INTRODUCTION

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Traditionally, the CaaX sequence motif has been defined as a cysteine (C) four amino acids from the C terminus, followed by two amino acids that are often aliphatic (aa), and a C-terminal amino acid (X). Proteins containing a CaaX sequence motif undergo a series of sequential posttranslational modifications that are important for their localization and function. Processing of most CaaX proteins, including Ras, Rho, G protein gamma subunit, and yeast a-factor, involves prenylation of the cysteine four amino acids from the C terminus, endoproteolytic removal of the three C-terminal amino acids, and carboxymethylation of the newly formed prenyl cysteine. The specific amino acids that comprise the CaaX sequence influence whether the 15-carbon farnesyl group or the 20-carbon geranylgeranyl group is attached to the cysteine and whether the aaX sequence is proteolytically removed. Considerable effort has been made to define the sequence features that influence prenylation by the farnesyltransferase and the geranylgeranyltransferase I (7, 8, 30, 35). More recently, two CaaX proteases, Afc1p and Rce1p, have been identified in yeast (4), and homologs of these enzymes have been found in mammals (12, 23, 32, 40; D. H. Wong, C. E. Trueblood, D. Dimster-Denk, J. W. Phillips, P. M. Lagaay, J. Rine, and M. N. Ashby, unpublished data). Since activated Ras function in yeast is attenuated when the aaX sequence is not removed (4), the CaaX proteases that process Ras may be targets for anticancer drug discovery. A number of CaaX protease inhibitors are currently being developed (10, 27).

There is relatively little information about substrate sequence requirements for the CaaX proteases. Even though the aaX sequence is removed from most CaaX proteins, including Ras and Rho proteins (11, 15, 16), some CaaX proteins, such as the alpha and beta subunits of phosphorylase kinase from rabbit muscle (17), retain the aaX sequence after farnesylation. For most CaaX proteins, the identity of the CaaX proteases responsible for cleavage has not been determined. Despite great interest in biochemical characterization of the CaaX proteolytic activity, only partial purification has been achieved (2, 10, 20, 31), presumably because the CaaX proteases are integral membrane proteins. A recent in vitro study examined the ability of a mammalian membrane fraction to proteolyze a large set of CaaX peptide substrates (20). Yet, it is not clear which CaaX protease was being assayed or whether more than one CaaX protease was present in the membrane fraction.

The isolation of the Saccharomyces cerevisiae AFC1 and RCE1 genes (4) clarified the identity of the various yeast CaaX protease activities that had been reported (3, 11, 18) and demonstrated that Afc1p and Rce1p have distinct but overlapping CaaX sequence specificities. Despite a lack of sequence similarity, both Afc1p and Rce1p process the a-factor CaaX sequence, CVIA. However, the CaaX sequences CAMQ and CTLM, when substituted into a-factor, can be processed only by Afc1p and Rce1p, respectively (4). Rce1p also proteolyzes the CaaX sequence of yeast Ras2 protein, CIIS, but apparently Afc1p does not (4).

In our study, a-factor variants with all possible single amino acids substitutions at either the a₁, a₂, or X position of the Ca₁a₂X sequence were expressed in MATa yeast strains that lacked one, both, or neither of the CaaX protease genes, AFC1 and RCE1. The processing of a-factor was measured by halo assay, a biological readout in which secretion of fully processed a-factor leads to growth arrest of a lawn of MAT cells. Halo data, together with in vitro farnesylation information, were used to determine which CaaX sequences were farnesylated and which were proteolyzed by Acf1p and Rce1p. In addition, a region of the Rce1p CaaX protease that affects substrate recognition was identified.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Plasmids. A series of plasmids encoding a-factor variants with single amino acid substitutions at either the a₁, a₂, or X position of the CaaX sequence were generated by insertion of PCR products (encoding the last seven amino acids of the a-factor precursor and the 3' flanking sequence) between the BamHI and EcoRI sites of YCpL-MFA1', a CEN LEU2 plasmid that carries the MFA1 promoter and coding sequence for the first 29 amino acids of the a-factor precursor (43). The BamHI site in the MFA1 gene of YCpL-MFA1' was created by site-directed mutagenesis of nucleotide 90 from C to T. The 5' oligonucleotides used in PCR with Perkin-Elmer Taq polymerase were derivatives of C-TGG-GAT-CCA-GCA-TGT-GTT-ATT-GCT-TAG-TTT-C that differ in the nucleotides present in the a₁, a₂, or X codon (in boldface). For the a₁, a₂, and X substitution series, the oligonucleotide synthesis included a mixture of all four nucleotides at the a₁, a₂, and X codons, respectively. The BamHI site that was used to clone the PCR products is underlined. The 3' oligonucleotide used in the PCR (TCA-CTG-TAT-ACG-GAA-TTC-TCA-TCA-GC) contained an EcoRI site (underlined) and was complementary to the 3' flanking sequence of the MFA1 gene, except for the C in boldface. After ligation into the YCpL-MFA1' BamHI-EcoRI vector fragment and transformation of Escherichia coli, individual plasmids were sequenced. Approximately 75% of the a₁, a₂, and X substitutions were obtained by sequencing about 60 plasmids from each of the three transformant pools. The remaining a-factor variants were constructed by a similar strategy using specific oligonucleotides. a-factor variants with G, A, V, L, I, S, T, D, N, E, Q, K, R, H, W, F, Y, P, C, and M at the a₁ position are encoded by plasmids pJR2047 to pJR2066, respectively. a-factor variants with G, A, V, L, I, S, T, D, N, E, Q, K, R, H, W, F, Y, P, C, and M at the a₂ position are encoded by plasmids pJR2067 to pJR2086, respectively. a-factor variants with G, A, V, L, I, S, T, D, N, E, Q, K, R, H, W, F, Y, P, C, and M at the X position are encoded by plasmids pJR2087 to pJR2106, respectively.

Strains. Strains used in these studies were constructed by standard genetic manipulations and grown on standard media (1). The AFC1 RCE1 (JRY5460), afc1 RCE1 (JRY5461), AFC1 rce1 (JRY5462), and afc1 rce1 (JRY5463) strains used for these studies are closely related to W303-1a (41) and have the following alleles: MATa his3 leu2 trp1 ura3 ram1^H83Y mfa1::hisG mfa2::hisG. The construction of these strains is described below. The mfa2::hisG::URA3::hisG and mfa1::hisG::URA3::hisG alleles were constructed and transformed sequentially into JRY2640 to create a mata1 mfa1::hisG mfa2::hisG ade2 leu2 lys2 ura3 can1 strain, JRY4276 (4). JRY4276 was transformed with MAT plasmid pJR157 to create strain JRY5390, which was then crossed to a MATa his3 leu2 trp1 ura3 afc1::HIS3 strain (JRY5315) (4). The resulting diploid was sporulated to obtain a MATa his3 leu2 trp1 ura3 afc1::HIS3 mfa1::hisG mfa2::hisG segregant that was then crossed to a W303 strain that had the MATa promoter deleted (matap) and had a MAT plasmid. This diploid yielded a matap his3 leu2 trp1 ura3 afc1::HIS3 mfa1::hisG mfa2::hisG segregant, JRY5459, which was transformed with a MAT plasmid and then crossed to JRY5316 (MATa his3 leu2 trp1 ura3 rce1::TRP1) (4). The AFC1 RCE1 (JRY5460), afc1 RCE1 (JRY5461), AFC1 rce1 (JRY5462), and afc1 rce1 (JRY5463) strains were segregants from the resulting diploid strain. These four strains were transformed with MFA1 plasmids (pJR2047 to pJR2106) that encode the a-factor variants with all possible single amino acids substitutions at either the a₁, a₂, or X position.

afc1 RCE1

afc1 rce1

afc1::HIS3

afc1::HIS3

afc1 RCE1

afc1 rce1

afc1 rce1

Halo assays. The relative levels of a-factor produced by various MATa strains were evaluated by pheromone diffusion (halo) assay. One microliter of a yeast cell pellet (approximately 10⁶ cells) was spotted onto a solid rich medium (YPD) plate containing 0.04% Triton X-100 that had been spread with a lawn (approximately 2 × 10⁶ cells) of the MAT sst2 strain, JRY3443. After 2 to 3 days of growth at 30°C, the relative amounts of a-factor produced by each MATa strain were evident from the size of the zone of growth inhibition (or halo) surrounding the MATa cells. The sst2 mutation facilitates measurement of a-factor production by making MAT cells more sensitive to a-factor-induced arrest (9). For the hydrophilic -factor peptide, halo diameter is directly proportional to the log of pheromone concentration (35). This simple relationship between halo size and pheromone concentration is not observed for a-factor because it is hydrophobic and does not diffuse freely through the medium. The addition of Triton X-100 increases the rate of diffusion of a-factor, thereby increasing the sensitivity range of the halo assay. The relative halo sizes are reflective of the amount of a-factor exported from the MATa cells.

Farnesyltransferase enzyme assays. The assay used for farnesylation of peptides was described previously (36). [³H] farnesyl diphosphate (9 µM, 80 µCi/µmol), various peptides of the sequence RTRCxxx (0 to 2.0 mM), and protein farnesyltransferase (116 nM) in buffer containing 50 mM Tris-HCl (pH 7.0), 5 mM MgCl₂, 5 mM dithiothreitol, and 0.04% (wt/vol) dodecyl--D-maltoside with a total volume of 50 µl were incubated for 4 to 20 min at 25°C. Background levels were determined by reactions containing no peptide. The reaction mixtures were then spotted on a 1- by 3-cm strip of phosphocellulose P81 filter paper (Whatman). The filter papers were immersed in a solution of 1:1 75 mM H₃PO₄-95% ethanol (10 ml/strip) and gently swirled on a rotary platform for 10 min, followed by two additional 10-min wash cycles with fresh wash solution. The individual wet strips were transferred to scintillation vials containing 10 ml of Cytoscint (ICN) and 0.5 ml of 6 M HCl. The concentration of farnesylated peptide for each RTRCxxx peptide was then calculated by subtraction of background radioactivity from the radioactivity for each peptide.

In vitro PCR mutagenesis. A DNA fragment containing the RCE1 open reading frame cloned into pRS315 LEU2 CEN vector was used as a template for PCR-based random mutagenesis of RCE1. The mutagenesis was performed in the PCR by increasing the final concentration of three of the four nucleotides to 1 mM, while maintaining the final concentration of the fourth dropout nucleotide at 0.1 mM. The dropout reaction mixtures were set up separately for all four nucleotide combinations. The final concentration of magnesium used in mutagenic PCR reactions was 7 mM. Commercially available M13 forward and reverse sequencing primers (New England Biolabs) were used at the final concentration of 1 µM in a 100 µl of RCE1 amplification reaction mixture. PCR products obtained with each dropout nucleotide mix were purified and pooled.

Identification of Rce1p variants with altered substrate specificity. The pRS315 LEU2 CEN vector (5 µg) was digested with restriction enzymes PstI and HindIII to generate a linear backbone. The pooled PCR product (10 µl) obtained in the mutagenic PCR was cotransformed with 0.5 µg of the linearized backbone into the afc1 rce1 mfa1 mfa2 yeast strain, JRY5463, which had been transformed with the CAMQ version of MFA1 on a CEN URA3 pRS316 plasmid (pJR1556). Each of six independent transformation reactions was subdivided and plated on five plates. After incubation at 30°C for 3 days, transformants (approximately 250/plate) were replica plated directly onto a lawn of  sst2 cells to identify strains containing the versions of RCE1 displaying improved processing of the a-factor-CAMQ substrate. Halos were visible after 36 h of incubation at 30°C. The second round of selection was performed in a similar fashion using the F189L RCE1 mutant as a template for mutagenesis.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Substrate specificities of Afc1p and Rce1p CaaX proteases. To investigate the protein substrate specificities of the Afc1p and Rce1p CaaX proteases, we examined the posttranslational processing of a-factor variants that had all possible single amino acid substitutions at either the a₁, a₂, or X position of the a-factor CaaX sequence, CVIA. Plasmids encoding all 57 single amino acid CaaX variants and wild-type a-factor were transformed into four MATa yeast strains that differ in their CaaX protease genes: AFC1 RCE1 (JRY5460), afc1 RCE1 (JRY5461), AFC1 rce1 (JRY5462), and afc1 rce1 (JRY5463). The relative levels of a-factor produced by these strains were evaluated by a-factor pheromone diffusion (halo) assay (Fig. 1). In this assay, secretion of fully processed a-factor leads to growth arrest of a lawn of MAT cells. The size of the a-factor halo, a zone of growth inhibition of MAT cells, reflects the amount of functional a-factor exported from the MATa cells. To be exported and functional, the 36-amino-acid a-factor precursor undergoes farnesylation of the cysteine four amino acids from the C terminus, followed by proteolytic removal of the aaX sequence, carboxylmethylation of the newly formed C terminus, and two N-terminal proteolytic cleavage events. Mature a-factor is a 12-amino-acid protein that is farnesylated and carboxylmethylated. As discussed below, the in vivo halo results provided valuable information about the specificities of the Afc1p and Rce1p CaaX proteases. Note that for a-factor CaaX sequence variants that produced no halos, interpretation of the a-factor halo results requires knowledge about the extent of farnesylation, since production of mature a-factor requires multiple processing steps and farnesylation must occur prior to the other steps. In vitro farnesylation data, presented below, aided in the interpretation of a-factor halo data for the CaaX sequence that produced no halos.

View larger version (76K): [in this window] [in a new window]   FIG. 1.   a-factor CaaX variants expressed in strains with and without AFC1 and RCE1 produce different amounts of mature a-factor. Plasmids encoding a-factor variants with all possible single amino acid substitutions at either the a1 (A), a2 (B), or X (C) position of the wild-type a-factor CaaX sequence, CVIA, were transformed into four MATa yeast strains that differ in their CaaX protease genes: AFC1 RCE1 (JRY5460), afc1 RCE1 (JRY5461), AFC1 rce1 (JRY5462), and afc1 rce1 (JRY5463). The relative levels of a-factor produced by these strains were evaluated by a-factor pheromone diffusion (halo) assay, a biological assay in which secretion of fully processed a-factor leads to growth arrest of MAT cells (see Materials and Methods). Biologically active a-factor exported from the MATa strains arrested growth of the MAT sst2 cells, forming a zone of growth inhibition (halo) that reflects the amount of a-factor produced.

afc1 rce1

afc1 rce1

N-terminal role of Afc1p in a-factor processing accounted for the absence a halo in afc1 strains carrying certain a-factor variants. In addition to its role in C-terminal proteolysis, Afc1p also plays an important role in maturation of a-factor (40). Afc1p appears to be directly involved in the N-terminal cleavage which removes the first seven amino acids of the a-factor precursor (40), although the precise nature of its N-terminal role is unknown. Both Afc1p and the N-terminal seven amino acids of the a-factor precursor are required for efficient processing of a-factor, even for a-factor variants with CaaX sequences, such as CTLM, that cannot be proteolyzed by Afc1p (5). Deletion of the N-terminal region of the a-factor-CTLM precursor reduces the halo observed in a wild-type strain to a small size, similar to the halo observed in an afc1 strain (5).

Farnesylation was limiting for some a-factor variants. A number of a-factor CaaX sequence variants produced no halo or a very small halo in the wild-type strain. Since farnesylation is required for production of mature a-factor and farnesylation is a prerequisite for CaaX proteolysis, two approaches were taken to determine whether farnesylation was limiting for a-factor production for these CaaX sequence variants: (i) the farnesyltransferase beta subunit was overproduced in strains carrying the a-factor variants and (ii) in vitro farnesylation assays were performed on a selected set of peptides.

View larger version (80K): [in this window] [in a new window]   FIG. 2.   Overexpression of the beta subunit of farnesyltransferase increased production of many a-factor CaaX variants expressed in AFC1 RCE1 and afc1 RCE1 strains. The AFC1 RCE1 strain (JRY5460) and an isogenic afc1 RCE1 strain (JRY6095) carrying plasmids encoding a-factor variants with all possible single amino acid substitutions at either the a1, a2, or X position of the wild-type a-factor CaaX sequence, CVIA, were assayed for a-factor production in the absence (no plasmid) and the presence (high-copy-number RAM1 plasmid) of overexpression of the wild-type farnesyltransferase beta subunit. The relative levels of a-factor produced by these strains were evaluated by a-factor pheromone diffusion (halo) assay.

afc1 RCE1

afc1 RCE1

afc1 RCE1

afc1 RCE1

a-factor CaaX variants with severe halo defects were poor substrates for in vitro farnesylation. To determine directly whether the primary defect in processing of certain CaaX variants was in farnesylation, in vitro farnesylation assays were performed. A selected set of peptides was synthesized that had CaaX sequences identical to each of the a-factor variants that produced no halo and to some of the a-factor variants that produced very small halos. Relative to the natural a-factor CaaX sequence, CVIA, each of the peptides tested was poorly farnesylated in vitro (Table 1); the k_m values were 8- to 800-fold higher, and the k_cat values were 5- to 300-fold lower, than for the wild-type CVIA. Thus, poor farnesylation of these a-factor CaaX variants was at least partially responsible for the low levels of mature a-factor in the in vivo halo assay. Peptides with D, K, or R at the a₂ position and the peptide with P at the X position were unreactive, even at peptide concentrations of 50 mM. A 10-fold increase in the concentration of the enzyme did not result in detectable farnesylation of these peptides. Clearly, for a-factor with D, K, and R at the a₂ position or P at the X position, the farnesylation defects were sufficient to account for the lack of a halo in vivo. Whether or not these substrates could be recognized by either CaaX protease is irrelevant since CaaX proteolysis is dependent on prenylation. The peptides with substitutions G, N, or E at the a₂ position and R at the X position were very poor farnesylation substrates, exhibiting 100- to 700-fold increases in K_m, 90- to 300-fold decreases in k_cat, and catalytic efficiencies (k_cat/K_m) between 10⁵ and 4 × 10⁵, compared to 0.8 for CVIA. Peptides with substitution of Y or P at the a₂ position exhibited 80- and 30-fold increases in K_m, 20- and 40-fold decreases in k_cat, and catalytic efficiencies of 10³ and 3 × 10³, respectively. Peptides with W, Y, or P at the a₁ position exhibited 8- to 10-fold increases in K_m, 5- to 10-fold decreases in k_cat, and catalytic efficiencies that ranged from 8 × 10³ to 2 × 10².

Some a-factor variants are poorly farnesylated and poorly proteolyzed. A more detailed comparison of the in vitro farnesylation data and the in vivo halo data suggested that certain a-factor CaaX variants that were inefficiently farnesylated were also poorly proteolyzed in vivo. The ability of CVNA and CVIR to produce detectable halos in the AFC1 RCE1 strain carrying high-copy-number RAM1 (Fig. 2) was remarkable given the very low catalytic efficiencies for farnesylation of CVNA and CVIR in vitro: 100- to 200-fold-higher K_m values and 100- to 300-fold-lower k_cat values, resulting in catalytic efficiencies more than 10,000-fold lower than for CVIA. Presumably, any substrates that were farnesylated more effectively than CVNA and CVIR in vitro and had comparable or smaller halos in vivo were poor substrates for CaaX proteolysis in vivo. Three a-factor CaaX variants (CWIA, CPIA, and CVPA) had lower K_m values and higher k_cat values than CVNA and CVIR, yet failed to produce a detectable halo. By deduction, these variants had a CaaX proteolysis defect in addition to their farnesylation defect. The catalytic efficiency for farnesylation of CVPA was about 10-fold higher than for CVNA and CVIR, suggesting that CVPA was proteolyzed somewhat less efficiently than CVNA and CVIR. The catalytic efficiencies for CWIA and CPIA farnesylation were 400- to 1,000-fold higher than for CVNA, strongly suggesting that the latter two sequences were at least partially farnesylated in vivo but were not proteolyzed by either CaaX protease. Two a-factor CaaX variants (CYIA and CVYA) produced halos that were comparable in size to CVNA and CVIR (Fig. 2), yet they had catalytic efficiencies that were 30- to 300-fold higher than for CVNA and CVIR. From this analysis, we deduce that CaaX proteolysis of CYIA and CVYA was partially defective. It is quite possible that, in addition to the established defects in farnesylation, CVGA and CVEA have a defect in CaaX proteolysis, since these CaaX sequences produced no halo and exhibited catalytic efficiencies that are only twofold lower than for CVNA.

a-factor variants were not substrates for geranylgeranyltransferase I. We considered the possibility that changes in the CaaX sequence would make a-factor variants substrates of the geranylgeranyltransferase I. In fact, previously published studies show that a-factor-CVIL is both farnesylated and geranylgeranylated in vivo, with both forms being exported and functional (6). However, a-factor-CVIL did not produce a halo in a ram1 strain, which lacks the  subunit of the farnesyltransferase (C. Trueblood, unpublished results). Taken together, these data suggest that farnesyltransferase is able to farnesylate and geranylgeranylate a-factor-CVIL to a physiologically significant extent and that a-factor is not accessible to geranylgeranyltransferase I in vivo. It is notable that CVIL and CVII halo sizes increased significantly when Ram1p was overproduced (Fig. 2A). Ram1p overproduction would be expected to increase farnesyltransferase activity and decrease geranylgeranyltransferase I activity due to competition for the  subunit, which is shared by the two prenyltransferases. These data were consistent with the supposition that the farnesyltransferase, rather than the geranylgeranyltransferase I, was responsible for functional prenylation of a-factor-CVIL and a-factor-CVII.

Predictive value of specificity data. There are 98 proteins encoded in the S. cerevisiae genome that have a cysteine as the fourth amino acid from the C terminus (http://genome-www.stanford.edu/cgi-bin/SGD/search). To test whether the single amino acid substitution series in a-factor had predictive value for determining which of the 98 potential yeast CaaX proteins can serve as substrates of Afc1p and Rce1p, several additional a-factor variants were constructed. Based on our results, substrates with serine or threonine at the a₁ position would be predicted to be proteolyzed by Rce1p but not by Afc1p, whereas substrates with serine or threonine at the a₂ position would be predicted to be proteolyzed by Afc1p. The yeast genome encodes 18 proteins with serine or threonine at the a₁ position. Nine of these proteins have an amino acid at the a₂ position (G, D, N, E, K, or R) that resulted in no halo in our substitution series (Fig. 1) and in very poor or no farnesylation in vitro (Table 1). These proteins are unlikely to be adequately prenylated in vivo and therefore were not considered candidates for further study. The nine remaining proteins were considered likely to be Rce1p-specific substrates. One of these CaaX sequences, CTLM from Ste18p (the gamma subunit of the heterotrimeric G protein), was previously placed on a-factor and found to be proteolyzed by Rce1p but not Afc1p (5), in agreement with our predictions. Similarly, a-factor variants with CSVM and CTVM were tested and found to be proteolyzed by Rce1p but not Afc1p, as predicted (Fig. 3). The yeast genome encodes three proteins with serine or threonine at the a₂ position that would be predicted to be proteolyzed by Afc1p. One of these three C-terminal sequences, CASQ, which is derived from Ydj1p (YNL064C), was tested in the context of a-factor and found to be proteolyzed by Afc1p but not Rce1p (Fig. 3), as predicted. Thus, to a first approximation, the information from the a-factor single amino acid substitution series appeared to be valuable in predicting which CaaX protease could proteolyze a-factor variants with CaaX sequences derived from bona fide proteins.

View larger version (54K): [in this window] [in a new window]   FIG. 3.   Afc1p and Rce1p CaaX proteases differ in the ability to proteolyze CaaX sequences with serine and threonine at the a1 and a2 positions. Plasmids encoding wild-type a-factor (CVIA) and a-factor variants with the CaaX sequences CSVM, CTVM, and CASQ were transformed into four MATa yeast strains that differ in their CaaX protease genes: AFC1 RCE1 (JRY5460), afc1 RCE1 (JRY6095), AFC1 rce1 (JRY5462), and afc1 rce1 (JRY5463). The relative levels of a-factor produced by these strains were evaluated by a-factor pheromone diffusion (halo) assay.

Mutations that alter Rce1p substrate specificity. The lack of obvious sequence similarity of Rce1p to known proteases has precluded predictions of the position of the Rce1p active site or substrate binding site. A genetic screen was performed to identify mutations in RCE1 that would alter Rce1p substrate specificity and allow proteolysis of a-factor-CAMQ, a substrate that was very poorly recognized by the wild-type Rce1p (4, 5). The logic behind the screen was that mutations altering the substrate recognition properties of the enzyme would identify key regions involved in substrate recognition and/or binding. RCE1 PCR products, generated by PCR-based random mutagenesis, and a linearized CEN LEU2 plasmid were cotransformed into a afc1 rce1 yeast strain that expressed a-factor-CAMQ as the only form of a-factor precursor (JRY5463 transformed with pJR1556). Transformants were obtained as a result of recombination between the ends of the RCE1 PCR product and the homologous ends of the linear plasmid. Mutant forms of Rce1p capable of cleaving a-factor-CAMQ were identified by their ability to produce mature a-factor and thereby form halos on a lawn of  sst2 cells. Two different single amino acid changes in Rce1p led to small but reproducible increases in the halo size (Fig. 4): a phenylalanine-to-leucine substitution at position 189 (F189L) and a glutamine-to-arginine substitution at position 201 (Q201R). The mutated RCE1 gene encoding F189L-Rce1p was subjected to a second round of PCR mutagenesis to search for an additional mutation that could further increase the a-factor-CAMQ processing. A change of the glutamic acid codon at position 139 to a lysine codon (E139K) led to a notable increase in halo size (Fig. 4).

View larger version (47K): [in this window] [in a new window]   FIG. 4.   Amino acid substitutions in Rce1p that increased proteolysis of a-factor-CAMQ but did not alter proteolysis of other a-factor CaaX variants. Plasmids encoding a-factor variants with the indicated CaaX sequences (CAMQ, CASQ, CAIA, CVMA, CVSA, and CVIQ) or wild-type a-factor (CVIA) were transformed into MATa afc1 rce1 yeast strains that differ in the RCE1 allele carried on a second plasmid: wild-type RCE1 (wild type) or a mutant allele with the indicated amino acid substitution (F189L, E139K F189L, or Q210R). The relative levels of a-factor produced by these strains were evaluated by a-factor pheromone diffusion (halo) assay.

afc1 rce1

afc1 rce1

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The goal of this work was to provide a comprehensive evaluation of the substrate specificities for the S. cerevisiae CaaX proteases, Afc1p and Rce1p, and to identify amino acids in Rce1p that affect substrate specificity. Part of the interest in CaaX proteases stems from their potential utility in modulating the activity of Ras proteins and other prenylated proteins that contribute to the growth and division of cancer cells. There is considerable interest in developing inhibitors against the human CaaX protease(s) that proteolyze the human N-, Ki-, and Ha-Ras proteins. Like farnesyltransferase inhibitors, which are currently being developed (13, 45), CaaX protease inhibitors could modulate Ras activity and thereby potentially function as anticancer agents. Even in cancer cells that do not carry activated Ras, Ras and other prenylated proteins, including RhoB, contribute to cell propagation and are potential targets for therapeutic intervention (24, 26, 39). Previously, there has been little information about the substrates and substrate specificities of the yeast and mammalian CaaX proteases.

Single amino acid substitution data serve as a predictive guide for which yeast CaaX sequences are prenylated and which are substrates of Afc1p and/or Rce1p. The in vivo halo and in vitro farnesylation data from single amino acid substitutions in the a-factor CaaX sequence are summarized in Table 2. Afc1p and Rce1p exhibited substantial overlap in specificity at the a₁, a₂, and X positions (Table 2, row 1). However, Rce1p was able to accept additional amino acids at the a₁ position that Afc1p was unable to accept (Table 2, row 2), and Afc1p was able to accept additional amino acids that Rce1p may not accept (Table 2, row 3). It was not possible to determine whether or not Rce1p was able to act on these latter a-factor variants, since the loss of the N-terminal role of Afc1p in a-factor processing, in combination with a partial defect in prenylation, reduced a-factor production below a detectable level. Both Afc1p and Rce1p accepted all amino acids at the X position that allowed efficient farnesylation. These results are in agreement with the observation that partially purified CaaX proteases can accept D-amino acids at the X position, but not at the a₁ or a₂ position (27), and can proteolyze farnesylated tripeptides nearly as well as farnesylated tetrapeptides (20, 27).

Deductions about which potential CaaX proteins in yeast are unlikely to be farnesylated. In vitro farnesylation data, together with the in vivo a-factor production (halo assay) data, suggest that at least 29 out of 98 yeast proteins with a cysteine four amino acids from the C terminus are unlikely to be farnesylated (Table 3). These 29 proteins have an amino acid at the a₂ position that resulted in very poor farnesylation (G, N, or E) or no farnesylation (D, K, or R) in vitro. Note that even among the remaining 69 proteins, some may be poorly prenylated due to the presence of W, Y, or P at the a₁ position and Y or P at the a₂ position, which exhibited a partial in vitro farnesylation defect. In addition, G or D at the a₁ position, A, S, T, Q, H, W, or F at the a₂ position, or D, E, K, or W at the X position may decrease farnesylation efficiency. In the a-factor substitution series, a-factor variants with these substitutions produced small halos that increased in size when the beta subunit of farnesyltransferase was overexpressed, supporting the idea that inefficient farnesylation is at least partially responsible for the low level of a-factor production from these variants (Fig. 2).

Yeast CaaX protease specificity information aids in the interpretation of mammalian CaaX protease observations. The yeast and mammalian CaaX proteases have proven extremely difficult to purify, and consequently in vitro studies of substrate specificity on purified proteases have not been done. A partially purified prenyl protein-specific endoprotease (PPEP) activity, which may include one or more CaaX proteases of unknown identity, was used in a recent specificity study (20). By comparing the in vitro specificity of the partially purified activity to the in vivo specificities of yeast Afc1p (Fig. 1), yeast Rce1p (Fig. 1), and human Rce1p expressed in yeast (Wong et al., unpublished), we deduce that the activity is likely to be that of a mammalian Afc1p homolog. One of the key results that led to this deduction was that yeast Rce1p (Fig. 1) and human Rce1p (Wong et al., unpublished) were able to cleave the a-factor variant with R at the a₁ position, whereas neither yeast Afc1p (Fig. 1) nor the PPEP activity (20) could cleave substrates with R at the a₁ position. Specificity studies have not been performed with the other partially purified enzymes (2, 10, 31), but one enzyme activity (31) is inhibited by o-phenanthroline, as has been observed for yeast Afc1p (4, 10a), whereas the other enzyme activity (10) is not sensitive to o-phenanthroline and has properties expected for Rce1p. Further studies will be necessary to clarify which of these activities are Afc1p, Rce1p, or other CaaX proteases.

In vivo cleavage of Ras proteins by Rce1p, but not Afc1p, is not explained by Afc1p CaaX sequence specificity constraints. Studies of human Rce1p protein in insect cells establish its ability to cleave farnesyl-Ki-Ras, geranylgeranyl-Ki-Ras, farnesyl-Ha-Ras, farnesyl-N-Ras, farnesyl-G1, and geranylgeranyl-Rap1 (32), which have the CaaX sequences CVIM, CVLS, CVLM, CVIS, and CQLL, respectively. In a complementary study, mouse fibroblasts that are homozygous for an rce1 deletion are unable to process farnesyl-Ki-Ras, geranylgeranyl-Ki-Ras, farnesyl-Ha-Ras, farnesyl-N-Ras, farnesyl-G1, or geranylgeranyl-Rap1 (23). By deduction, the mouse Afc1p homolog appears not to proteolyze these substrates.

A substrate recognition domain in Rce1p. Amino acid substitutions in Rce1p that improved its ability to process a-factor-CAMQ, without affecting processing of other a-factor variants, provided information about the region of Rce1p likely to be involved in substrate recognition. The F189L and Q201R amino acid substitutions in Rce1p each independently increased Rce1p processing of a-factor-CAMQ. F189 is conserved in both the human and Schizosaccharomyces pombe homologs of Rce1p, which share amino acid identity of only 32% with each other (Fig. 5). A conserved histidine that residues between F189 and Q201 (H194 in yeast Rce1p and H208 in human Rce1p) is required for proteolytic activity of yeast (10a) and human (Wong et al., unpublished) Rce1p. Together, these data strongly suggest that this region of Rce1p is involved in substrate binding. This same region contains a conserved HxxE motif which has been proposed to be a Zn binding site (12), by analogy to a previously described class of metalloproteases (33). However, the HxxE sequence is not critical to Rce1p function because human Rce1p remains functional when either histidine 211 or glutamate 214 is replaced with alanine (Wong et al., unpublished). Substitution of the analogous histidine of yeast Rce1p reduced the specific activity about 10-fold (10a). Note also that zinc chelators, such as o-phenanthroline, do not appear to inhibit Rce1p, raising doubt about the role of zinc in Rce1p function (5, 10a).

View larger version (62K): [in this window] [in a new window]   FIG. 5.   Alignment of Rce1p homologs from S. cerevisiae (4), human (32), and S. pombe (accession numbers: Swiss-Prot Q10071 and GenBank AL035064.1) sequences were aligned with the ClustalW 1.7 program (42), accessed through the BCM sequence launcher (http://www.hgsc.bcm.tmc.edu/SearchLauncher/). Identities between homologs are shaded in black, and similarities are shaded in gray. The region spanning amino acids 147 to 257 of S. cerevisiae is most highly conserved region among the three homologs, with pairwise percent identities of 41% (S. cerevisiae-human), 44% (human-S. pombe), and 35% (S. cerevisiae-S. pombe) and percent similarities of 50 to 58%. The figure was prepared using BOXSHADE (http://www.isrec.isb-sib.ch:8080/software/BOX_form.html).