From the Institut für Pharmazeutische Biologie,
Martin-Luther-Universität Halle-Wittenberg, Hoher Weg 8, D-06120
Halle/Saale, Germany, the § Max-Planck-Forschungstelle
"Enzymologie der Proteinfaltung," Weinbergweg 22, D-06120
Halle/Saale, Germany, and the Cancer Biology Program,
Department of Medicine, Beth Israel Deaconess Medical Center, Harvard
Medical School, HIM 1047, Boston, Massachusetts 02215
Received for publication, August 3, 2000, and in revised form, December 7, 2000
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ABSTRACT |
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A functionally Pin1-like peptidyl-prolyl
cis/trans isomerase (PPIase1) was isolated from
proembryogenic masses (PEMs) of Digitalis lanata according
to its enzymatic activity. Partial sequence analysis of the purified
enzyme (DlPar13) revealed sequence homology to members of
the parvulin family of PPIases. Similar to human Pin1 and yeast Ess1,
it exhibits catalytic activity toward substrates containing
(Thr(P)/Ser(P))-Pro peptide bonds and comparable inhibition kinetics
with juglone. Unlike Pin1-type enzymes it lacks the phosphoserine or
phosphothreonine binding WW domain. Western blotting with
anti-DlPar13 serum recognized the endogenous form in
nucleic and cytosolic fractions of the plant cells. Since the
PIN1 homologue ESS1 is an essential
gene, complementation experiments in yeast were performed. When
overexpressed in Saccharomyces cerevisiae DlPar13 is almost as effective as hPin1 in rescuing the temperature-sensitive phenotype caused by a mutation in ESS1. In contrast, the human
parvulin hPar14 is not able to rescue the lethal phenotype of this
yeast strain at nonpermissive temperatures. These results suggest a function for DlPar13 rather similar to parvulins of the
Pin1-type.
Human Pin1 belongs to the parvulin family of peptidyl-prolyl
cis/trans isomerases (EC 5.2.1.8) (1). Regarding substrate specificity there exists the parvulin subfamily of the Pin1-type. Enzymes of this subfamily show a striking preference for peptide and
protein substrates containing phosphorylated side chains of serine or
threonine residues preceding the proline position (2-6). Phosphorylation of (Ser/Thr)-Pro motifs by proline-directed proteine kinases occurs as part of regulatory processes during cell division and
signal transduction events. For hPin1 a role in control of mitosis was
suggested (2). Consistent with the observed substrate specificity,
hPin1 generates catalytically a substrate conformation of
phosphoproteins productive in the dephosphorylation by protein phosphatase 2A (7). Besides the catalytic preference of hPin1 for
phosphorylated substrates, the direct interaction with numerous mitotic
phosphoproteins has been previously demonstrated (2, 3, 8-11). The
major docking site for these phosphoproteins has been found in the WW
domain of hPin1 (8). WW domains are generally known as small protein
interaction modules with the binding preference to proline-rich peptide
motifs (12, 13).
Most of other identified parvulins of the Pin1-type contain an
N-terminal WW domain, e.g. Ess1/Ptf1 from
Saccharomyces cerevisiae (14, 15), mPIN1 from Mus
sp. (16), Dodo from Drosophila melanogaster (17, 18),
xPin1 from Xenopus laevis (19), PINA from
Aspergillus nidulans (9), and SspI from
Neurospora crassa (20). One exception is a recently
described parvulin from Arabidopsis thaliana
(AtPin1) (6). For this enzyme a preference for
phosphorylated oligopeptide substrates has been shown. However,
currently there is no data concerning the physiological role of this enzyme.
The function of Pin1-type parvulins appears to be conserved in several
organisms. The only existing yeast homologue, Ess1/Ptf1, is
encoded by an essential gene (14, 15). A mutation leading to a single
amino acid exchange in the protein causes a temperature-sensitive phenotype and cells exhibit terminal mitotic arrest at nonpermissive temperature. The lack of Ess1/Ptf1 function under this condition can be
readily replaced by hPin1 (21) or by D. melanogaster Dodo
(18), supporting the idea of a related function. It remains an open
question whether the WW domain is a crucial condition of the rescuing
function. However, for the homologous protein xPin1 in X. laevis a requirement for proper function of the replication checkpoint has been demonstrated (19).
Here we describe the isolation of a small parvulin from PEMs of
D. lanata according to its PPIase activity toward
phosphorylated peptide substrates, which demonstrates for the first
time the PPIase activity of an endogenous parvulin of the Pin1-type.
The substrate specificity and inhibition kinetics were analyzed in more
detail. Similar to other plant homologues such as the A. thaliana
AtPin1 and the Malus domesticus MdPin1 (43) this enzyme does not comprise a WW or any other domain adjacent to the catalytical core. We therefore addressed the question if this special plant enzyme
can also functionally replace the yeast Ess1/Ptf1.
Enzyme Assay--
For general screening purposes the
protease-coupled PPIase microplate assay was performed (22). The
specificity constants kcat/Km
were determined according to Fischer et al. (23). Substrates
listed in Table I were kindly provided by
M. Schutkowski (Halle). Inhibition characteristics toward
naphthoquinones were determined according to Hennig et al.
(24). The sensitivity of the PPIase assay was calculated with the
equation [lowest detectable PPIase concentration] = 4*ko/kcat/KM), with ko as the uncatalyzed first-order rate constant. The factor 4 results from both, the sample volume was half of the total
volume and the limit of detection is the 2-fold of
ko. We calculated the sensitivity of the
assay for DlPar131
to about 0.3 nM at 7 °C, by using the substrate
Ac-AS(OPO3H2)PY-NH-Np with a
ko of about 0.0026 s Purification of the DlPar13--
Cells of Digitalis
lanata Ehrh. strain VIII (25) were grown as PEMs (26) and stored
after harvesting at Subcellular Localization--
Subcellular fractionation and
measurement of marker enzyme activity (alcohol dehydrogenase,
cytytochrome c reductase, and succinate dehydrogenase) was
performed as described by Qi et al. (29). Nuclei were
stained with 4,6-diamidino-2-phenylindole solution (Partec,
Göttingen, Germany) and observed with light microscopy (data not shown).
Amino Acid Sequence Analysis--
DlPar13
(approximately 30 pmol) was digested with 0.1 µg of endoprotease
Lys-C (Roche Molecular Biochemicals, Mannheim, Germany) in 25 mM Tris, pH 8.5, for 60 min at 30 °C. The molecular
masses of the obtained fragments were determined by matrix-assisted
laser desorption time-of-flight mass spectrometry and sequenced using sequencer 476A (Applied Biosystems, Weiterstadt, Germany) according to
the manufacturers instructions or as described by Pfeifer et al. (30). All obtained partial sequences were compared with entries of the Swiss-Prot and EMBL data bases using the program BLAST
2.0 and FASTA 3.0 (31, 32).
Amplification of a cDNA Fragment by RT-PCR--
cDNA was
prepared from poly(A)+ RNA of D. lanata PEMs
using the SuperscriptTM Preamplification System (Life
Technologies, Inc., Karlsruhe, Germany). For RT-PCR the two
degenerative primers (Amersham Pharmacia Biotech, Freiburg, Germany),
sense 5'-CGNGGCGGNGAYCTNGG-3' and antisense 5'-TTRATGATGTGNACKCC-3',
were used. PCR amplification was performed under the following
conditions: 5 min 95 °C, 35 cycles of 1 min 95 °C, 1 min
55 °C, and 1 min 72 °C, and at least 10 min at 72 °C. The
resulting 128-base pair PCR fragment was cloned into a TOPO T/A-vector
(Invitrogen, Groningen, The Netherlands), multiplied, and sequenced
(33).
cDNA Library Screening, Overexpression, and Purification of
DlPar13--
A
Recombinant DlPar13 was purified according to the
manufacturers instructions for His-tagged protein expression (Qiagen)
and used for antiserum production in rabbit (pab-productions,
Herbertshausen, Germany). Immunoblot analysis was performed with
horseradish peroxidase-coupled anti-rabbit secondary antibodies (Sigma)
and the ECL Western blotting detection reagents (Amersham Pharmacia
Biotech, Braunschweig, Germany).
Yeast Complementation Analysis--
The yeast
temperature-sensitive strain YPM2 (11) was transformed with the
expression vector pBC100 containing the respective gene under the
transcriptional control of a galactose inducible promoter. The coding
sequence for DlPar13 was subcloned in-frame with the
hemagglutinin epitope tag into a pBC100 vector. Simultaneous the human
PAR14 gene was subcloned into the pBC100 vector. Gene expression in the stably transformed YPM2 strains was controlled by
Western blot analysis of the cell lysates from cultures grown in
inducing medium at permissive temperature (23 °C) using a monoclonal anti-hemagglutinin epitope antibody (12CA5). For the complementation analysis cells of the respective strain selected on the appropriate medium at permissive temperature were resuspended in 10 mM
Tris buffer, pH 7.5, 1 mM EDTA and the optical density
adjusted to A600 = 1.0. The suspensions (5 µl)
and three 10-fold dilutions were applied to agar plates containing
minimal media supplemented with the appropriate carbohydrate source to
induce or repress gene expression. As controls two YPM2 strains
transformed with the vector only and a hPin1 pBC100 construct,
respectively, were used.
Isolation of DlPar13--
The chromatographic isolation of a
phospho-specific parvulin from cells grown as PEMs of D. lanata Ehrh. strain VIII was carried out by applying a sensitive
PPIase assay for detection of phosphorylation-specific enzyme activity
in crude protein solutions. According to the ratio of enzymatic
activity toward the side chain phosphorylated substrate Ac-Ala-Ser(OPO3H2)-Pro-Tyr-NH-Np and the
unphosphorylated counterpart, the phospho-specific protein was enriched
19-fold after anion exchange chromatography and dye affinity
chromatography. This almost perfectly coincides with the purification
factor of 22.7 calculated from the specific PPIase activity toward the
phosphorylated substrate, indicating a very low probability of other
phospho-specific PPIase activities in these cells. About 0.5 µg of
homogenous DlPar13 was isolated from a single peak with the
retention time of 17.2 min in the reversed phase high performance
liquid chromatography (nucleosil C18) using 320 mg of total cell
protein as starting material.
For identification, the molecular mass of the protein was determined at
12,846 Da by matrix-assisted laser desorption time-of-flight mass
spectrometry (data not shown). Since the N terminus was not accessible
to Edman degradation the protein was digested with endoprotease Lys-C
yielding eight proteolytic fragments (data not shown). A data base
search using the obtained partial sequence information revealed
homology to the catalytic domains of parvulins.
Subcellular Distribution of DlPar13--
The nuclear fraction and
the mitochondrial, microsomal, ribosomal, and cytosolic supernatant
fractions of the D. lanata PEMs extract were examined for
DlPar13 content. Activities of three enzymes were
used as subcellular markers. The DlPar13 was detectable in
both nuclear and cytosolic fractions (Fig.
1). The marker enzymes for mitochondrial
(succinate dehydrogenase) and microsomal (cytochrome c
reductase) fractions indicate that both fractions contain a mixture of
proteins of the particular compartment (Table
II). By using Western blot analysis the
amount of DlPar13 was negligible in the microsomal fraction
but has a considerable magnitude in the mitochondrial fraction. The
presence of mitochondrial marker enzymes in microsomal fractions has
already been described for subcellular fractionating of A. thaliana preparations using a similar preparation technique (29).
Our results indicate that DlPar13 does not occur in
microsomes but could not definitively exclude a minor amount of
DlPar13 in mitochondria.
Cloning of DlPAR13--
Using the obtained partial sequence data,
oligonucleotide primers were designed and used for RT-PCR with D. lanata mRNA as template. With an amplified 128-base pair PCR
fragment as probe a cDNA library derived from PEMs of D. lanata was screened. The resulting 354-base pair full-length
cDNA clone encoding for 118 amino acids was subcloned into the
procaryotic expression vector pQE30. Based on the amino acid
composition a theoretical isoelectric point of 8.9 and a molecular mass
of 12,834 Da was calculated. The amino acid sequence either derived
from the cDNA clone (Fig. 2) or the
128-base pair PCR fragment was identical, but showed a deviation in
three amino acid residues (S79P, D91E, and G95A) when compared with the
original partial sequence of DlPar13. Since these data was
verified in repeated experiments and sequencing errors can be excluded,
the existence of two or more isoforms of this phosphorylation-specific
parvulin in D. lanata is conceivable. As the complete amino
acid sequence of the isolated DlPar13 is unknown we only can
assume that more exchanges are likely to exist between the two
suggested isoforms. The occurrence of multiple enzyme forms seem not to
be unusual for plant parvulins because of the presence of several
homologous EST sequences of parvulin genes in Lycopersicon
esculentum (AW621901, AW621939, AW945046) and in Glycine
max (AW308915, AW397670, AW761425, AI507774).
Using a specific DlPar13 antibody raised against the
recombinant protein, the concentration of the endogenous
DlPar13 in the cell homogenates of PEMs was estimated at 5 ng of protein/mg of total protein by Western blot analysis using the
recombinant protein as an internal standard (data not shown). Based on
Northern blot analysis the transcription level of the
DlPAR13 within a 2-year-old foxglove plant was compared
(data not shown). In all analyzed tissues (1- and 2-year-old roots,
stems, 1- and 2-year-old rosette leaves, flowers, and seedlings) the
specific mRNA transcripts were detectable with similar concentrations.
Amino Acid Sequence--
DlPar13 shares a high degree
of homology to the parvulins of the Pin1-type (Fig. 2). This similarity
is especially high with the plant enzyme AtPar13 (6) revealing 73%
identity of both amino acid sequences. The degree of homology to the
PPIase domains of other Pin1-like enzymes were found to be 53% with
hPin1 (accession number Q13526 (21)), 51% with Ptf1 from
S. cerevisiae (accession number P22696 (14, 15)) and
SspI from N. crassa (accession number AJ0006023
(20)), respectively, and 47% with Dodo from D. melanogaster
(accession number P54353 (18)). Less sequence homology exists to other
parvulins with rather unspecific substrate recognition pattern
including hPar14 with 28% identity (accession number AB009690 (34,
35)) and ECPar10 from E. coli (accession number
P39159 (36, 37)) with 32% identity to DlPar13. In both
enzymes some amino acid residues of the supposed substrate binding
pocket differ from the conserved region of the phosphorylation-specific proteins as illustrated in Fig. 2. Based on data of the crystal structure of hPin1 (5) and site-directed mutagenesis experiments (11),
it was supposed that a basic cluster of the two arginine residues
Arg68 and Arg69 coordinates the side chain
phosphate group of the substrate. Both residues together with the third
basic residue, Lys63 in hPin1, are conserved among the
Pin1-type parvulins. In DlPar13 the specific sequence
features exist as Lys14, Arg19, and
Arg20.
PPIase Activity--
The substrate specificity of the recombinant
DlPar13 toward a set of oligopeptide derivatives (Table I)
indicates a preference for phosphorylated Ser or Thr side chains in the
position preceding proline. For example, the
kcat/Km value for the
substrate Ac-Ala-Ser(OPO3H2)-Pro-Tyr-NH-Np was
determined with 15.9 µM
To further characterize this enzyme activity, inhibition studies with
the parvulin-specific irreversible inhibitor juglone were performed
(Fig. 3). The mechanism of enzyme
inactivation comprises the covalent binding of the inhibitor molecule
to a highly conserved cysteine residue (Cys113 in hPin1
(24)) followed by partial unfolding of a region in the active site of
the proteins, and the subsequent loss of enzymatic activity. The
specific target thiol group is Cys68 present in the amino
acid sequence of the D. lanata parvulin and incubation of
this enzyme with a 10-fold excess of juglone renders it inactive with a
similar slow kinetics of inactivation as was shown for hPin1 and yeast
Ptf1 (24).
In contrast to juglone, the structurally related naphthoquinone
plumbagin did not cause enzyme inactivation. This is consistent with
the proposed mechanism of action of juglone toward parvulins (24).
Thus, this result supports the structural relationship of
DlPar13 to the parvulin family of PPIases. No inhibition of the enzyme activity was observed with a 1000-fold excess of cyclosporin A or FK506 at nanomolar concentrations of DlPar13.
Functional Assay--
The strong preference for phosphorylated
(Ser/Thr)-Pro motifs in substrates and the lack of the WW domain raised
the question of whether the plant enzyme is able to replace the
function of hPin1-like PPIases. To approach this, we used the
temperature-sensitive YPM2 strain of S. cerevisiae known to
be mutated in the ESS1 gene locus which leads to a G127D
amino acid substitution in the Ess1/Ptf1 protein (11) (Fig. 2). It has
been previously shown that hPin1 can functionally replace Ptf1 by
preventing terminal mitotic arrest of the cells under restrictive
temperatures (21). For our experiment, cDNA encoding for
DlPar13 was subcloned in a yeast expression vector under the
control of the galactose inducible promoter in-frame with the sequence
of a hemagglutinin epitope tag for comparative antibody detection of
the protein with other proteins used in this system. In the same set of
experiments hPin1 and hPar14 were used as control. Interestingly, at
nonpermissive temperatures DlPar13 was clearly able to
restore the function of Ptf1 almost as well as hPin1 (Fig.
4). Conversely, the temperature
sensitivity of the yeast cells was not abolished by hPar14. These
results clearly distinguish DlPar13 from small
phosphorylation-independent parvulins and support the idea that
DlPar13, despite lacking the WW domain, can function
similarly to the hPin1-like parvulins.
In cell extracts of PEMs the PPIase activity of cyclophilins is
dominating (38) and interferes with the detection of other PPIase
activities. We detected in cell lysates of PEMs of D. lanata a 90-fold higher total PPIase activity toward the unphosphorylated substrate Ac-Ala-Ser-Pro-Tyr-NH-Np than toward the side chain phosphorylated peptide derivative
(Ac-Ala-Ser(OPO3H2)-Pro-Tyr-NH-Np). Consistently, inhibition by 90% of the PPIase activity toward the
unphosphorylated substrate was observed using cyclosporin A and the
remaining enzymatic activity was almost completely suppressed by FK506.
There was no inhibition by juglone on the PPIase activity. In contrast,
the PPIase activity toward the phosphorylated substrate was inhibited
by cyclosporin A to only 50%. In this case, the remaining activity was
completely sensitive to juglone. By applying this differential PPIase
assay as a detection method we isolated the phospho-specific
DlPar13. Thus the catalytic activity of an authentic
parvulin of the Pin1-type has been demonstrated for the first time. The
subsite specificity of this enzyme was compared with data of the
purified recombinant protein and was found to be identical.
In contrast to other parvulins of the hPin1 subfamily the isolated
plant enzyme does not contain a WW domain. However, it does consist of
a parvulin catalytic core with striking similarity to the Pin1-type
subfamily of the parvulins. For example, DlPar13 shares 47%
identity with the PPIase domain of hPin1 and only 28% sequence
identity was found with hPar14, which does not exhibit the
phosphorylation-dependent substrate specificity.
As shown in Table I the side chain phosphorylated substrates were
preferred by parvulins of the Pin1-type and under the assay conditions
the WW domain had no influence on the substrate specificity. Lu
et al. (8) reported on the important role of the WW domain for binding of (Ser(P)/Thr)-Pro motifs of mitosis-specific proteins and
for the function of hPin1 in vivo (8). Obviously, for the survival of the ESS1 mutant yeast strain (YPM2) lacking the
function of Ptf1 under nonpermissive temperatures, there is no
requirement for the WW domain when overexpressing the
phosphorylation-specific parvulin DlPar13. Indeed it was
shown in a similar approach that the PPIase domain of hPin1 is
sufficient for rescuing the temperature-induced lethal phenotype (7).
These results indicate that although the WW domain is normally required
for hPin1 to perform its essential function, it is the PPIase domain
that carries out the essential function. Recently, the indispensable
function of the PPIase domain of the X. laevis homologue
xPin1 for its supposed role in regulation of the replication checkpoint
has also been demonstrated (19). In this case a PPIase inactive mutant
form of xPin1 which still comprises the WW domain and thus has protein
binding activity was no longer active in restoring the proper function
of the replication checkpoint. In addition, the failure of hPar14 to
prevent lethality under nonpermissive temperatures in our yeast
complementation experiments shows that the phospho-specific PPIase
activity is the key for the essential role of hPin1-like enzymes.
The absence of a WW domain seems not to be unusual among plant
parvulins, and the known three proteins from D. lanata, M. domesticus (43), and A. thaliana (6) contain only the
highly conserved catalytic core. There is also no indication of
phosphorylation-specific parvulins containing a WW interaction module
from sequence data of other plant species, yet several isoforms have
been found in the EST data base. Experimentally, by applying a PPIase
assay which is sensitive enough to detect 10-fold lower enzyme
activities as was measured for the endogenous DlPar13, we
failed to detect a second phosphorylation-specific PPIase activity in
cell lysates of D. lanata. Possibly, the described small
form of the hPin1-type parvulins is the only expressed parvulin type in
plant species and hence could reflect the general requirement of
phosphorylation-specific PPIase activity in eukaryotes.
There are some differences when comparing DlPar13 with other
hPin1-type parvulins regarding the intracellular concentration and
distribution. Similar to hPin1 (21), DlPar13 appears
nucleus-located. However, we observed a considerable amount of the
enzyme in the cytosolic fraction too (Fig. 1). Similarly, a cytosolic
localization could not be excluded for SspI (20). These
findings are at variance to the results for Pin1. Recently, Rippmann
et al. (39) reported that the Pin1-WW domain is responsible
to direct the localization of Pin1 into nuclear speckles. Why the
DlPar13 does not need the WW domain for nuclear localization
remains an open question.
The amount of endogenous DlPar13 was determined to be about
5 ng/mg of total cell protein which is considerably lower than found
for other Pin1-type parvulins. In N. crassa hyphae the
SspI content was measured with 0.05-0.1% of total cellular
protein (20), within in the concentration range of Cyp20 (40) and FKBP13 (41). High concentrations were observed for hPin1 in HeLa cells
and xPin1 in X. laevis egg extracts with 0.5 (10) and 1 (190) µM, respectively, which is a concentration range
frequently found for cyclophilins and FKBPs in higher eukaryotes
(42).
The phosphorylation-specific PPIases in D. lanata, M. domesticus, and A. thaliana lacking a WW domain appear
to be an evolutionary specialization of plants. The question remains of
how these PPIases overcome the role of the WW domain as protein-protein
interaction module. The hypothesis of Landrieu et al. (6),
that the hydrophobic
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and a
kcat/Km value of 15,900 mM
1 s
1 (Table I).
Comparison of the specificity constants kcat/KM of
DlPar13, hPin, and Ptf1 for several peptide substrates
70 °C. Frozen PEMs (30 g) were resuspended in
90 ml of 2 mM MES buffer, pH 6.8, containing 1 mM EDTA, 1 mM dithiothreitol, and 0.3% Triton
X-100 for 20 min, followed by homogenization using a Potter homogenizer (Glass Col, Terre Haute, IN) for 3 × 2 min with 1,000 rpm. After centrifugation at 25,000 × g for 45 min at 4 °C (L8
60 M, Beckman, Unterschleißheim, Germany), the supernatant
was dialyzed against 2 mM MES buffer, pH 8.0, 1 mM dithiothreitol, and 1 mM EDTA to a final
conductivity of 300 µS cm
1 and applied onto an anion
exchange column (DEAE-EMD-Fractogel, 150 × 20 mm, Merck,
Darmstadt, Germany). After an affinity chromatography step (Fractogel
TSK AF-Blue, 80 × 15 mm, Merck) the fractions containing the
highest phospho-specific activity were further separated by reverse
phase HPLC (Nucleosil 300-5 C18, 125 × 3 mm, Macherey-Nagel,
Düren, Germany) applying a linear gradient of 30-50%
acetonitrile in 0.1% trifluoroacetic acid for 40 min at 40 °C with
a flow rate of 0.5 ml min
1. The protein concentration was
determined by the method of Bradford (27) or from the absorbance at 280 nm using the molar extinction coefficient derived from the amino acid
sequence (28).
-ZAP cDNA library (Stratagene, Heidelberg,
Germany) constructed from poly(A)+ RNA of PEMs of D. lanata served as a template for screening. Approximately 250,000 plaque-forming units were screened using the
[
-32P]dATP-labeled PCR fragment described above as a
probe. The three positive plaques were further purified by new rounds
of plating and screening and afterward in vivo excised and
sequenced. Further subcloning was performed using PCR to introduce a 5'
KpnI and a 3' HindIII site to the coding
sequence. The construct was inserted in-frame with the coding sequence
for an N-terminal hexahistidine tag into the pQE30 expression vector (Qiagen).
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DISCUSSION
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Fig. 1.
Immunoblotting of DlPar13 in
subcellular fractions. Aliquots (10 µg) of subcellular fractions
from PEMs of D. lanata were analyzed by SDS-polyacrylamide
(20%) gel and immunoblotted with an anti-DlPar13 polyclonal
antibody.
Subcellular distribution of DlPar13 and organelle marker enzyme
activities in PEMs of D. lanata
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Fig. 2.
Alignment of DlPar13 with
the prototypic Escherichia coli Par10
(ECPar10 (36, 37)), the human (hPar14 (34, 35)), and
the (Thr(P)/Ser)-Pro specific parvulins from A. thaliana
(ATPin1 (6)), human (hPin1 (21)), S. cerevisiae (SCPtf1 (14, 15)), N. crassa (NCSspI (20)), and D. melanogaster (DMDodo (18)). The core
of the N-terminal WW domain of the proteins is boxed and the
conserved residues shown by the crystal structure of human Pin1 (5) to
be within the active site of the PPIase moiety are indicated by
asterisks. Residues that are identical in at least four of
the eight proteins are printed in bold letters. The
hydrophobic 1-element identified in hPin1 (5) is indicated
above. Underlined is the corresponding amino acid sequence
of the authentic DlPar13. The observed amino acid deviations
(S79P, D91E, G95A, see "Results") are not shown.
1 s
1
and hence is in the order of the specificity constants of other Pin1-related parvulins (3, 11, 20). Toward the unphosphorylated substrate the catalytic activity of DlPar13 is reduced by a
factor of 10,000. This is even less enzymatic activity than was
determined for hPin1 and Ptf1 toward this unphosphorylated substrate.
It should be mentioned that we observed the same ratio of catalytic activity toward both substrates for the authentic DlPar13.
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Fig. 3.
Inhibition of DlPar13 by
juglone and plumbagin. The PPIase actvity was determined in a
protease coupled assay using Suc-Ala-Glu-Pro-Phe-NH-Np as substrate.
The enzyme (2 µM) was incubated with a 10 M
excess of juglone ( ) and plumbagin (
), respectively, in 35 mM Hepes buffer, pH 7.8, containing 1 µM
bovine serum albumin at 10 °C. For the PPIase activity assay the
preincubated protein was diluted to a final concentration of 28 nM. The data for Ess1/Ptf1(
) and hPin1 (
) were taken
from Hennig et al. (24). The first-order rate constant for
the inhibition of DlPar13 was calculated at 3 × 10
4 s
1. First-order rate constants for
Ess1/Ptf1 and for hPin1 were calculated at 5.3 × 10
4 s
1 and 1.5 × 10
4
s
1.
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Fig. 4.
DlPar13 can suppress the
temperature-sensitive phenotype of the yeast strain YPM2 under
nonpermissive temperatures. The strain was either transformed with
expression vector alone or vector containing the coding sequence of
DlPar13, hPin1, or hPar14 under control of a galactose
inducible promoter. After selection on appropriate media at permissive
temperatures cells of the respective stably transformed strains were
transferred to agar plates containing the indicated carbohydrate
source. Data were obtained after 3-6 days of incubation at permissive
or nonpermissive temperatures. From left to right, 10-fold dilutions of
the initial cell suspension of an A600 = 1.0 were applied to the plates.
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ABSTRACT
INTRODUCTION
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1 helix (Fig. 2) might take over the function
of the WW domain would be an explanation. Our results of better
discrimination between phospho- and unphosphorylated substrates by
DlPar13 in comparison with hPin1 and Ptf1 supports also the
speculations of Yao et al. (43), that these enzymes in
plants overcome the WW domain lack by improvement of the substrate affinity.
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ACKNOWLEDGEMENTS |
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We thank A. Peterson (Biozentrum Halle, D-06120 Halle/Saale, Germany) for the cDNA library and DNA sequencing. We are grateful to A. Schierhorn for the determination of the molecular masses (Max-Planck-research unit "Enzymologie der Proteinfaltung", D-06120 Halle/Saale, Germany). B. Schöne is gratefully acknowledged for skilled technical assistance.
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FOOTNOTES |
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* 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.
The nucleotide sequence(s) reported in this paper for D. lanata parvulin-like PPIase DlPar13 has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ133755.
¶ To whom correspondence should be addressed.
Published, JBC Papers in Press, December 15, 2000, DOI 10.1074/jbc.M007005200
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ABBREVIATIONS |
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The abbreviations used are: DlPar13, Digitalis lanata phospho-specific parvulin; NH-Np, 4-nitroanilide; PPIase, peptidyl-prolyl cis/trans isomerase; PEMs, proembryogenic masses; Suc, succinyl; RT-PCR, reverse transcriptase-polymerase chain reaction; MES, 4-morpholineethanesulfonic acid.
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