Mutations in a Peptidylprolyl-cis/trans-isomerase
Gene Lead to a Defect in 3'-End Formation of a Pre-mRNA in
Saccharomyces cerevisiae*
Jean
Hani
§,
Birte
Schelbert¶,
Anne
Bernhardt¶,
Horst
Domdey
,
Gunter
Fischer¶,
Karin
Wiebauer
, and
Jens-U.
Rahfeld¶
From the
Genzentrum der
Ludwig-Maximilians-Universität München,
Feodor-Lynen Strasse 25, 81377 München and
¶ Forschungstelle "Enzymologie der Proteinfaltung" der
Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V.,
Kurt-Mothes Strasse 3, 06120 Halle/Saale, Germany
 |
ABSTRACT |
In a genetic screen aimed at the identification
of trans-acting factors involved in mRNA 3'-end
processing of budding yeast, we have previously isolated two
temperature-sensitive mutants with an apparent defect in the 3'-end
formation of a plasmid-derived pre-mRNA. Surprisingly, both mutants
were rescued by the essential gene ESS1/PTF1 that encoded a
putative peptidylprolyl-cis/trans-isomerase (PPIase) (Hani,
J., Stumpf, G., and Domdey, H. (1995) FEBS Lett. 365, 198-202). Such enzymes, which catalyze the
cis/trans-interconversion of peptide bonds N-terminal of
prolines, are suggested to play a role in protein folding or
trafficking. Here we report that Ptf1p shows PPIase activity in
vitro, displaying an unusual substrate specificity for peptides
with phosphorylated serine and threonine residues preceding proline.
Both mutations were found to result in amino acid substitutions of
highly conserved residues within the PPIase domain, causing a marked
decrease in PPIase activity of the mutant enzymes. Our results are
suggestive of a so far unknown involvement of a PPIase in mRNA
3'-end formation in Saccharomyces cerevisiae.
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INTRODUCTION |
Despite intensive efforts to unravel the complex process of
mRNA 3'-end formation in Saccharomyces cerevisiae, the
list of participating factors still awaits its completion.
We have recently isolated a gene complementing the phenotype of two
temperature-sensitive yeast mutants that were impaired in mRNA
3'-end formation. This gene, designated PTF1
(processing/termination factor 1; identical with the previously
described ESS1 (1)), encodes a protein that, by virtue of
sequence similarity, was identified as a
peptidylprolyl-cis/trans-isomerase
(PPIase)1 (2). PPIases are
ubiquitous enzymes that catalyze the interconversion from
cis to trans of peptide bonds preceding a proline
and are thought to accelerate this often rate-limiting step in the
folding of a number of proteins in vivo (3-6).
PPIases are divided in three families, based on their sensitivities
toward two clinically relevant immunosuppressants: the cyclosporin
A-binding proteins (cyclophilins), the FK506-binding proteins, and a
third family, named after the Escherichia coli protein
parvulin, which is not inhibited by either of the two drugs (for review
see Refs. 3-6). In addition, the members of each family are
characterized by conserved but distinct amino acid domains. By this
criterion, PTF1 was predicted to belong to the parvulin
family of PPIases (2).
Although disruption of PPIase genes did not generally impair cell
growth (7-8), PTF1 was the first PPIase gene shown to be essential for cell viability (1). In fact, PTF1 is the only essential PPIase gene in S. cerevisiae as demonstrated more
recently by the viability of a yeast mutant lacking the remaining 12 PPIases, members of the other two immunosuppressant binding families.
(8). So far, the only other example of an essential PPIase is the
recently discovered PIN1, a human protein, that is structurally and
functionally related to Ptf1p (9-10).
In this paper we describe the genetic screen that led to the isolation
of PTF1 and the phenotypes of the two temperature-sensitive strains carrying mutations in this gene. Moreover, we demonstrate that
Ptf1p displays PPIase activity in vitro and that this
activity is drastically reduced in the two mutant PTF1 proteins
isolated at nonpermissive temperatures. The intriguing observation that yeast cells harboring mutant PTF1 proteins are defective in the 3'-end
formation of a plasmid-encoded pre-mRNA invites speculations on the
possible role of a PPIase in mRNA 3'-end processing and/or transcription termination in budding yeast.
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EXPERIMENTAL PROCEDURES |
Strains--
E. coli XL 1 Blue (recA1 endA1
gyrA96 thi-1 hsdR17 supE44 relA1 lac [F'proAB
lacIqZ
M15 Tn10(tet)]) (Stratagene) was
used for cloning procedures. E. coli BL21 (DE3)
(F
ompT rB
mB
(DE3)) (Novagen) was used
for expression of Ptf1p.
Yeast DH484 cells (MATa ade2-1 leu2-3 leu2-112
can1-100 trp5-48 ura4-11 lys1-1) were used for the production of
mutants (11). DBY874 cells (MAT
, his5-2 (o),
leu2-1 (o), can1-100 (o), ura3-52, trp5-48 (o)) were used for mating with the mutant YPM2 (12). EJ101(20B-12-1) (MAT
, trp1,
pro1-126, prb1-112, pep4-3,
prc1-126) was used for the production of yeast whole cell
extracts (13). W303 (MATa/MAT
,
ade2/ade2 his3/his3,
leu2/leu2, trp1/trp1,
ura3/ura3) was used as a diploid control
(14).
Plasmid Constructions--
The screening plasmid pJH702CEN was
constructed with plasmid pPZ[LEU2] (15) as basis vector in
which the TRP1 gene was replaced by the LEU2
gene. In the single BamHI site of pPZ[LEU2] the
following fragments were inserted: a 730-bp long
BamHI/BglII fragment containing the
ACT1 promoter plus flanking sequences, a modified
ADH1 terminator without stop codons, and a 3073-bp
BamHI fragment from pMC1871 (Pharmacia, Freiburg, Germany)
containing the complete lacZ gene. The modified
ADH1 terminator consisted of a 97-mer synthetic DNA fragment
with bases exchanged at the three stop codons to provide an open
reading frame through the whole DNA fragment (see Fig. 4).
The control plasmid pJH712CEN contained, instead of the modified
ADH1 terminator sequence, a 28-bp synthetic DNA fragment connecting in frame the ACT1 sequence with the
lacZ gene (Fig. 4).
For the generation of pGALPTF1, which contains the PTF1 gene
under control of the GAL1 promoter, the EcoRI
fragment of YIpH-GESS (1) carrying the ESS1 gene under
GAL1 control was purified and inserted into pBluescript II
KS
(16-17). From this construct, a 60-nt-long fragment
which still contained a part of the 5' non-transcribed region was
deleted by site-specific mutagenesis according to Kunkel (18).
For the overexpression of a mutated parvulin gene, the corresponding
DNA was amplified by PCR from pSEP38 (19) with the primer MP1 (5'-GCA
GGA TCC GAT GAC GAT GAC AAA GCA AAA ACA GCA GCA GCA C-3') which
contains an enterokinase cutting site and a BamHI
restriction site and the primer MP3 (5'-CGG GCG AGC TCG GTA AAG CTA-3')
which contains a SacI restriction site. The resulting 532-bp
fragment was cloned into pKSII
. Site-specific mutagenesis
was carried out as described by Kunkel (18) with the oligonucleotide
primers MP48 (5'-CAG GCA AAC GCG GCG ATG ATT TAG GTG AAT TCC-3') and
MP83 (5'-GCA CAC CCA GTT CTC ATA TCA CAT CAT TAA G-3'). The mutagenesis
led to amino acid exchanges at position 48 from Gly to Asp
(corresponding to the mutant Ptf1p-2) and at position 83 from Gly to
Ser (corresponding to the mutant Ptf1p-5). The mutated DNA fragments
were inserted into pQE30 which harbors a His6-tag coding sequence.
The construction of the plasmids pHD509s (ACT1 terminator),
pHD511s (CYC1 terminator), pHD512s (YPT1
terminator), pSH101 (ADH1 terminator 143 nt), pSH102
(ADH1 terminator 80 nt) has been described in Heidmann
et al. (20). The 3'-terminal fragments of ADH1
had a length of 143 (pSH101) or 80 nt (pSH102). The 3'-terminal region of ACT1 consisted of a 354-nt-long Sau3AI
fragment (pHD509s), the CYC1 region consisted of a
241-nt-long Sau3AI fragment (pHD511s), and the
YPT1 region of a 494-nt-long Sau3AI fragment (pHD512s).
EMS Mutagenesis and Tetrad Analysis of Yeast Cells--
EMS
mutagenesis was performed as described by Lawrence (21). The production
of diploid yeast cells, the sporulation analysis, and tetrad analysis
were done according to Haber and Halvorson (22) and Sherman and Hicks
(23).
RNA Analysis--
Overnight cultures of YPM2 and of DH484 (200 ml), transformed with the selection plasmid pJH702CEN, were grown in
selective medium to an A600 = 1 at 23 °C. A
100-ml aliquot was removed for RNA preparation, and the remaining 100 ml were mixed with the same amount of medium prewarmed to 50 °C.
Incubation was continued at 37 °C for 6 h when another 100 ml
were removed for RNA preparation. The last aliquot of 100 ml was taken
after another 13 h. Total RNA was prepared from the collected
yeast cells by the hot phenol method as described by Köhrer and
Domdey (24). Poly(A)+ RNA was isolated with an mRNA
purification kit purchased from Amersham Pharmacia Biotech. Then all
RNA aliquots were treated with RNase-free DNase to remove traces of
remaining DNA.
For Northern blot analysis, 20 µg of glyoxal-treated total RNA was
separated on a 1.5% agarose gel and transferred to a Hybond N nylon
membrane (Amersham Pharmacia Biotech).
For the quantitation of processed and unprocessed chimeric
ACT1-ADH1-lacZ transcripts, RT-PCR
products were generated with the primers JH24 (5'-AGA TTT TTC ACG CTT
ACT G-3'), JH25 (5'-TAA GAA ATT CGC TTA TTG A-3'), JH26 (5'-AAG CGA ATT
TCT TAT GAT T-3'), and JH27 (5'-GGT TAC GTT GGT GTA GAT G-3') (see Fig.
3) in the presence of [32P]dATP. Radioactivity on the
nylon membrane was measured with a PhosphorImager.
Production of Recombinant Ptf1p--
The coding region of
PTF1 was amplified by PCR with the synthetic
oligonucleotides JH34 (5'-AGG AAC ATA TGC CAT CTG ACG TAG CAT CG-3')
and GS2 (5'-AGG AAG GAT CCG AGG TGG AGA AGC AAA TGC C-3') and inserted
into the E. coli expression vector pET15b (25-26) which
includes the codons for an oligohistidine tag (Novagen). The expressed
protein therefore contained an oligohistidine tag at its N terminus,
followed by a thrombin recognition site. For the production of
authentic Ptf1p without the oligohistidine tag, the E. coli
expression vector pET11d was used (Novagen).
For the production of the mutated proteins Ptf1-2p (corresponding to
the mutant Ptf1p from YPM2) and Ptf1-5p (corresponding to the mutant
Ptf1p from YPM5), the PCR-amplified construct was modified by
site-specific mutagenesis according to Kunkel (18) with the synthetic
oligonucleotides JH37 (5'-TCT CCC GAA CCA GCC TAG GTC GTC GCC TCG CTT
GTA TG-3') for production of Ptf1-2p and JH38 (5'-ACC TAC CCG CTT GAT
TAC ATG AAC ACT GCT TCC TGA TTC AAC-3') for production of Ptf1-5p.
E. coli BL21(DE3) (Novagen) cells harboring the expression
plasmids were grown at room temperature to an
A600 of 0.6 and then induced with 4 mM isopropyl-1-thio-b-D-galactopyranoside at
37 °C for another 4 h.
Protein Purification--
For the purification of the
oligohistidine-tagged fusion proteins, cells were harvested by
centrifugation, resuspended in 2 mM Tris buffer,
pH 8.0, and ruptured in an SLM Aminco French pressure cell. The cell
lysate was stirred with 0.1% (v/v) Benzonase for 15 min at 4 °C and
centrifuged at 20,000 × g for 30 min at 4 °C. The
supernatant was applied to a Ni-nitrilotriacetic acid column (1 × 4 cm, Qiagen), equilibrated with 2 mM Tris buffer, pH 8.0. The column was washed with 100 ml of equilibration buffer to remove
unbound protein. Bound protein was eluted by a linear gradient from 0 to 0.5 M imidazole in 60 ml of 2 mM Tris
buffer, pH 8.0. The Ptf1p-containing fractions were detected by
Coomassie stained SDS-polyacrylamide gel electrophoresis and
concentrated with a Filtron OMEGACELL, exclusion size 10,000 Da.
Samples (1 ml) were applied to a Superdex 75 gel filtration column
(1.6 × 60 cm, Amersham Pharmacia Biotech), equilibrated with 10 mM HEPES buffer, pH 7.8, containing 150 mM KCl,
1.5 mM MgCl2, and 0.5 mM dithioerythritol. The flow rate was 0.8 ml/min. Fractions containing Ptf1p were pooled, and protein was dialyzed overnight against 3 liters
of 10 mM HEPES buffer, pH 7.5, at 6 °C. Ion exchange chromatography was performed for further protein purification using a
Fractogel EMD SO3
650(M) column
(1 × 6 cm) (Merck). The column was equilibrated with 10 mM HEPES buffer, pH 7.5. Protein was applied to the column at a flow rate of 1.5 ml/min, after which the column was washed with
100 ml of equilibration buffer. PPIase-containing fractions were
obtained by running a linear gradient from 0 to 1 M KCl in 100 ml of 35 mM HEPES buffer, pH 7.5. The fractions were
pooled and dialyzed for 3 h against 2 liters of 10 mM
HEPES buffer, pH 7.5, and applied to a Fractogel TSK AF-Blue column
(1 × 6 cm) equilibrated with dialysis buffer. Homogenous Ptf1p
was obtained by running a linear gradient from 0 to 2 M KCl
in 60 ml of 10 mM HEPES buffer, pH 7.5.
For the purification of authentic heterologously expressed Ptf1p, the
same procedure was used with the exception of the initial chromatographic step. The supernatant derived from ultracentrifugation was passed through a Fractogel EMD DEAE-650(M) column (2.5 × 20), equilibrated with 2 mM Tris buffer, pH 8.0. Ptf1p passed
unbound through the column and was applied to gel filtration as
described above.
For the purification of the mutated recombinant parvulins, the
harvested cells were resuspended in 2 mM Tris buffer, pH
8.0, containing a protease inhibitor mix as recommended by the
manufacturer (Complete Protease Inhibitor Mixture Tablets, Boehringer
Mannheim), lysed by French press, and ultracentrifuged as described for
Ptf1p. The supernatant was applied to a Ni-nitrilotriacetic acid
column, and His-tagged proteins were purified as described for Ptf1p. The eluted parvulin-containing fractions were dialyzed two times for
1 h against 3 liters of 10 mM HEPES buffer, pH 7.5, containing 1 mM dithiothreitol and the protease inhibitor
mix. Ion exchange chromatography using a Fractogel EMD
SO3
650(M) column (1 × 6 cm)
(Merck) was carried out for further purification as described above.
The recombinant proteins were eluted with a linear gradient from 0 to 2 M KCl in 10 mM HEPES buffer, pH 7.5.
Production of Polyclonal Antibodies Against Recombinant
Ptf1p--
Recombinant purified Ptf1p expressed in E. coli
was used for production of polyclonal antibodies in rabbits. The
immunization was done according to the instructions supplied with the
Ribi Adjuvant System.
Western Blot Analysis--
SDS-polyacrylamide gel
electrophoresis was performed according to Laemmli (27). The protein
was transferred onto nitrocellulose according to Haid and Suissa (28).
Immunoreactions were carried out with the ECL Western blotting kit from
Amersham Buchler.
PPIase Assay--
For the proteolytic assay, measurements were
carried out as described previously (29), using Suc-Ala-Xaa-Pro-Yaa-4NA
(where NA is nitroanilide) as enzyme substrate. Xaa and Yaa denote
variable aminoacyl residues at this position.
With respect to the prolyl bond, such peptide substrates exist in an
equilibrium of about 5-20% cis- and 80-95%
trans-confomers. Commonly known endopeptidases like
chymotrypsin, trypsin, or subtilisin split off the C-terminal 4NA
residue only in the trans population of these
proline-containing substrates. Thus, in the presence of sufficient
amounts of protease in the reaction mixture, the trans
population is rapidly cleaved, whereas the cis population remains intact (first rapid phase). The following slow isomerization reaction is accelerated by PPIases, resulting in the cleavable trans substrate (second, slow phase). The time course of
4-nitroaniline release was determined by monitoring the absorbance at
390 nm in a Hewlett-Packard 8452 diode array UV-visible
spectrophotometer in 0.5-s intervals for a total of 4 min. Reported
data are given as the mean value of three to five measurements.
A disadvantage of the protease-coupled assay is the requirement for
high concentrations of helper proteases in order to obtain the
two-phase reaction described above. This assay could therefore only be
used for proteins that proved to be resistant toward attack from these
enzymes, at least for the duration of the experiment. The intactness of
each putative PPIase after the reaction was examined on Western blots.
In the case of recombinant Ptf1p, thrombin and trypsin cleaved off the
oligohistidine tag at the inserted thrombin cleavage site but did not
further digest Ptf1p. Subtilisin did not attack the protein under the
conditions of PPIase measurements, whereas chymotrypsin digested Ptf1p
completely within 2 min of incubation (2). Thus, in the PPIase assay of
Ptf1p, trypsin (0.08 and 0.02 mg/ml) was used for cleavage of the
Lys-4NA and Arg-4NA bonds, respectively, and subtilisin (0.04 mg/ml),
for cleaving the Phe-, Tyr- Leu- and Met-4NA bonds. Substrates were purchased from Bachem (Heidelberg) or synthesized according to Schutkowski et al. (30). Stock solutions of various
substrates were prepared in dimethyl sulfoxide.
For inhibition studies, the Suc-Ala-Phe-Pro-Phe-4NA was used as
substrate for parvulin and Suc-Ala-Ala-Pro-Arg-4NA as substrate for
Ptf1p. FK506 was a gift from Fujisawa Pharmaceutical Co., Osaka. Stock
solutions of the inhibitors were prepared in 50% ethanol. The
incubation time was 5 min for FK506 and 15 min for cyclosporin A. Three
independent experiments were performed.
Protease-free Assay--
Because of the unexpected sensitivity
of the mutant proteins Ptf1-2p and -5p toward proteases, PPIase
activity was measured using a modification of the assay described by
Kofron et al. (31) and others
(32).2 This assay exploits
the difference in the absorption coefficients of cis and
trans conformers of the substrates Suc-Ala Ala
Pro-(NO2)Tyr-4-fluoranilide at 430 nm.
 |
RESULTS |
Identification and Characterization of Yeast Mutants Defective in
Pre-mRNA 3'-End Formation--
In an effort to identify
trans-acting factors participating in the 3'-end processing
reaction of yeast pre-mRNAs, a genetic selection system was
established, in which a defect in this reaction could be recognized by
the appearance of blue-colored colonies. The system was based on a
fusion construct, pJH702CEN, composed of a 3'-end truncated
ACT1 gene joined to the lacZ gene by a
minimum-sized ADH1 3'-end formation site in a centromere
vector (Fig. 1). The ADH1-derived sequences had been modified such that an open
reading frame was maintained throughout the originally noncoding
3'-region of the ADH1 gene and that translation of
read-through transcripts would result in a lacZ fusion
protein. Stable read-through transcripts were expected to occur only in
plasmid-carrying cells in which the formation of mRNA 3'-ends was
impaired.

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Fig. 1.
Representation of the centromere plasmid
constructs used in the genetic screen. A, schematic of the
ACT1-ADH1-lacZ fusion construct. The small rectangular
arrows show the transcription start sites. ATG marks
the translation start site; poly(A) indicates the
polyadenylation site. B, nucleotide and corresponding amino
acid sequences of the linker region connecting the truncated
ACT1 gene with the lacZ gene in pJH702CEN and
pJH712CEN. pJH702CEN, the original sequence was changed
through inversions of adjacent nucleotides at the three ADH1
stop codons (arrows) to generate an open reading frame.
pJH712CEN, the ADH1 termination region was
replaced by a short 7-nt-long oligonucleotide linker (boxed)
connecting the open reading frame of the truncated ACT1 gene
with the lacZ gene. The ACT1- and
lacZ-derived sequences are written in lowercase,
and the inserted sequences are written in uppercase.
pAI and pAII mark two of the three original
polyadenylation sites of the ADH1 gene.
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Transformation of wild type yeast with pJH702CEN (Fig. 1) yielded, as
expected, white colonies on 5-bromo-4-chloro-3-indolyl b-D-galactopyranoside-containing medium, as the
plasmid-derived transcripts were processed and polyadenylated at the
ADH1 3'-end formation site. In contrast, a positive control
construct (pJH712CEN, Fig. 1), in which the 3'-end truncated
ACT1 gene was directly connected with the lacZ
gene, gave rise to blue colonies on 5-bromo-4-chloro-3-indolyl b-D-galactopyranoside containing selective medium (data
not shown).
After EMS mutagenesis of 1.9 million yeast cells containing the
selection plasmid pJH702CEN, three viable mutants (yeast
processing mutants YPM2, YPM3, and YPM5) that
displayed the expected blue-color phenotype were isolated at 23 °C.
Two of them, YPM2 and YPM5, additionally showed temperature-sensitive
growth at 30 and 37 °C, respectively. The results of several control
experiments demonstrated that the mutations leading to the specific
phenotypes were chromosomal mutations (data not shown) as follows. (i)
The plasmids isolated from the mutants and retransformed into wild type
yeast led to a wild type phenotype, i.e. white colonies on
5-bromo-4-chloro-3-indolyl b-D-galactopyranoside-containing
plates. (ii) The DNA sequence of the ADH1 3'-end formation
site and the beginning of the lacZ gene was not altered in
the mutants. (iii) Mutants, which had been grown on non-selective
medium until they had lost their plasmid, turned blue again after
re-transformation with the original selection plasmid.
Shifting the YPM2-mutant cells from 23 °C to the nonpermissive
temperature of 37 °C led to a significant decrease of the amount of
poly(A)+ RNA within 2 h after the shift (Fig.
2).

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Fig. 2.
Gel electrophoresis of poly(A)
(lanes 1-4) and poly(A)+ RNA (lanes
5-8) isolated from YPM2 cells grown at 23 °C (lanes 1 and 5) and then shifted to 37 °C (lanes 2-4
and 6-8). RNA from equal amounts of cells was
loaded on a 2% agarose gel.
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To demonstrate that the blue phenotype of the mutants coincided with
the presence of the chimeric ACT1-ADH1-lacZ
read-through transcripts in the mutant cells, RT-PCR was performed with
total RNA isolated from the mutant YPM2 and wild type yeast grown at 23 and 37 °C, respectively. As a positive control, RNA from wild type
yeast DH484, which had been transformed with the test plasmid, was
isolated under identical conditions. Two sets of primer pairs were used
as follows: the primers JH24 and JH25 amplified all ACT1-ADH1-lacZ-derived transcripts
(Fig. 3A, product I), whereas the primers JH26 and JH27 were designed to amplify only those transcripts that had not been cleaved at the ADH1-derived
polyadenylation site (Fig. 3A, product II). The
radioactively labeled RT-PCR products, separated on agarose gels (Fig.
3B) were quantitated by scanning with a PhosphorImager. In
wild type cells, read-through transcripts (Fig. 3A, product
II) comprised only 2.5 and 1.5% of the total amount of plasmid-encoded
ACT1 transcripts (Fig. 3A, product I) at 23 °C
and 37%, respectively. In contrast, 10-fold higher levels of
read-through transcripts, i.e. 30%, were detected in the
mutant cells at 37 °C, with 13% already present at the permissive
temperature of 23 °C.

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Fig. 3.
Quantitative RT-PCR of transcripts derived
from the chimeric transcription unit contained in pJH702CEN in wild
type yeast (DH484) and mutant (YPM2). A, the positions of
the two primer pairs and their amplification products are shown.
Horizontal arrows represent the oligonucleotides used for
RT-PCR; I, RT-PCR product representing all transcripts
(3'-processed and non-processed); II, RT-PCR product
representing only non-processed transcripts. B, native
polyacrylamide gel electrophoresis of the RT-PCR products. The
temperatures at which the cells were grown (23 °C, and shifted for
6 h to 37 °C, respectively) are indicated.
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In order to test whether the observed increase in the amount of
read-through transcripts from the selection plasmid in YPM2 was
restricted to the special template, the transcripts of three other
yeast genes, ACT1, CYC1, and YPT1,
were examined on Northern blots. To facilitate the analysis, YPM2 cells
were transformed with plasmids containing 3'-terminal fragments of
these genes inserted between the ACT1 promoter and the
terminator of either ACT1 or ADH1 (Fig.
4 (20)). As a control, ADH1
was included with either a complete or truncated version of its
3'-terminal region (Fig. 4). The latter was very similar to the one
used in the mutant screen.

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Fig. 4.
Schematic presentation of the constructs,
which were examined for their ability to yield mature mRNA
3'-ends. The cloning of these constructs was described in Heidmann
et al. (10). Horizontal arrows indicate the
orientation of the insertions. SH16 and SH18
indicate regions where the corresponding oligonucleotides hybridize.
Restriction sites are as follows: BHI, BamHI;
DI, DraI; ERV, EcoRV,
HIII, HindIII; and SI,
SpHI.
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The plasmid constructs and positions of the oligonucleotide probes SH16
and SH18 are shown schematically in Fig. 4. The two probes enabled the
distinction between transcripts ending in the inserted fragment and
unprocessed products ending within the downstream adjacent
ADH1 or ACT1 terminal region.
Additionally, both probes could also detect endogenous ACT1
mRNA. The corresponding signal was used as a standard for the amount of RNA isolated from YPM2 cells that differed only in the DNA
constructs with which they had been transformed. Northern blot analysis
was carried out with total RNA from cells that had been grown at either
the permissive or the nonpermissive temperature (Fig.
5). The same blot was successively probed
with SH16 (Fig. 5A) and SH18 (Fig. 5B).

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Fig. 5.
Northern blot analysis of RNA transcripts
from cells that had been transformed with one of the constructs shown
in Fig. 4 and that had been grown at either 23 °C or shifted to
37 °C. In each lane, 10 µg of total RNA of YPM2 cells were
separated. M, DNA length standard digested with
HindIII. A, hybridization with oligonucleotide
SH16 (see Fig. 4). B, hybridization with oligonucleotide
SH18 (see Fig. 4).
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Hybridization performed with SH16 and RNA that had been synthesized at
23 °C resulted mostly in strong signals, corresponding well in size
to transcripts that had initiated at the plasmid-derived ACT1 promoter and ended at the polyadenylation sites within
the respective, gene-specific 3'-end insertions (Fig. 5A, lanes
1-5, and legend). In each lane, the endogenous ACT1
mRNA appeared above those signals. As judged by this internal
standard, the amount of total RNA in lanes 3 and
8 had apparently been underestimated, resulting in only weak
signals for the YPT1 transcript at both temperatures.
Hybridization of RNA isolated from cells cultured at 37 °C with the
same probe led to generally less intense signals for each of the
gene-specific transcripts and also for the endogenous ACT1 mRNA (Fig. 5A, lanes 6-10). As the amount of
total RNA loaded was kept the same, this result reflected again the
significant reduction of poly(A)+ RNA in mutant cells grown
at the non-permissive temperature (see Fig. 2).
Any read-through transcripts that extended into the second 3'-terminal
insertion of each DNA construct was expected to hybridize also to SH18
(Fig. 4), as this probe, in addition to endogenous ACT1
mRNA, could only light up transcripts that had failed to be
processed within the gene-specific 3'-end insertions. To facilitate the
evaluation, the blot in Fig. 5B was drastically overexposed as exemplified by the increased intensity of the internal
ACT1 standard.
The presence of a disproportionally strong signal at the position of
the endogenous ACT1 mRNA in lane 8 of Fig.
5B clearly demonstrated that the YPT1 transcript,
present in mutant cells grown at 37 °C, was a genuine read-through
transcript. A similarly clear result was obtained for the
ADH1 transcript expressed from the DNA construct carrying
the truncated version of the ADH1 3'-terminal element; a
read-through transcript was visible at 37 °C (lane 9) and
at 23 °C (lane 4). Interestingly, the presence of the
complete ADH1 3'-end formation site totally suppressed the
formation of longer transcripts which seems also to be true for the
ACT1 and CYC1 constructs. The shorter RNA
species, detected in the overexposed autoradiogram of Fig.
5B, lanes 1 and 3, and 6 and 8, are presumably transcripts initiating at cryptic
promoter sites downstream of the ACT1 promoter.
To establish whether the mutant phenotype could also morphologically be
distinguished from wild type cells, mutant and wild type protoplasts
were inspected under the microscope. This analysis revealed that
protoplasts of the mutant YPM2, maintained at the non-permissive
temperature for 10 h, had about double the diameter of wild type
protoplasts (data not shown).
In addition, immunofluorescence microscopy with
-tubulin antibodies
showed that the spindles in YPM2 cells were very large, compared with
the ones seen in the wild type and occurred also in cells that did not
show budding (data not shown). This observation indicates that the
mutants were arrested in the late anaphase I.
Consistent with the observed lethal phenotype of PTF1
mutants, tetrad analysis of diploid YPM2/DBY874 yeast reproducibly
yielded a 2:2 co-segregation of the expression of
-galactosidase
together with the temperature sensitivity (data not shown).
Identification of the PTF1 Locus and Characterization of Wild Type
and Mutant Genes--
Complementation of the temperature sensitivity
of both YPM2 and YPM5 with two different genomic libraries resulted in
the cloning and isolation of the same genomic region and the
identification of PTF1 (2). Its predicted gene product is
characterized by a PPIase domain with homology to E. coli
parvulin and a WW domain (W denotes the invariant tryptophans), which
is assumed to mediate protein-protein interactions (34).
The mutant PTF1 gene locus that caused the temperature
sensitivity was isolated by PCR amplification of genomic DNA from the mutants. The amplified products were either sequenced directly or after
cloning in a Bluescript vector. In YPM2, a single point mutation led to
a change from the glycine residue at position 127 to aspartic acid. The
same was true for YPM5, where again a single nucleotide difference
caused an amino acid change, this time from a glycine at position 163 to a serine. On the DNA level, both mutations resulted from transitions
of a guanine to adenine. More importantly, both mutations were located
within the highly conserved regions of PPIases (Fig.
6).

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Fig. 6.
Schematic of the transcription unit of
PTF1. The amino acid sequence of the region containing
the sequence disparities of the mutants YPM2 and YPM5 in comparison to
the wild type strain DH484 are shown below. Hyphens
represent sequence identities of the mutants to DH484. Motif 1 and
motif 2 indicate the conserved PPIase motifs.
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Coexpression of wild type Ptf1p in each mutant strain restored cell
growth at non-permissive temperatures. Moreover, in contrast to results
from a previous publication (1), overexpression of Ptf1p in wild type
yeast did not lead to cell death (data not shown).
Western Blot Analysis and Mass Determination of Ptf1p--
A
polyclonal antiserum raised against denatured Ptf1p was obtained from
rabbit by immunization with the recombinant His-tagged protein that was
described previously (2). Using this antiserum, one of two signals,
corresponding to proteins of 70 (Fig. 7,
lanes 4 and 6) and 23 kDa (Fig. 7, lanes
3 and 5), respectively, appeared on Western blots
prepared with proteins contained in yeast whole cell extracts or
fractions thereof. The appearance of either species depended on the
method of extract preparation. In extracts, prepared with glass beads,
a single species of 70 kDa was identified, which is in obvious contrast
to the predicted molecular mass of Ptf1p (19,404 Da). This species
survived even the most stringent denaturation protocol applied to the
protein sample prior to gel electrophoresis. However, when the extract
was prepared in the same manner as for the in vitro 3'-end
processing reactions (35), the only signal visible matched the expected
molecular mass of Ptf1p (about 23 kDa). Surprisingly, after
fractionation of the same extract with 40% ammonium sulfate, the
23-kDa species was found in the supernatant, whereas the 70-kDa signal
appeared in the pellet. So far, we can only hypothesize about the
nature of the latter protein species. It might represent Ptf1p in an
unusually stable multimeric state or in tight complex with another
protein, possibly its target. A third possible explanation that the
observed high molecular weight protein resulted from cross-reaction
with the Ptf1p antiserum can be largely excluded, since in a computer
search we did not detect any Ptf1p-related sequences within the
complete yeast genome (36).

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Fig. 7.
Immunoblot with anti-Ptf1p serum of
recombinant Ptf1p expressed in E. coli and of reactive
proteins contained in fractionated and unfractionated yeast
extracts. Lane 1, 15 ng of recombinant
His6-tagged Ptf1p; lane 2, 15 ng of recombinant
Ptf1p cleaved with thrombin (note: 3 amino acids originating from the
plasmid construct remain); lane 3, 80 µg of yeast whole
cell extract; lane 4, 40 µg of protein of the 40%
ammonium sulfate precipitate of the yeast whole cell extract;
lane 5, 20 µg of supernatant of the 40% ammonium sulfate
precipitation of the yeast whole cell extract; lane 6, whole
cell yeast extract of the mutant YPM2.
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The molecular mass of the recombinant proteins was analyzed by
electrospray mass spectrometry. The resulting values of 21,438 Da for
the oligohistidine-tagged protein and 19,263 Da for the one without tag
agree well with the predicted sizes and provide no indication for a
post-translational modification in E. coli besides removal
of the starting Met residue.
PPIase Activity of Recombinant Ptf1p--
A protease-coupled assay
(see Refs. 37 and 38 and "Experimental Procedures") was employed to
determine the PPIase activity of wild type Ptf1p isolated from E. coli cells that expressed the recombinant, His-tagged protein.
cis/trans-Isomerization of the proline bond in synthetic
tetrapeptide substrates was measured spectroscopically, and activity
was expressed as kcat/KM.
The values for PPIase activity of Ptf1p and its substrate specificity
are summarized in Table I. Using the
standard PPIase substrate Suc-Ala-Ala-Pro-Phe-4NA, only moderate
enzymatic activity was detected
(kcat/KM = 5.9 × 103 M
1 s
1). Using a
series of peptides, which differed in the residues flanking a single
proline, the highest specificity constant was obtained with
Suc-Ala-Glu-Pro-Phe-4NA
(kcat/KM = 4.2 × 106 M
1 s
1). This
almost 1000-fold higher enzymatic activity of Ptf1p toward the latter
substrate in which glutamic acid, a phosphorylated serine surrogate,
preceded proline, led us to investigate peptides containing
phosphorylated Ser, Thr, or Tyr residues in the equivalent position. As
depicted in Table I, the enzyme showed the highest increase in activity
over the standard value when a phosphorylated serine
(kcat/KM = 1.7 × 107 M
1 s
1) or, to a
lesser extent, a phosphorylated threonine
(kcat/KM = 2 × 106 M
1 s
1) occurred
N-terminal to the proline moiety. However, phosphorylated tyrosine in
this position did not enhance the values obtained with the standard
substrate.
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Table I
PPIase activity and substrate specificity of Ptf1p
Measurements were carried out at 35 mM HEPES buffer, pH
7.8, at 10 °C in a protease-coupled assay (see "Experimental
Procedures"). Activitues are expressed as
kcat/KM. The values for the
standard and optimal peptide substrates are in boldface.
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Other PPIases like human FKPB12, human CYP18, trigger factor, and more
importantly, E. coli parvulin showed a decrease rather than
an increase of their specificity constants when tested with the
phosphorylated
substrates.3
In order to rule out that the His-tag of the recombinant protein might
have affected the activity and specificity of the enzyme, an authentic
His-tag-free recombinant Ptf1p was expressed in E. coli and
purified to homogeneity. The integrity of the protein was confirmed by
high performance liquid chromatography and mass spectrometry. No
differences were found between the enzymatic activities of
oligohistidine-tagged and the authentic His-tag-free recombinant Ptf1p
(data not shown).
PPIase Activity of Recombinant Ptf1p from the Yeast Mutants YPM2
and YPM5--
As described above, one single, although distinct, amino
acid change in Ptf1p led to temperature sensitivity of the yeast mutants YPM2 and YPM5 (Fig. 6). To test the PPIase activity of the
mutant forms, the corresponding recombinant proteins were expressed in
E. coli and isolated from cells grown at non-permissive temperature (37 °C). The mutant proteins turned out to be very unstable and sensitive toward all proteases used for activity measurements. Therefore, activity was determined in a protease-free assay (see "Experimental Procedures" and Ref. 32).2
The data in Table II demonstrate that the
PPIase activity of both mutants was strongly reduced as compared with
the wild type protein. Moreover, the degree of temperature sensitivity
correlated directly with the relative decrease in enzymatic activities
of the corresponding enzymes, i.e. Ptf1p from the mutant
YPM2 (Ptf1-2p) which showed temperature sensitivity at 30 °C was
about half as active as Ptf1p from YPM5 (Ptf1-5p) which is still viable
at 30 °C.
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Table II
Comparison of specific activities of wild type and mutant Ptf1p
Measurements were performed at 35 mM MES buffer, pH 6.1, at
10 °C in a protease-free assay (see "Experimental Procedures")
using Suc-Ala-Ala-Pro-(NO2)Tyr-4FA as substrate.
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To establish whether the mutations at the conserved Gly residues in
Ptf1p affected also the activity of another PPIase within the same
family, the equivalent Gly substitutions were introduced into E. coli parvulin, resulting in the parvulin mutants
Gly48-Asp, corresponding to Ptf1-2p, and
Gly83-Ser, comparable to Ptf1-5p. Like the Ptf1p mutants,
both parvulin mutant proteins were found to be very unstable. Most
notably, as shown in Table III, the
presence of either mutation virtually abolished the enzymatic activity
of parvulin (less than 0.1%).
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Table III
PPlase activity of parvulin proteins carrying the same mutations as
Ptf1-2p (Gly48-Asp) and Ptf1-5p (Gly83-Ser)
Measurements were performed at 35 mM HEPES buffer, pH 7.8, at 10 °C in a protease-coupled assay (see "Experimental
Procedures") using Suc-Ala-Phe-Pro-Phe-4NA as substrate.
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 |
DISCUSSION |
The most curious aspect of PTF1, the first essential
PPIase gene identified in S. cerevisiae, is the fact that it
was discovered in a genetic screen aimed at the identification of
factors involved in the mRNA 3'-end processing pathway in bakers'
yeast (2). Moreover, PTF1 was the only gene found in this
screen to complement the two independently isolated, EMS-induced
temperature-sensitive mutants, YPM2 and YPM5, with an apparent defect
in the 3'-end formation of a plasmid-encoded pre-mRNA. Isolation
and sequence determination of the PTF1 genes from both
mutants revealed a single, albeit distinct, point mutation at the DNA
level, each resulting in a change of highly conserved amino acid
residues within the PPIase domain. These changes were accompanied by a
drastic decrease in PPIase activity. Coexpression of wild type Ptf1p in
either mutant was sufficient to restore growth at non-permissive temperatures.
Our mutant screen was based on the detection of plasmid-derived
transcripts arising from lack of cleavage and polyadenylation at a
truncated ADH1 terminator region, located between yeast
actin sequences and the bacterial lacZ gene (Fig. 1 and
text). Still, with the notable exception of YPT1, no such
read-through events were observed in the mutant strains, when the
truncated ADH1 terminator region was replaced by its
complete version, or by equivalent sequences from the ACT1
and CYC1 genes (Fig. 4).
A similar "diffuse" effect on mRNA 3'-end formation was
reported by Russnak et al. (39). The ref2-1 mutants,
which the authors identified, showed differences in mRNA 3'-end
processing only for transcripts encoded by certain, artificially
designed plasmids. By this criterion, the authors defined "weak"
and "strong" polyadenylation sites, a definition which had been
introduced earlier by Irniger et al. (40). Moreover it was
suggested that impairment of processing efficiency should not occur
without a negative effect on termination frequency in yeast cells, as
in this organism, unlike in higher eucaryotes, transcription and
polyadenylation are tightly coupled. In fact, it might even have been
impossible to distinguish in our screen whether the primary defect had
occurred in transcription termination or at the level of
endonucleolytic cleavage and polyadenylation because of their expected
common consequences. Any failure in transcription termination should
also interfere with the transcription initiation of the gene following
immediately downstream, as in yeast there is generally little space
between polyadenylation sites and the transcription start site of the
adjacent gene (41). Additionally, read-through transcripts should not
be stable in either scenario, whether they fail to be processed at all
or lack the 5'-cap, once they are cleaved at their respective poly(A) sites but transcription continues. The very first consequence would be
a rapid decline in mRNA accumulation, which is what we observed in
the YPM2 mutant at non-permissive temperatures as early as 2 h
after the shift (Fig. 2, lane 6).
The multiple phenotypes of the mutant strains described before could be
reversed by coexpression of a gene (PTF1 (2)) encoding a
peptidylprolyl-cis/trans-isomerase. Whereas the previous
identification of Ptf1p as a putative PPIase resulted from its sequence
similarity with the E. coli protein parvulin (29), two
eucaryotic counterparts of Ptf1p have been discovered in the meantime:
DODO from Drosophila melanogaster (42) and PIN1 (9) from
human cells. All three proteins are clearly distinct from the
prokaryotic members of the Parvulin family of PPIases and have recently
been suggested to be named products of the dodo gene family
(43). In addition to their high degree of structural identity, these
proteins are also functional homologues as demonstrated by the fact
that the human PIN1, as well as the fly DODO, complemented the lethal
phenotype of ESS1/PTF1 disrupted yeast cells (9, 42).
Moreover, intact PPIase activity of PIN1 was necessary for successful
complementation. Yet, whereas PTF1 and PIN1 were shown to be
essential for growth of yeast and HeLa cells, respectively, total
removal of the fly gene did not impair development of the mutant
insects (42). Whether the differences in importance of these related
genes in their natural host organisms reflect differences in their
functions or in host cell requirements remains to be seen.
Unusual for PPIases, Ptf1p revealed a distinct substrate specificity
with respect to the amino acid residues preceding the prolyl-peptide
bond. The highest activity was achieved with peptides containing
phosphorylated Ser/Thr moieties at this position. Most remarkably, the
activity toward the optimal substrate
(Ac-Ala-(PO3H2)Ser-Pro-Tyr-4NA) was enhanced up
to 3000-fold over the value obtained with the standard substrate. This
rather unique substrate specificity is also shared by the human PIN1
(9), very likely reflecting the nature of in vivo targets
(discussed below).
In agreement with the putative function of Ptf1p as a PPIase in
vivo, its mutated forms in YPM2 and YPM5 displayed significantly reduced activities in vitro compared with the wild type protein.
Taken together, our results clearly correlate a PPIase activity with
efficient pre-mRNA 3'-end processing and/or transcription termination in S. cerevisiae. Thus, the most compelling
question arising from our studies concerns the nature of the putative
involvement of a PPIase in these processes. In the absence of more
experimental clues, we speculate that Ptf1p interacts with components
of the mRNA transcription complex, if only at the final stages of
RNA transcription. Upon the appropriate trigger (or at the time of entry), Ptf1p might induce a conformational switch in either the accessory proteins or the polymerase itself, ultimately causing the
dissociation of RNA polymerase II from the DNA template,
i.e. transcription termination.
The discovery of a PPIase of the cyclophilin family, shown to interact
with the C-terminal domain of mammalian polymerase II in a yeast
two-hybrid system (44), seems to lend support to the postulated
interaction of Ptf1p with the transcription machinery in yeast. More
importantly, the former interaction depended on the presence of
phosphoepitopes on the C-terminal domain, as extensive treatment of the
yeast extracts with phosphatases resulted in complete loss of this
interaction. As the C-terminal domain, which consists of multiple
tandem repeats of a heptapeptide Tyr-Ser-Pro-Thr-Ser-Pro-Ser, is highly
conserved between yeast and mammals, this domain represents an ideal
candidate for a PPIase, with specificity toward phosphorylated serines
N-terminal to prolines.
Moreover, two recent publications (45-46), in which another
cyclophilin, USA-CyP (a member of the cyclosporin A-binding PPIase family), was shown to be tightly associated with the spliceosomal (U4/U6.U5) tri-small nuclear ribonucleoprotein in HeLa cells, also
support the proposed role of Ptf1p in the dissociation of the
transcription machinery from the DNA template. In one of the papers
(45), the authors discuss the intriguing possibility that the role of
USA-CyP in the human spliceosome is not to act as a chaperone in the
folding or assembly steps but rather to assist in the disassembly of
spliceosomes. This view was spurred by the example of one of the best
characterized cyclophilins, cyclophilin-A, for which there is
convincing evidence that it exerts its function by binding specifically
to the HIV-1 capsid protein (CA), destabilizing interactions between
the CA molecules and thus facilitating the disassembly of the CA core
(47-49). Nevertheless, as much as it is tempting to draw parallels
from the above examples to the physiological function of Ptf1p, its
putative role in RNA transcription (and/or termination) has first to
await experimental confirmation.
Finally, we should like to consider again PIN1, the functional and
structural human homologue of yeast Ptf1p. When underexpressed, both
proteins induced mitotic arrest of their respective host cells,
suggesting a role in cell cycle regulation. In agreement with this
notion, PIN1 has been found to be part of the nuclear speckle (33), a
large protein complex, which contains several other mitosis-related
proteins and, most notably, some splicing factors. Given the fact that
PIN1 substitutes functionally for the putative pre-mRNA 3'-end
processing factor ESS1/PTF1 in yeast cells, the presence of
Pin1 in the nuclear speckle, the components of which are supposedly
involved in cell cycle regulation, is rather intriguing. It may be that
factors such as these serve as checkpoints for the integrity of the
mRNA maturation process thus acting as a link between pre-mRNA
3'-end processing and cell cycle regulation.
 |
ACKNOWLEDGEMENTS |
We are grateful to M. Schukowski for the
synthesis of Suc-Ala-Ala-Pro-(NO2)Tyr-4-fluoranilide and
other peptides and B. Janowski for performing measurements.
 |
FOOTNOTES |
*
This work was supported by a grant of the
Boehringer-Ingelheim Stiftung and by donations from Hoechst AG, Wacker
Chemie, and Boehringer Mannheim GmbH.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Present address: Munich Information Centre for Protein Sequences,
Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 München, Germany.
To whom correspondence should be addressed. Tel.: 49 89 74017403; Fax: 49 89 74017448.
2
B. Janowski, S. Wöllner, M. Schutkowski,
and G. Fischer, submitted for publication.
3
B. Schelbert, A. Bernhardt, G. Fischer, and
J.-U. Rahfeld, unpublished results.
 |
ABBREVIATIONS |
The abbreviations used are:
PPIase, peptidylprolyl-cis/trans-isomerase;
Suc, succinimidyl;
bp, base pair(s);
nt, nucleotide(s);
EMS, ethyl methanesulfonate;
RT-PCR, reverse transcriptase-polymerase chain reaction;
NA, nitroanilide..
 |
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