residue that is located at the beginning of an alpha helix that contains two hydrophobic residues proposed to be critical for the positioning of the isoprenoid substrate. Additionally, cysteine 309 adjacent to glycine 308 corresponds to cysteine 299 of the rat FTase which coordinates the catalytic Zn2+ ion (26). Substitution of a glycine residue with a threonine residue can reduce the conformational flexibility of a polypeptide chain due to the additional stearic hindrance of the threonine side chain. The alpha  helix secondary structure that positions cysteine 309 for Zn2+ coordination and isoprenoid binding is likely to be disrupted by the limited conformations of the polypeptide chain which are imposed by the presence of the threonine side chain.

The third class of beta  subunit mutants that were produced using this strategy possessed altered isoprenoid and protein substrate specificities. The ram1p-74D substitution is located in a short loop between helix 1 and helix 2 of the rat FTase, and it is positioned away from the putative active site of the enzyme (25). The wild type residue is isoleucine. It is unlikely that this residue interacts directly with the CAAX box of the protein substrate. The substitution of a charged residue for a branched chain hydrophobic amino acid possibly effects the local structure of the enzyme sufficiently to perturb the substrate binding pocket. Mechanistically, the coupled changes in both protein and isoprenoid substrate preferences of this mutant suggest that the particular amino acid at the X position of the CAAX sequence and isoprenoid selected to modify that protein substrate are tightly interrelated. Although the mutant enzyme modifies Ras-CIIS with farnesyl nearly as well as the wild type Ram1p FTase, it cannot farnesylate either Ras-CIIM or Ras-CIIL. The geranylgeranylation activity of this mutant is not hindered for either protein substrate and in the case of Ras-CIIM is significantly increased. The placement of this mutation outside the binding pocket of the enzyme and its unique substrate preferences suggest that regions outside the isoprenoid binding pocket can influence the substrate preferences for the FTase enzyme.

The enhanced ability of the ram1p-206DDLF mutant to distinguish between Fpp and GGpp suggests that these residues are important for distinguishing between the different sized isoprenoids. Examination of the rat FTase crystal structure places this substitution in a position that lines the putative isoprenoid binding pocket (25). Arginine 211 of the beta  subunit is positioned opposite the Zn2+ ion and may be important for coordination of the pyrophosphate group of the isoprenoid. The ram1p-206DDLF substitution is located in the highly conserved region and may serve to lock the position of the conserved arginine so that only Fpp is functionally positioned into the isoprenoid binding pocket.

The ram1p-351FSKN mutant FTase provided the most interesting insight into the substrate recognition by the FTase. This mutant enzyme showed that both isoprenoid substrates and protein substrates can be distinguished accurately with only a few changes to the amino acid sequence of the wild type enzyme. This enzyme behaves as a "perfect" FTase, it farnesylates only non-leucine-ending substrates, and it cannot geranylgeranylate any of the substrates tested. Steady-state kinetic analysis was employed to investigate the unique substrate preferences of the ram1p-351FSKN at a kinetic level.

Table I depicts the enzymatic rate constants for both the wild type and the ram1p-351FSKN mutant FTases. The ram1p-351FSKN FTase was shown to have a reduced affinity (increased Km) for the GGpp and Ras-CIIM substrate combination (17.5 and 1.87 µM, respectively) versus the wild type FTase (3.59 and 0.28 µM, respectively). Additionally, the ram1p-351FSKN has a reduced affinity for Ras-CIIL relative to the wild type enzyme (22.8 versus 10.2 µM, respectively). Interestingly, with Ras-CIIL as a protein substrate the most dramatic effect is observed with the Kcat values, the catalytic turnover rates at saturating concentrations of substrates. The wild type enzyme can turnover product, farnesylated-Ras-CIIL, nearly six times faster than the ram1p-351FSKN mutant FTase (0.35 versus 0.06, respectively). Together these data support the findings from Fig. 3, A and B, where the ram1p-351FSKN mutant FTase cannot geranylgeranylate the Ras-CIIM substrate, nor can it farnesylate the Ras-CIIL substrate under the standard assay conditions.

Based on the kinetic data in Table I, a model of substrate preference can be proposed. In an ordered Bi-Bi reaction system, the first substrate, the isoprenoid, must bind to the active site of the enzyme. Comparison of the Km for isoprenoid for the wild type enzyme with Fpp indicates that the affinity of the enzyme for this substrate is only slightly dependent on the Ras-CAAX substrate that is used in the reaction. Clearly, GGpp is a less favored substrate. To form a productive binary complex the isoprenoid must remain bound to the enzyme. Table I indicates that Fpp is released from the enzyme more readily when Ras-CIIS is the protein substrate than when either Ras-CIIM or Ras-CIIL is the protein substrate. Isoprenoid binding and release constitute the first step of the catalysis reaction, the binding of the isoprenoid to form an active enzyme-isoprenoid binary complex. The second step in the ordered Bi-Bi reaction would be the binding of the CAAX protein substrate. Table I indicates that a protein preference order is Ras-CIIS >>  Ras-CIIM > Ras-CIIL with Fpp as the isoprenoid substrate. The next step in substrate preference and isoprenyl transfer is at the level of catalytic rate, where the rates are Fpp-Ras-CIIS > GGpp-Ras-CIIM >>  Fpp-Ras-CIIM > Fpp-Ras-CIIL. Clearly, these rates themselves do not represent the transfer activities found in Fig. 2. The substrate preference of the wild type Ram1p and the ram1p-351FSKN mutant enzymes is apparently the result of a complex interrelation among the affinity and release for the isoprenoid substrate, the affinity for the CAAX substrate, and the catalytic rate for the transfer reaction.

The limited substrate specificity of the ram1p-351FSKN FTase mutant provided a genetic tool with which to address the question of the physiological role of the substrate overlap between the FTase and the GGTase-I. S. cerevisiae requires a functional FTase for growth at elevated temperatures. The discovery of a "perfect" FTase (ram1p-351FSKN) that can only farnesylate non-leucine-ending protein substrates allowed us to determine which property of the FTase is essential for viability. The ram1p-74D FTase mutant, which has only limited farnesylation activity and normal geranylgeranylation activity, provided a readily available control for these experiments. The poor farnesylation activity of the ram1p-74D FTase mutant suggests that it is unlikely to farnesylate those substrates that are required for growth at elevated temperatures. The mating assays indicate that only farnesylation of a limited set of -CAAX substrates is necessary for normal mating. Although these two mutants have been tested with only a limited set of Ras-CAAX substrates, these studies indicate that farnesylation, and not geranylgeranylation, is required for growth of yeast at elevated temperatures and a functional mating pathway. We observe from these studies that not only are there residues that specify isoprenoid preference (ram1p-206DDLF) and protein preference (ram1p-74D), but there are also regions of the beta  subunit in which both isoprenoid and protein substrate preferences are determined (ram1p-351FSKN). These studies have shown that the recognition of isoprenoid substrate and protein substrate is interrelated, suggesting that the specific isoprenoid bound to the enzyme influences which CAAX substrate will be bound. The availability of these mutant enzymes with specific isoprenoid and protein preferences also provides specific genetic tools for determination of the role that farnesylation or geranylgeranylation of particular proteins may play in normal cellular processes.

    FOOTNOTES

* 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.

** To whom correspondence should be addressed: 1044 W. Walnut St., Rm. 351, Indianapolis, IN 46202. Tel.: 317-274-7565; Fax: 317-274-7592; E-mail: mark_marshall{at}iucc.iupui.edu.

1 The abbreviations used are: FTase, farnesyltransferase; GGTase, geranylgeranyltransferase; Fpp, farnesyl phosphate; GGpp, geranylgeranyl diphosphate; ADH, alcohol dehydrogenase.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
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