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INTRODUCTION |
The m7GpppN cap structure of eukaryotic mRNA is formed by
three enzymatic reactions as follows. (i) The 5' triphosphate end of
the nascent pre-mRNA is hydrolyzed to a diphosphate by RNA 5'
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.
The mRNA capping apparatus differs in significant respects in
fungi, metazoans, protozoa, and viruses (1). Hence, the cap-forming enzymes are potential targets for antifungal, antiviral, and
antiprotozoal drugs that would interfere with capping of pathogen
mRNAs but spare the mammalian host capping enzymes. Mammals encode
a two-component capping system consisting of a bifunctional
triphosphatase-guanylyltransferase polypeptide (named Mce1p in the
mouse and Hce1p in humans) and a separate methyltransferase polypeptide
(Hcm1p in humans) (2-8). The budding yeast Saccharomyces
cerevisiae encodes a three-component system consisting of separate
triphosphatase (Cet1p), guanylyltransferase (Ceg1p), and
methyltransferase (Abd1p) gene products (9-12). Cet1p and Ceg1p
interact in trans to form a bifunctional heteromeric enzyme
(13). Although the physical association of the triphosphatase and
guanylyltransferase activities is common to budding yeast (in
trans) and mammals (in cis), the structures and
catalytic mechanisms of the yeast and mammalian RNA triphosphatases are completely different (14-17).
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, 25-27), 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, 25-27). 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.
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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 CaCET1(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 3' 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 3' 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 A600 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
80 °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 µg/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
80 °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 3' 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. N-terminal 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 (2µ 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 A600 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
80 °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 µg/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 µg 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
80 °C.
Yeast Strains--
YBS50 (MATa leu2 ade2 trp1
his3 ura3 can1 ceg1::hisG cet1::LEU2
p360-CET1/CEG1) is deleted at the chromosomal loci encoding RNA
triphosphatase and guanylyltransferase. YBS40 (MATa
leu2 ade2 trp1 his3 ura3 can1
abd1::hisG p360-ABD1) and YBS10 (MAT
ura3 trp1 lys2 his3 leu2 abd1::LEU2 p360-ABD1) are
deleted at the chromosomal locus encoding cap methyltransferase. YBS52
(MATa leu2 ade2 trp1 his3 ura3 can1
ceg1::hisG cet1::LEU2 abd1::KAN
p360-CET1/CEG1/ABD1) is deleted at the chromosomal loci encoding all
three components of the yeast capping apparatus.
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RESULTS |
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).

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Fig. 1.
In vivo deletion analysis of
C. albicans RNA triphosphatase. The amino acid
sequence of S. cerevisiae (Sce) Cet1p from
residues 241-291 is aligned with the homologous segment of C. albicans (Cal) CaCet1p. The margins of the serial
N-terminal deletion alleles of CaCET1 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+ 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:
++ indicates wild type colony size at all temperatures; + 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.
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In the present in vivo experiments, CaCET1
expression was driven by the TPI1 promoter, and
CGT1 expression was driven by the strong constitutive
GPD1 promoter. The temperature-sensitive (ts) phenotype of the CaCET1(203-520) triphosphatase
mutant (
202) was completely suppressed by GPD-CGT1. The
temperature-sensitive (ts) phenotype was also suppressed
when GPD-CEG1 was present in cis on a
CaCET1(203-520) plasmid (Fig. 1). The new and instructive findings were that GPD-CGT1 suppressed the lethality of the
more extensively truncated
216,
222, and
228 mutations of
Candida triphosphatase (Fig. 1). Yeast cells containing
GPD-CGT1 plus CaCET1(217-520),
CaCET1(223-520), or CaCET1(229-520) formed wild type-sized colonies on rich medium at 16, 25, 30, and 37 °C (not shown). The segment of CaCet1p from residues 203 to 228 corresponds to
the guanylyltransferase-binding site of S. cerevisiae Cet1p (30). Apparently, the overexpression of Candida
guanylyltransferase can compensate for the loss of key contacts between
the triphosphatase and guanylyltransferase. The lethal
216 and
222 mutations of Candida triphosphatase were also
suppressed by GPD-CEG1 (Fig. 1).
The lethal CaCET1(257-520) allele (
256) was weakly
complemented by GPD-CGT1 at 25 °C. Cells containing
CaCET1(257-520) plus GPD-CGT1 formed tiny
colonies on rich medium at 25° and were unable to grow at either
15° or 37 °C, i.e. they were cold-sensitive and
temperature-sensitive (Fig. 1). The
266 allele was lethal at all
temperatures, even when cotransformed with GPD-CGT1.
Genetic and Biochemical Analysis of Candida RNA
Guanylyltransferase--
The C. albicans CGT1 gene encodes
a 449- amino 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(1-408), and
CGT1(1-387) mutants were viable, whereas
CGT1(1-367) was lethal (Fig.
2A).

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Fig. 2.
Deletion analysis of Candida
guanylyltransferase. A, yeast strain YBS50
(ceg1 ) was transformed with CEN TRP1 plasmids
containing the indicated genes. Trp+ isolates were streaked
on agar containing 0.75 mg/ml 5-FOA. The plate was photographed after
incubation for 3 days at 30 °C. B, polypeptide
composition. Aliquots (4 µg) of the nickel-agarose preparations of
wild type Cgt1p and truncation mutants C 23, C 41, C 62, and
C 82 were analyzed by SDS-PAGE. A Coomassie Blue-stained gel is
shown. The positions and sizes (in kDa) of marker proteins are
indicated on the left. C, guanylyltransferase activity.
Reaction mixtures (20 µl) contained 50 mM Tris HCl (pH
8.0), 5 mM DTT, 5 mM MgCl2, 0.17 µM [ 32P] GTP, and 0.5 µg of the
indicated enzyme preparations. The reaction products were resolved by
SDS-PAGE. An autoradiograph of the dried gel is shown. The positions
and sizes (in kDa) of prestained marker polypeptides are indicated on
the left. WT, wild type.
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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 His10-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 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-[32P]GMP adduct (EpG) when the protein
fractions were incubated with [
-32P]GTP and magnesium.
The activity detected in the soluble lysate (Fig. 3B,
lane S) was adsorbed to Ni2+-agarose and eluted
in the 100 and 200 mM imidazole fractions in parallel with
the recombinant Cgt1p polypeptide (Fig. 3B).

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Fig. 3.
Purification and guanylyltransferase activity
of recombinant Cgt1p. A, nickel-agarose chromatography.
The polypeptide compositions of the soluble lysate of
isopropyl-1-thio- -D-galactopyranoside-induced
BL21(DE3)/pET-Cgt1 cells (lane S), the nickel-agarose
flow-through (lane F), and wash (lane W)
fractions, and the 50, 100, 200, and 500 mM imidazole
eluate fractions were analyzed by SDS-PAGE. The polypeptides were
visualized by staining with Coomassie Blue dye. The positions and sizes
(kDa) of coelectrophoresed marker polypeptides are indicated on the
left. The recombinant Cgt1p is indicated by an arrow on the
right. B, guanylyltransferase activity. Reaction mixtures
(20 µl) contained 50 mM Tris HCl (pH 8.0), 5 mM DTT, 5 mM MgCl2, 0.17 µM [ -32P] GTP, and 1 µl of the soluble
lysate or the indicated nickel-agarose fractions. The reaction products
were resolved by SDS-PAGE. An autoradiograph of the dried gel is shown.
The positions and sizes (in kDa) of prestained marker polypeptides are
indicated on the left. The enzyme-[32P]GMP complex
(EpG) is denoted by the arrow on the right.
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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.

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Fig. 4.
Sedimentation of Candida
guanylyltransferase. Aliquots (30 µg) of the
nickel-agarose preparations of wild type Cgt1p (panel A),
C 62 (panel B), or C 82 (panel C) were mixed
with catalase (25 µg), bovine serum albumin (BSA) (25 µg), and cytochrome c (cyt c, 25 µg) in 0.2 ml of buffer G (50 mM Tris HCl (pH 8.0), 100 mM
NaCl, 1 mM EDTA, 2 mM DTT, 0.05% Triton
X-100). The mixtures were layered onto a 4.8-ml 15-30% glycerol
gradient containing buffer G. The gradients were centrifuged in a
Beckman SW50 rotor at 50,000 rpm for 16 h at 4 °C. Fractions
(0.2 ml) were collected from the bottoms of the tubes. Aliquots (20 µl) of odd-numbered fractions were analyzed by SDS-PAGE along with
samples of the input protein mixtures (lane L) for each
gradient. Polypeptides were visualized by staining with Coomassie Blue
dye.
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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-[32P]GMP adduct during a
10-min reaction at 37 °C with 5 µM
[
32P]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 MgCl2 (not shown). Cgt1p-[32P]GMP formation in the presence of manganese was
optimal at 1.25 mM MnCl2 (not shown). The yield
of Cgt1p-[32P]GMP increased with GTP concentration up to
2.5 µM GTP and leveled off at 5 µM GTP (not
shown). Kinetic analysis showed that Cgt1p-[32P]GMP
formation in the presence of 5 µM GTP increased with time up to 2 min and plateaued at 5 min (data not shown). Approximately 9%
of the input Cgt1p polypeptide was labeled with [32P]GMP
during the in vitro reaction with 5 µM GTP.
Effects of Cgt1p Deletions on Binding to
Cet1(232-265)--
S. cerevisiae RNA triphosphatase
(Cet1p) and RNA guanylyltransferase (Ceg1p) interact in vivo
and in vitro to form a bifunctional mRNA capping enzyme
complex. Although it is not known if CaCet1p and Cgt1p exist as a
complex in vivo in C. albicans, the findings of
Yamada-Okabe et al. (26) that 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 retained 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.

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Fig. 5.
Effects of Cgt1p deletions on binding to
Cet1(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 µg 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 µl 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 µl 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.
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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+ 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.

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Fig. 6.
Characterization of Candida
cap methyltransferase. A, complementation of
abd1 . Yeast strain YBS10 (abd1 ) was
transformed with CEN TRP1 plasmids containing the indicated
genes. Trp+ isolates were streaked on agar containing 0.75 mg/ml 5-FOA. The plate was photographed after incubation for 3 days at
30 °C. B, protein purification. Aliquots (3.5 µg) of
the peak glycerol gradient fractions of wild type Ccm1p
(WT), Ccm1(137-474)p ( 136), Ccm1(151-474)p ( 150),
and Ccm1(175-474)p ( 174) were analyzed by SDS-PAGE. A Coomassie
Blue-stained gel is shown. The positions and sizes (in kDa) of marker
proteins are indicated on the left. C, methyltransferase
activity. Reaction mixtures (10 µl) containing 50 mM
Tris-HCl (pH 7.5), 5 mM dithiothreitol, 50 µM
AdoMet, 20 fmol of 32P cap-labeled poly(A), and recombinant
Ccm1p as specified were incubated at 37 °C for 10 min. The reaction
products were digested with nuclease P1 and analyzed by
polyethyleneimine-cellulose TLC as described (Saha et al.
(8)). The extent of cap methylation [m7GpppA/(m7GpppA + GpppA)] was determined by scanning the chromatogram with a
phosphorimager. D, overexpression of Ccm1(151-474)p
partially restores function in vivo. Strain YBS10
(abd1 ) was transformed with 2µ TRP1 plasmids
containing the CCM1 or CCM1(151-474) [ 150]
genes. Trp+ isolates were streaked on agar containing 0.75 mg/ml 5-FOA. 5-FOA-resistant isolates were tested for growth on YPD
agar. The YPD plate was photographed after incubation for 4 days at
30 °C.
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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 His10-tagged 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 32P 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 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 ~5
µM 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 2µ vector under the
control of the constitutive TPI1 promoter. abd1
cells bearing the 2µ 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 2µ
CCM1(151-474) cells formed uniformly small colonies
compared with 2µ CCM1 cells (Fig. 6D). 2µ
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 2µ 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 (2-4, 31).
The CTD, which is unique to polymerase II, is composed of a tandemly
repeated heptad motif (consensus sequence = 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 capping 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 28-mer 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.

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Fig. 7.
Binding of Candida cap
methyltransferase to CTD phosphopeptides. The 28-amino acid CTD
peptide ligand consisted of a tandem array of 4 heptad repeats, as
shown, with either no modification (no PO4) or with
phosphoserine introduced at position 2 (Ser2-PO4),
position 5 (Ser5-PO4), or positions 2 and 5 (Ser2-PO4 Ser5-PO4) of each
heptad repeat. An N-terminal biotin anchors the CTD peptide to a
streptavidin-coated magnetic bead. A, affinity
chromatography was performed by mixing 4 µg of Ccm1 136 with 0.5 mg
of CTD peptide beads (estimated to contain 375-450 pmol of the
unmodified or serine-phosphorylated peptide, as specified) in 50 µl
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 µl 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 µg
of Ccm1 136, Ccm1 150, or Ccm1 174 to 0.5 mg of streptavidin
beads containing the Ser-2-PO4 Ser-5-PO4 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.
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We found that the biologically active Candida
methyltransferase domain Ccm1
136 bound to either the
Ser-5-PO4 CTD or the Ser-2-PO4 CTD peptide.
About half of the input protein was retained on the beads. This binding
was specific for CTD-PO4, 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-PO4/Ser-5-PO4 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 capping-specific
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.
A CEN TRP1 expression plasmid (pCan-CAP-1) was constructed
that contained three C. albicans genes under the control of
S. cerevisiae promoters, as follows:
CaCET1(179-520) controlled by the TPI1 promoter;
CGT1 driven by the GPD1 promoter; CCM1
controlled by the TPI1 promoter. Another CEN TRP1
expression plasmid (pCan-CAP-2) was constructed that contained
CaCET1(179-520) controlled by the TPI1 promoter,
CGT1 driven by the GPD1 promoter, and
CCM1(137-474) controlled by the TPI1 promoter.
pCan-CAP-1 and pCan-CAP-2 were transformed into the S. cerevisiae
cet1
ceg1
abd1
triple-deletion strain YBS52 (8), the growth of which is sustained by a CEN URA3
CET1 CEG1 ABD1 plasmid. Plasmid shuffle experiments showed that
YBS52 cells transformed with either pCan-CAP-1 and pCan-CAP-2 or with
an "all-Saccharomyces" capping enzyme expression plasmid (CEN TRP1 CET1 CEG1 ABD1) grew on 5-FOA, whereas plasmids
containing only CET1, CEG1, or ABD1
did not complement growth on 5-FOA (not shown).
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 2µ HCM1(121-476)
plasmids (Fig. 8).

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Fig. 8.
Complete replacement of the
Saccharomyces capping apparatus by Candida
enzymes. Yeast strain YBS52 (cet1
ceg1 abd1 ) was transformed with
TRP1 plasmids as specified below and then selected on 5-FOA.
FOA-resistant isolates were streaked on a YPD agar plate. The plate was
photographed after incubation for 3 days at 30 °C. ABD1 CET1
CEG1 was transformed with the three S. cerevisiae genes
on a single CEN plasmid. CaCET1(179-520),
CGT1 CCM1(137-474), and CaCET(179-520) CGT1
CCM1 were transformed with the indicated sets of three C. albicans genes on CEN plasmids pCAN-CAP-2 and
pCAN-CAP-1. MCE1 HCM1(121-476) was cotransformed with
CEN MCE1 and 2µ HCM1(121-476).
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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 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
guanylyltransferase-binding 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 remodeling 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
cap-forming enzymes should facilitate the elucidation of structure
activity relationships in vitro for inhibitory compounds.