Functional Replacement of the Essential ESS1 in Yeast by the Plant Parvulin DlPar13*

A functionally Pin1-like peptidyl-prolylcis/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 thePIN1 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)(3)(4)(5)(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).
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.

EXPERIMENTAL PROCEDURES
Enzyme Assay-For general screening purposes the protease-coupled PPIase microplate assay was performed (22). The specificity constants k cat /K m 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] ϭ 4*k o /k cat /K M ), with k o 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 k o . We calculated the sensitivity of the assay for DlPar13 1 to about 0.3 nM at 7°C, by using the substrate Ac-AS(OPO 3 H 2 )PY-NH-Np with a k o of about 0.0026 s Ϫ1 and a k cat /K m value of 15,900 mM Ϫ1 s Ϫ1 (Table I).
Purification of the DlPar13-Cells of Digitalis lanata Ehrh. strain VIII (25) were grown as PEMs (26) and stored after harvesting at Ϫ70°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 ϫ 2 min with 1,000 rpm. After centrifugation at 25,000 ϫ 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 S cm Ϫ1 and applied onto an anion exchange column (DEAE-EMD-Fractogel, 150 ϫ 20 mm, Merck, Darmstadt, Germany). After an affinity chromatography step (Fractogel TSK AF-Blue, 80 ϫ 15 mm, Merck) the fractions containing the highest phospho-specific activity were further separated by reverse phase HPLC (Nucleosil 300 -5 C18, 125 ϫ 3 mm, Macherey-Nagel, Dü ren, 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 Ϫ1 . 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).
Amino Acid Sequence Analysis-DlPar13 (approximately 30 pmol) was digested with 0.1 g 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).
cDNA Library Screening, Overexpression, and Purification of Dl-Par13-A -ZAP cDNA library (Stratagene, Heidelberg, Germany) constructed from poly(A) ϩ RNA of PEMs of D. lanata served as a template for screening. Approximately 250,000 plaque-forming units were screened using the [␣-32 P]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 5Ј KpnI and a 3Ј 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).
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 A 600 ϭ 1.0. The suspensions (5 l) 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.

RESULTS
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(OPO 3 H 2 )-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 g 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  (15). PPlase measurements were performed in a protease coupled assay as described under "Experimental Procedures." As isomer-specific proteases ␣-chymotrypsin (250 g/ml, top six peptides) and trypsin (25 g/ml, bottom two peptides) were used. b ND, not determined.
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 Arg 68 and Arg 69 coordinates the side chain phosphate group of the substrate. Both residues together with the third basic residue, Lys 63 in hPin1, are conserved among the Pin1-type parvulins. In DlPar13 the specific sequence features exist as Lys 14 , Arg 19 , and Arg 20 .
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 k cat /K m value for the substrate Ac-Ala-Ser(OPO 3 H 2 )-Pro-Tyr-NH-Np was determined with 15.9 M Ϫ1 s Ϫ1 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.
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 (Cys 113 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 Cys 68 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 pro- moter 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.

DISCUSSION
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(OPO 3 H 2 )-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 temperatureinduced 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.
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 M) was incubated with a 10 M excess of juglone (q) and plumbagin (f), respectively, in 35 mM Hepes buffer, pH 7.8, containing 1 M 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(OE) and hPin1 (E) were taken from Hennig et al. (24). The first-order rate constant for the inhibition of DlPar13 was calculated at 3 ϫ 10 Ϫ4 s Ϫ1 . First-order rate constants for Ess1/Ptf1 and for hPin1 were calculated at 5.3 ϫ 10 Ϫ4 s Ϫ1 and 1.5 ϫ 10 Ϫ4 s Ϫ1 .   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 A 600 ϭ 1.0 were applied to the plates.
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 phosphorylationspecific 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) M, 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 ␣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.