Characterization of the mRNA capping apparatus of Candida albicans.

The mRNA capping apparatus of the pathogenic fungus Candida albicans consists of three components: a 520- amino acid RNA triphosphatase (CaCet1p), a 449-amino acid RNA guanylyltransferase (Cgt1p), and a 474-amino acid RNA (guanine-N7-)-methyltransferase (Ccm1p). The fungal guanylyltransferase and methyltransferase are structurally similar to their mammalian counterparts, whereas the fungal triphosphatase is mechanistically and structurally unrelated to the triphosphatase of mammals. Hence, the triphosphatase is an attractive antifungal target. Here we identify a biologically active C-terminal domain of CaCet1p from residues 202 to 520. We find that CaCet1p function in vivo requires the segment from residues 202 to 256 immediately flanking the catalytic domain from 257 to 520. Genetic suppression data implicate the essential flanking segment in the binding of CaCet1p to the fungal guanylyltransferase. Deletion analysis of the Candida guanylyltransferase demarcates an N-terminal domain, Cgt1(1-387)p, that suffices for catalytic activity in vitro and for cell growth. An even smaller domain, Cgt1(1-367)p, suffices for binding to the guanylyltransferase docking site on yeast RNA triphosphatase. Deletion analysis of the cap methyltransferase identifies a C-terminal domain, Ccm1(137-474)p, as being sufficient for cap methyltransferase function in vivo and in vitro. Ccm1(137-474)p binds in vitro to synthetic peptides comprising the phosphorylated C-terminal domain of the largest subunit of RNA polymerase II. Binding is enhanced when the C-terminal domain is phosphorylated on both Ser-2 and Ser-5 of the YSPTSPS heptad repeat. We show that the entire three-component Saccharomyces cerevisiae capping apparatus can be replaced by C. albicans enzymes. Isogenic yeast cells expressing "all-Candida" versus "all-mammalian" capping components can be used to screen for cytotoxic agents that specifically target the fungal capping enzymes.

The m7GpppN cap structure of eukaryotic mRNA is formed by three enzymatic reactions as follows. (i) The 5Ј triphosphate end of the nascent pre-mRNA is hydrolyzed to a diphosphate by RNA 5Ј triphosphatase, (ii) the diphosphate end is capped with GMP by GTP:RNA guanylyltransferase, and (iii) the GpppN cap is methylated by AdoMet:RNA (guanine-N7) methyltransferase (1). RNA capping is essential for cell growth.
Our mutational analyses of S. cerevisiae Cet1p, Ceg1p, and Abd1p have resulted in the delineation of minimal catalytic domains for each protein and the identification of catalytically important amino acid side chains that comprise the triphosphatase, guanylyltransferase, and methyltransferase active sites (16, 18 -24). Genes and/or cDNAs encoding homologues of Cet1p, Ceg1p, and Abd1p have been isolated from other fungal species, including Schizosaccharomyces pombe and Candida albicans (8,20,(25)(26)(27), but the enzymes have not been well characterized biochemically, and functional domains have not been defined.
In considering ways to identify antifungals that target cap formation, one wishes to focus on the capping apparatus from a clinically significant human pathogen such as C. albicans. The triphosphatase (CaCet1p), guanylyltransferase (Cgt1p), and methyltransferase (Ccm1p) components of the Candida capping apparatus have now been identified (8,(25)(26)(27). Here, we define a minimal functional domain of the RNA triphosphatase CaCet1p capable of complementing a S. cerevisiae cet1⌬ mutant. Analysis of the guanylyltransferase Cgt1p includes the demarcation of functional domain boundaries and studies of the binding of recombinant Cgt1p to its docking site on yeast triphosphatase. We also present studies of the cap methyltransferase Ccm1p including delineation of a minimal functional domain and biochemical characterization of recombinant Ccm1p and truncated version thereof. We show that Ccm1p binds to the phosphorylated C-terminal domain (CTD) 1 of RNA polymerase II and that binding is enhanced when the CTD is phosphorylated on both Ser-2 and Ser-5. Finally, we construct yeast strains in which the entire S. cerevisiae capping apparatus is replaced by C. albicans enzymes. The availability of isogenic yeast cells expressing all-Candida versus all-mammalian capping components should facilitate screening in vivo for antifungals that block cap formation.

EXPERIMENTAL PROCEDURES
Yeast Expression Vectors for Candida RNA Triphosphatase-Yeast CEN TRP1 plasmid vectors containing CaCET1, CaCET1(203-520), or CaCET1(217-520) under the control of the yeast TPI1 promoter were described previously (13). New N-terminal deletion mutants CaCET1(223-520), CaCET1(229 -520), CaCET1(257-520), and Ca-CET1(267-520) were constructed by PCR amplification with mutagenic sense-strand primers that introduced an NdeI restriction site and a methionine codon in lieu of the codons for Thr-222, Asn-228, or Lys-266 or an NdeI restriction site at the Met-257 codon. The PCR products were digested with NdeI and BamHI, then inserted into pYN132 (CEN TRP1). The inserts of each deletion plasmid were sequenced to verify that no unwanted coding changes were introduced during amplification and cloning.
Yeast CGT1 Expression Plasmids-The CGT1 open reading frame encoding C. albicans mRNA guanylyltransferase was amplified from a genomic library (28) by PCR using oligonucleotide primers designed to introduce an NdeI restriction site at the translation start codon and a BamHI site 3Ј of the stop codon. The PCR product was digested with NdeI and BamHI and inserted into yeast expression vector pYN132. In this vector, expression of the C. albicans guanylyltransferase is under the control of the yeast TPI1 promoter. C-terminal truncation mutants of CGT1 were constructed by PCR amplification with mutagenic antisense-strand primers that introduced stop codons in lieu of the codons for Gln-427, Arg-409, Arg-388, or Lys-368 along with a BamHI site 3Ј of the new stop codon. The PCR products were digested with NdeI and BamHI, then inserted into pYN132. The inserts of each plasmid were sequenced to verify that no unwanted coding changes were introduced during amplification and cloning.
Expression and Purification of Recombinant Cgt1p-NdeI-BamHI restriction fragments containing the wild type CGT1 gene or C-terminal truncation mutants were excised from the respective yeast expression vectors and inserted into the bacterial expression plasmid pET16b. The resulting plasmids, pET-Cgt1, pET-Cgt1-C⌬23, pET-Cgt1-C⌬41, pET-Cgt1-C⌬62, and pET-Cgt1-C⌬82, were introduced into Escherichia coli BL21(DE3). Cultures (500-ml) derived from single transformed colonies were grown at 37°C in Luria-Bertani medium containing 0.1 mg/ml ampicillin until the A 600 reached 0.5. The cultures were placed on ice for 20 min and then adjusted to 0.4 mM isopropyl-1-thio-␤-D-galactopyranoside and 2% (v/v) ethanol. Incubation was continued for 20 h at 17°C with constant shaking. Cells were harvested by centrifugation, and the pellets were stored at Ϫ80°C. All subsequent procedures were performed at 4°C. Thawed bacteria were resuspended in 25 ml of lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% sucrose). Cell lysis was achieved by the addition of 50 g/ml lysozyme and 0.1% Triton X-100. The lysates were sonicated to reduce viscosity, and insoluble material was removed by centrifugation. The soluble lysates were applied to 1-ml columns of nickel nitrilotriacetic acid-agarose that had been equilibrated with lysis buffer containing 0.1% Triton X-100. The columns were washed with 5 ml of lysis buffer containing 0.1% Triton X-100 and then eluted stepwise with 2-ml aliquots of a buffer solution (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 mM DTT, 10% glycerol, 0.05% Triton X-100) containing 50, 100, 200, 500, and 1000 mM imidazole. SDS-PAGE analysis showed that the recombinant Cgt1p proteins were recovered in the 100 and 200 mM imidazole eluate fractions. The peak fractions containing the recombinant proteins were pooled, adjusted to 5 mM EDTA, and dialyzed against buffer containing 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 2 mM DTT, 5 mM EDTA, 10% glycerol, 0.05% Triton X-100. The enzyme preparations were stored at Ϫ80°C.
Yeast CCM1 Expression Plasmids-The CCM1 open reading frame encoding C. albicans cap methyltransferase was amplified from genomic clone pCCM1-6.6 (8) by PCR using oligonucleotide primers designed to introduce an NdeI restriction site at the translation start codon and a XhoI site 3Ј of the stop codon. The PCR product was digested with NdeI and XhoI and inserted into yeast expression vector pYN132 (CEN TRP1), placing expression of the C. albicans cap methyltransferase gene under the control of the yeast TPI1 promoter. Nterminal deletion mutants of CCM1 were constructed by PCR amplification with mutagenic sense-strand primers that introduced an NdeI restriction site and a methionine codon in lieu of the codon for Ser-136 or Val-150 or an NdeI restriction site at the Met-175 codon. The PCR products were digested with NdeI and XhoI, then inserted into pYN132. The inserts of each plasmid were sequenced to verify that no unwanted coding changes were introduced during amplification and cloning. KpnI-XhoI fragments containing the CCM1 gene and the N-terminal deletion mutants were then transferred from their respective CEN plasmids to the yeast high copy vector pYX232 (2 TRP1), with methyltransferase expression under the control of the TPI1 promoter.
Expression and Purification of Recombinant Ccm1p-NdeI-XhoI restriction fragments containing the wild type CCM1 gene and N-terminal deletion mutants were excised from the respective yeast CEN expression vectors and inserted into the bacterial expression plasmid pET16b. The resulting plasmids, pET-Ccm1, pET-Ccm1⌬136, pET-Ccm1⌬150, and pET-Ccm1⌬174, were introduced into E. coli BL21(DE3). Cultures (1 liter) derived from single transformed colonies were grown at 37°C in Luria-Bertani medium containing 0.1 mg/ml ampicillin until the A 600 reached 0.5. The culture was adjusted to 0.2 mM isopropyl-1-thio-␤-D-galactopyranoside and 2% ethanol, and incubation was continued for 16 h at 18°C. Cells were harvested by centrifugation, and the pellet was stored at Ϫ80°C. All subsequent procedures were performed at 4°C. Thawed bacteria were resuspended in 50 ml of buffer L (50 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 10% sucrose). Cell lysis was achieved by the addition of lysozyme and Triton X-100 to final concentrations of 50 g/ml and 0.1%, respectively. The lysate was sonicated to reduce viscosity, and insoluble material was removed by centrifugation. The soluble extract was mixed for 1 h with 2 ml of nickel nitrilotriacetic acid-agarose resin that had been equilibrated with buffer L. The suspension was poured into a column and washed with buffer L followed by IMAC buffer (20 mM Tris HCl (pH 7.9), 0.5 M NaCl, 1 mM phenylmethylsulfonyl fluoride, 10% glycerol) containing 30 mM imidazole. The column was then eluted stepwise with IMAC buffer containing 50, 200, and 500 mM imidazole. The polypeptide compositions of the column fractions were monitored by SDS-PAGE. The recombinant Ccm1p proteins were recovered in the 200 mM imidazole eluate. Aliquots of the nickel-agarose fraction (60 g of protein) were applied to 4.8-ml 15-30% glycerol gradients containing 0.5 M NaCl in 50 mM Tris HCl (pH 8.0), 1 mM EDTA, 2 mM DTT, 0.1% Triton X-100. The gradients were centrifuged for 12 h at 50,000 rpm in a Beckman SW50 rotor. Fractions (0.2 ml) were collected from the bottoms of the tubes. The fractions were stored at Ϫ80°C.

Genetic Interaction between CaCet1p and Fungal
Guanylyltransferases-We reported previously that either full-length CaCET1 or N-terminal deletion alleles CaCET1(179-520), CaCET1(196-520), or CaCET1(203-520) complemented growth of a cet1⌬ mutant of S. cerevisiae, whereas the more extensively truncated CaCET1(217-520) gene did not complement (13). CaCET1(203-520) cells displayed a temperature-sensitive growth phenotype that was suppressed by overexpression of CEG1. These experiments delineated a short peptide segment between residues 203 and 216 that is required for CaCet1p function in vivo and also implied a functional interaction between the Candida triphosphatase and the endogenous Saccharomyces guanylyltransferase.
Here we tested complementation of the cet1⌬ mutation by a new series of truncated CaCET1 alleles alone versus complementation by truncated CaCET1 alleles plus the wild type Candida guanylyltransferase gene CGT1 present on the same CEN plasmid (Fig. 1). Note that biochemical studies of the purified recombinant proteins encoded by CaCET1 alleles ⌬202, ⌬216, ⌬222, ⌬228, and ⌬256) showed that the N-terminal truncated versions of CaCet1p were fully active in ␥-phosphate hydrolysis in vitro (29).
Genetic and Biochemical Analysis of Candida RNA Guanylyltransferase-The C. albicans CGT1 gene encodes a 449amino acid polypeptide with extensive sequence similarity to S. cerevisiae Ceg1p (25). The active-site lysine that becomes covalently bound to GMP during the nucleotidyltransferase reaction is located at position 67 in Cgt1p and position 70 in Ceg1p. Because even small deletions from the N terminus of Ceg1p result in loss of function in vivo and in vitro (19), we presumed that N-terminal deletions of Cgt1p would also be deleterious. Therefore, we constructed a series of C-terminal deletion alleles of CGT1 that were cloned into CEN vectors under the control of the TPI1 promoter and then tested by plasmid shuffle for complementation of the S. cerevisiae ceg1⌬ mutant. The CGT1(1-426), CGT1 , and CGT1(1-387) mutants were viable, whereas CGT1(1-367) was lethal ( Fig. 2A).
Full-length Cgt1p and the C-terminal deletion mutants Cgt1(1-426)p (C⌬23), Cgt1(1-408)p (C⌬41), and Cgt1(1-387)p (C⌬62), and Cgt1(1-367)p (C⌬82) were expressed in bacteria as His 10 -tagged fusions and purified from soluble bacterial lysates by nickel-agarose column chromatography. The elution profile of full-length Cgt1p is shown in Fig. 3. SDS-PAGE analysis are denoted below the aligned sequences. Yeast strain YBS20 (cet1⌬) was transformed with CEN TRP1 plasmids containing (i) CaCET1 or the indicated deletion mutants driven by the TPI1 promoter, (ii) CaCET1 or the indicated deletion mutants plus CGT1 driven by the GPD1 promoter, or (iii) CaCET1 or the indicated deletion mutants plus CEG1 driven by the GPD1 promoter. Trp ϩ isolates were streaked on agar plates containing 0.75 mg/ml 5-FOA. Growth was scored after 7 days of incubation at 25 and 30°C. Lethal alleles (scored as a minus sign) were those that failed to form colonies on 5-FOA at either temperature. For the viable alleles, individual colonies were picked from the 5-FOA plates and patched on YPD agar. Two isolates of each mutant were streaked on YPD agar at 16, 25, 30, 34, and 37°C. Growth was assessed as follows: ϩϩ indicates wild type colony size at all temperatures; ϩ indicates that the strains formed small colonies at the permissive temperature. Temperature-sensitive (ts) strains failed to form colonies at 37°C. Cold-sensitive (cs) strains failed to form colonies at 16°C. showed that a 55-kDa polypeptide corresponding to Cgt1p was recovered in the 100 and 200 mM imidazole eluate fractions (Fig. 3A). Guanylyltransferase activity was assayed by the formation of a covalent enzyme-[ 32 P]GMP adduct (EpG) when the protein fractions were incubated with [␣-32 P]GTP and magnesium. The activity detected in the soluble lysate (Fig. 3B, lane S) was adsorbed to Ni 2ϩ -agarose and eluted in the 100 and 200 mM imidazole fractions in parallel with the recombinant Cgt1p polypeptide (Fig. 3B).
The SDS-PAGE analysis of the nickel-agarose fractions of the full-length and truncated Cgt1p proteins revealed similar extents of purification and the expected increments in electrophoretic mobility (Fig. 2B). Mutants C⌬23, C⌬41, and C⌬62 retained guanylyltransferase activity, whereas the C⌬82 protein was unable to form the protein-GMP complex (Fig. 2C). Thus, there was a clear correlation between the in vivo lethality and the loss of catalytic activity elicited by deletion of the Cgt1p segment from positions 367 to 387.
Full-length recombinant Cgt1p was sedimented in a 15-30% glycerol gradient with internal standards catalase, bovine serum albumin, and cytochrome c (Fig. 4A). Note that the catalase and Cgt1p polypeptides migrated identically during SDS-PAGE. The more rapidly sedimenting polypeptide corresponds to catalase, whereas the component sedimenting as a discrete peak between bovine serum albumin and cytochrome c corresponds to Cgt1p. The guanylyltransferase activity profile was coincident with the slower sedimenting Cgt1p protein in fractions 17 to 21 (not shown). Thus, we conclude that Candida guanylyltransferase is a monomeric enzyme.
The catalytically active C⌬62 protein (clearly resolved from catalase during SDS-PAGE) also sedimented as a discrete monomeric peak between bovine serum albumin and cytochrome c (Fig. 4B). The same was true of the C⌬82 mutant (Fig. 4C), which suggests that the loss of catalytic activity accompanying the incremental deletion was not caused by gross misfolding and aggregation of the C⌬82 polypeptide.
Further characterization of the guanylyltransferase reaction was performed using the peak glycerol gradient fraction of wild type Cgt1p. Formation of the covalent Cgt1p-[ 32 P]GMP adduct during a 10-min reaction at 37°C with 5 M [␣ 32 P]GTP was absolutely dependent on a divalent cation cofactor. Either magnesium or manganese at 2.5 mM could satisfy the cofactor requirement, but calcium, cobalt, copper, and zinc could not (data not shown). Activity was proportional to magnesium concentration up to 2.5 mM and reached a plateaued at 2.5-10 mM CaCet1p interacts with either Cgt1p or Ceg1p in a two-hybrid reporter assay in S. cerevisiae are at least consistent with the existence of a bifunctional complex in Candida. We recently localized the guanylyltransferase binding function of S. cerevisiae Cet1p to a short segment from residues 232 to 265 (30). This domain is conserved in the C. albicans RNA triphosphatase CaCet1p and is essential for in vivo function when CaCet1p is expressed in S. cerevisiae (Fig. 1).
The experiment in Fig. 5 shows that the recombinant wild type C. albicans guanylyltransferase Cgt1p bound nearly quantitatively to a biotinylated 34-amino acid synthetic peptide Cet1(232-265) coupled to streptavidin-coated beads. The input Cgt1p was eluted with SDS from the peptide-containing beads along with the smaller streptavidin polypeptide (Fig. 5, lane B). Cgt1p did not bind at all to streptavidin beads alone (30). The catalytically active truncated proteins C⌬62 also bound to the immobilized Cet1(232-265) peptide (Fig. 5). The instructive finding was that the catalytically inactive C⌬82 protein re-tained the capacity to recognize the triphosphatase peptide ligand (Fig. 5). We conclude that the binding site for the triphosphatase resides within the N-terminal 367 amino acids of Cgt1p. Further truncation of Cgt1p from the C terminus resulted in extensive proteolysis when the recombinant polypeptide was expressed in bacteria; this problem impeded our efforts to delineate the C-terminal margins of the triphosphatase-binding site on Cgt1p.
Deletion Analysis of Candida Cap Methyltransferase Delineates a Functional Domain-The CCM1 gene was isolated from a C. albicans library by screening for complementation of the conditional growth defect of S. cerevisiae abd1-ts mutants (8).
To test whether CCM1 could fully replace ABD1, the complete CCM1 coding sequence was cloned into a yeast CEN TRP1 plasmid such that its expression was under the control of the constitutive S. cerevisiae TPI1 promoter. The CCM1 plasmid was introduced into a yeast strain in which the chromosomal ABD1 locus was deleted. Growth of the abd1⌬ strain is contingent on maintenance of an extrachromosomal ABD1 gene on a CEN URA3 plasmid. Trp ϩ transformants were plated on medium containing 5-fluoroorotic acid (5-FOA) to select against the URA3 ABD1 plasmid. Control cells transformed with a TRP1 ABD1 plasmid grew on 5-FOA, whereas cells transformed with the TRP1 vector were incapable of growth on 5-FOA. Cells bearing the CCM1 plasmid grew on 5-FOA (Fig.  6A). Thus, the C. albicans cap methyltransferase was functional in lieu of the endogenous S. cerevisiae enzyme.
Several N-terminal deletion mutants of CCM1 were cloned into the CEN TRP1 vector and tested for function in vivo by plasmid shuffle. CCM1(137-474) complemented growth of the abd1⌬ strain on 5-FOA. However, the more extensively truncated alleles CCM1(151-474) and CCM1(175-474) were lethal in vivo (Fig. 6A). Based on an alignment of the amino acid sequences of the Ccm1p and Abd1p polypeptides (24), the viable N-terminal ⌬136 deletion of Ccm1p would be analogous to a deletion of 108 amino acids from the N terminus of Abd1p. The lethal N-terminal ⌬150 and ⌬174 deletions of Ccm1p correspond to ⌬122 and ⌬146 deletions of Abd1p. Prior studies showed that deleting 109 amino acids from the N terminus of Abd1p had no effect on yeast cell growth, whereas deletion of 142 or 155 residues was lethal (23). Thus, the N-terminal margins of the functional domains of the Candida and Saccharomyces cap methyltransferases are fairly similar.
Purification and Characterization of Recombinant Ccm1p-Full-length Ccm1p and the N-terminal deletion mutants Ccm1(137-474)p (⌬136), Ccm1(151-474)p (⌬150), and Ccm1(175-474)p (⌬174) were expressed in bacteria as His 10tagged fusions and purified from soluble bacterial lysates by nickel-agarose chromatography, followed by glycerol gradient sedimentation. SDS-PAGE analysis of the polypeptide compositions of the peak glycerol gradient fractions of Ccm1p, ⌬136, ⌬150, and ⌬174 is shown in Fig. 6B. The apparent sizes of the predominant polypeptides corresponding to Ccm1p and serially truncated versions thereof are in good agreement with the calculated sizes of the His-tagged gene products.
RNA (guanine-N7-)-methyltransferase activity of the Ccm1p preparation was detected by the conversion of 32 P cap-labeled poly(A) to methylated cap-labeled poly(A) in the presence of AdoMet. The reaction products were digested to cap dinucleotides with nuclease P1 and then analyzed by polyethyleneimine-cellulose thin layer chromatography, which resolves the GpppA cap from the methylated cap m7GpppA. The radiolabeled product synthesized by Ccm1p comigrated with m7GpppA generated in a parallel reaction mixture containing purified recombinant vaccinia virus cap methyltransferase (not shown). Methylation of capped poly(A) varied linearly with  (232-265). The sequence of the Cet1(232-265) peptide is shown. An N-terminal biotin anchors the peptide to a streptavidin-coated magnetic bead. Affinity chromatography was performed by mixing 4 g of Cgt1p, C⌬62, or C⌬82 with 0.6 mg of Cet1 peptide beads (estimated to contain 390 -540 pmol of peptide) in 50 l of binding buffer (25 mM Tris HCl (pH 8.0), 50 mM NaCl, 1 mM DTT, 5% glycerol, 0.03% Triton X-100). After incubation for 20 -30 min on ice, the beads were concentrated by microcentrifugation and then washed 3 times with 0.5-ml aliquots of binding buffer. After the third wash, the beads were resuspended in 50 l of binding buffer. Aliquots of the input protein fraction (L) (equivalent to 40% of total material loaded), the free-unbound fraction (F) (40% of the first supernatant), and the bead-bound fraction (B) (40% of the SDS eluate of the beads) were analyzed by SDS-PAGE. A Coomassie Blue-stained gel is shown. The guanylyltransferase and streptavidin polypeptides are indicated on the right. WT, wild type. input Ccm1p protein and was nearly quantitative at saturation (Fig. 6C). Cap methylation depended on inclusion of S-adenosylmethionine in the reaction mixture. Half-maximal activity was observed at ϳ5 M AdoMet (not shown). S-Adenosylhomocysteine did not support cap methylation and was inhibitory in the presence of AdoMet (not shown).
The specific activity of ⌬120 in cap methylation was similar to that of full-length Ccm1p, whereas ⌬150 was one-fifth as active, and ⌬174 was inactive (Fig. 6C). The failure of ⌬150 to sustain growth of abd1⌬ cells when expressed in a single copy despite retention of partial activity in vitro raised the possibility that cell growth requires a threshold level of cap methyltransferase activity. To test this hypothesis, we increased the gene dosage of the ⌬150 mutant by cloning the CCM1(151-474) gene into a high-copy 2 vector under the control of the constitutive TPI1 promoter. abd1⌬ cells bearing the 2 CCM1(151-474) plasmid did form small colonies on 5-FOA (not shown). When tested for growth on rich medium (YPD) at 30°C, the 2 CCM1(151-474) cells formed uniformly small colonies compared with 2 CCM1 cells (Fig. 6D). 2 CCM1(151-474) cells also grew slowly at 25 and 37°C (not shown). These results are consistent with a threshold requirement for cap methyltransferase activity in vivo. Note that the catalytically defective ⌬174 mutant failed to support growth of abd1⌬ cells even when introduced on a 2 plasmid (not shown). We infer that the segment from residues 151 to 174 is essential for cap methylation.
Candida Cap Methyltransferase Binds to the Phosphorylated CTD of RNA Polymerase II-In vivo, the mRNA capping reactions occur cotranscriptionally, i.e. the substrates for the capping enzymes are nascent RNA chains engaged within RNA polymerase II elongation complexes. Targeting of cap formation to transcripts made by polymerase II is achieved through direct physical interaction of components of the capping apparatus with the phosphorylated CTD of the largest subunit of polymerase II (2)(3)(4)31). The CTD, which is unique to polymerase II, is composed of a tandemly repeated heptad motif (consensus sequence ϭ YSPTSPS). The CTD undergoes a cycle of extensive phosphorylation and dephosphorylation, which is coordinated with the transcription cycle (32). Cyclin-dependent protein kinases are implicated in CTD phosphorylation at positions Ser-2 and Ser-5 of the heptad repeats.
Ho and Shuman (33) employed synthetic CTD phosphopeptides to delineate the requirements for the interaction of cap-ping enzymes with the CTD. They found that mammalian guanylyltransferase binds to 28-mer CTD peptides containing phosphoserine at either position 2 or position 5 of all four heptad repeats but not to unphosphorylated CTD peptides.
Here we have analyzed the binding of Candida cap methyltransferase to CTD Ser-2 and Ser-5 phosphopeptides; we also extended the binding studies to include a synthetic 28-mer CTD phosphopeptide ligand that is phosphorylated on both Ser-2 and Ser-5 of each heptad repeat. An N-terminal biotin moiety was added during chemical synthesis so that the peptides could be linked to streptavidin beads for affinity chromatography purposes (Fig. 7). CTD phosphopeptide-containing beads and control beads containing an unphosphorylated 28mer CTD peptide were incubated with purified recombinant Ccm1⌬136 protein. The beads were recovered by centrifugation and held in place with a magnet while the supernatant-containing free methyltransferase was withdrawn. The beads were washed with buffer, and the bead-bound material was eluted from the beads with 1% SDS. The input methyltransferase protein (L) and the free (F) and bead-bound (B) fractions were then analyzed by SDS-PAGE (Fig. 7A). The streptavidin polypeptide was stripped off the beads by 1% SDS and recovered in every bound eluate fraction.
We found that the biologically active Candida methyltransferase domain Ccm1⌬136 bound to either the Ser-5-PO 4 CTD or the Ser-2-PO 4 CTD peptide. About half of the input protein was retained on the beads. This binding was specific for CTD-PO 4 , because the methyltransferase did not bind at all to the beads containing unphosphorylated CTD peptide (Fig. 7A). The salient finding was that the methyltransferase bound more avidly to the CTD when Ser-2 and Ser-5 were phosphorylated than when either Ser2 or Ser5 were phosphorylated singly (Fig. 7A). This experiment shows that the affinity of the cap methyltransferase for the CTD can be modulated by altering the phosphorylation array.
Ccm1⌬136 and Ccm1⌬150 bound to the Ser-2-PO 4 /Ser-5-PO 4 CTD, whereas Ccm1⌬174 did not (Fig. 7B). We conclude from that the Ccm1p segment from amino acids 151 to 174 is required both for catalysis of cap methylation and for interaction of the methyltransferase with the CTD.
Complete Replacement of the Saccharomyces Capping Apparatus by Candida Enzymes-A plausible strategy for cappingspecific antifungal drug discovery is to identify compounds that inhibit the growth of yeast cells containing fungus-encoded capping activities without affecting the growth of otherwise identical yeast cells bearing the mammalian capping enzymes. Ideally, the fungal capping enzymes that sustain growth of the tester yeast cells would be those encoded by a clinically significant fungal pathogen. To achieve this scenario, we sought to construct S. cerevisiae strains in which the entire S. cerevisiae capping apparatus was replaced by enzymes from the pathogenic fungus C. albicans.
5-FOA-resistant isolates were then streaked on YPD plates at 30°C. Using colony size as a rough estimate of growth, it is surmised that cells containing an all-Candida capping apparatus grew as well as the cells containing an all-Saccharomyces capping apparatus (Fig. 8). Also shown on this plate is the growth of cells containing an all-mammalian capping system encoded by CEN MCE1 plus 2 HCM1(121-476) plasmids (Fig. 8). DISCUSSION The present study defines the essential domains of all three components of the C. albicans mRNA capping apparatus: the triphosphatase CaCet1p, the guanylyltransferase Cgt1p, and the methyltransferase Ccm1p. We used in vivo complementation of S. cerevisiae cet1⌬, ceg1⌬, or abd1⌬ mutants as a means of demarcating functional domain boundaries for the Candida proteins. Characterization of purified recombinant Candida capping enzymes and truncated versions thereof reveals (i) that for C-terminal deletions of Cgt1p, there is a clear correlation between retention of guanylyltransferase activity in vitro and complementation in vivo, (ii) that serial N-terminal deletions of CaCet1p elicit lethality in vivo before loss of catalytic activity in vitro. We infer that protein segments flanking the catalytic domain of CaCet1p mediate important ancillary functions in vivo. Our studies provide new insights into the influence of CTD phosphorylation on its binding to the fungal cap methylating enzyme. They also highlight how the physical and repeat. An N-terminal biotin anchors the CTD peptide to a streptavidin-coated magnetic bead. A, affinity chromatography was performed by mixing 4 g of Ccm1⌬136 with 0.5 mg of CTD peptide beads (estimated to contain 375-450 pmol of the unmodified or serinephosphorylated peptide, as specified) in 50 l of binding buffer (50 mM Tris HCl (pH 8.0), 50 mM NaCl, 1 mM DTT, 5% glycerol, 0.03% Triton X-100). After incubation for 30 min on ice, the beads were concentrated by microcentrifugation and then washed 3 times with 0.5 ml aliquots of binding buffer. After the third wash, the beads were resuspended in 50 l of binding buffer. Aliquots of the input protein fraction (L) (equivalent to 40% of total material loaded), the free-unbound fraction (F) (40% of the first supernatant), and the bead-bound fraction (B) (40% of the SDS eluate of the beads) were adjusted to 1% SDS and then analyzed by SDS-PAGE. A Coomassie Blue-stained gel is shown. The positions and sizes (kDa) of coelectrophoresed marker polypeptides are indicated on the left. The Ccm1⌬136 and streptavidin polypeptides are indicated on the right. B, affinity chromatography was performed by adsorbing 4 g of Ccm1⌬136, Ccm1⌬150, or Ccm1⌬174 to 0.5 mg of streptavidin beads containing the Ser-2-PO 4 Ser-5-PO 4 CTD peptide. Aliquots of the input protein fraction, the free-unbound fraction, and the bead-bound fraction were analyzed by SDS-PAGE as described above. functional properties of the capping enzymes are conserved among fungi and how the differences between fungal and metazoan capping enzymes, especially the triphosphatase, can be exploited pharmacologically.
Triphosphate-Guanylyltransferase Interactions-The C-terminal catalytic domain of CaCet1p from residue 257 to 520 suffices for phosphohydrolase activity in vitro (29). N-terminal deletions of 216 to 228 amino acids of CaCet1p eliminate the high affinity guanylyltransferase-binding site (30) and are lethal in vivo. This result is in keeping with the proposal that fungal guanylyltransferase chaperones the triphosphatase to the RNA polymerase II elongation complex (13). The finding that guanylyltransferase overexpression suppressed the growth defects of strains expressing CaCet1p N-terminal deletion mutants ⌬202, ⌬216, ⌬222, and ⌬228 suggests that a second "low affinity" guanylyltransferase-binding site exists distal to amino acid 229. The putative secondary site apparently does not promote sufficient interaction between the truncated Candida triphosphatase and the endogenous pool of Ceg1p to sustain growth. However, increased guanylyltransferase expression can sustain growth by driving guanylyltransferase binding to the low affinity site by mass action. Yeast cell growth apparently requires a threshold level of RNA triphosphatase and guanylyltransferase activities that is severalfold lower than the level in wild type yeast cells (16,19). Thus, it is probably not necessary to completely restore the wild type level of triphosphatase-guanylyltransferase interaction to sustain cell growth. The efficacy of CGT1 suppression declined when the CaCet1p segment from residue 229 to 256 was removed, to the point that the cells grow very slowly at 25°C and were both cold-sensitive (cs) and temperature-sensitive (ts) (Fig. 1). This result suggests that the peptide region from 229 to 256, which is not present in the S. cerevisiae triphosphatase (Fig. 1), may comprise part of the proposed low affinity guanylyltransferasebinding site on the Candida triphosphatase.
Cap Methyltransferase Interaction with Phosphorylated CTD-Previous studies of the binding of the S. cerevisiae cap methyltransferase to the RNA polymerase II CTD employed an immobilized ligand composed of recombinant GST-CTD that was enzymatically phosphorylated in vitro using HeLa extract as the source of CTD kinase. The fusion protein contained 15 copies of the YSPTSPS heptad sequence and an average of 3 serine-phosphates per GST-CTD polypeptide (2). Our present study of the cap methyltransferase-CTD interaction employed a defined 4-heptad CTD ligand in which the number and position of the phosphates were known and subject to manipulation. We have used the synthetic CTD phosphopeptides to establish that (i) the interaction of cap methyltransferase with the phosphorylated CTD described initially for S. cerevisiae is conserved in the pathogenic fungus C. albicans, (ii) Candida cap methyltransferase binds to CTD phosphorylated on either Ser-2 or Ser-5 but binds more avidly when both Ser-2 and Ser-5 are phosphorylated, and (iii) the N-terminal domain of Ccm1p is not required for binding to the phosphorylated CTD.
Ser-5 and Ser-2 are both extensively phosphorylated in vivo, and various CTD kinases differ in their site preference (34 -36). Zhou et al. (36) showed recently that the phosphorylation site specificity of the mammalian CTD kinase P-TEFb is altered by its interaction with the human immunodeficiency virus Tat protein. P-TEFb phosphorylates Ser-2 of the CTD heptad in the absence of Tat, but it phosphorylates both Ser-2 and Ser-5 when Tat is present (36). The observation of better binding of the Candida cap methyltransferase when both CTD serines are phosphorylated is, to our knowledge, the first example of a specific "added value" incurred by simultaneous modification of both positions. This raises the prospect that dynamic remodel-ing of the CTD phosphate array may regulate the efficiency or timing of cotranscriptional cap methylation and the longevity of the association of the cap methyltransferase with the transcription elongation complex.
Tools for Antifungal Drug Discovery-The availability of isogenic yeast strains containing Candida versus mammalian capping systems provides an attractive means of drug discovery aimed at blocking cap formation in pathogenic fungi. Any compound that is selectively cytotoxic to the all-Candida strain but not to the all-mammalian strain is likely to be a specific inhibitor of fungal capping. Secondary screens for cytotoxicity comparing strains in which only a subset of the Candida capping activities are replaced by a mammalian enzyme could pinpoint which of the gene products is targeted by such a compound. The availability of recombinant versions of all three Candida capforming enzymes should facilitate the elucidation of structure activity relationships in vitro for inhibitory compounds.