Dolichol phosphate mannose synthase from the filamentous fungus Trichoderma reesei belongs to the human and Schizosaccharomyces pombe class of the enzyme

Joanna S. Kruszewska, Markku Saloheimo2, Andrzej Migdalski, Peter Orlean3, Merja Penttilä2 and Grazyna Palamarczyk1

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02 106 Warsaw, Poland, 2VTT Biotechnology P.O. Box 1500, FIN-02044 VTT, Finland, and 3University of Illinois, Department of Biochemistry, 600 South Mathews Ave., Urbana, IL 61801, USA

Received on February 7, 2000; revised on April 17, 2000; accepted on April 25, 2000.


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Dolichol phosphate mannose (DPM) synthase activity, which is required in N-glycosylation, O-mannosylation, and glycosylphosphatidylinositol membrane anchoring of protein, has been postulated to regulate the Trichoderma reesei secretory pathway. We have cloned a T.reesei cDNA that encodes a 243 amino acid protein whose amino acid sequence shows 67% and 65% identity, respectively, to the Schizosaccharomyces pombe and human DPM synthases, and which lacks the COOH-terminal hydrophobic domain characteristic of the Saccharomyces cerevisiae class of synthase. The Trichoderma dpm1 (Trdpm1) gene complements a lethal null mutation in the S.pombe dpm1+ gene, but neither restores viability of a S.cerevisiae dpm1-disruptant nor complements the temperature-sensitivity of the S.cerevisiae dpm1-6 mutant. The T.reesei DPM synthase is therefore a member of the "human" class of enzyme. Overexpression of Trdpm1 in a dpm1+::his7/dpm1+ S.pombe diploid resulted in a 4-fold increase in specific DPM synthase activity. However, neither the wild type T.reesei DPM synthase, nor a chimera consisting of this protein and the hydrophobic COOH terminus of the S.cerevisiae DPM synthase, complemented an S.cerevisiae dpm1 null mutant or gave active enzyme when expressed in E.coli. The level of the Trdpm1 mRNA in T.reesei QM9414 strain was dependent on the composition of the culture medium. Expression levels of Trdpm1 were directly correlated with the protein secretory capacity of the fungus.

Key words: dpm1 gene/dolichol phosphate mannose synthase/Trichoderma reesei


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The saprophytic fungus Trichoderma reesei secretes a wide range of hydrolytic enzymes such as cellulases and hemicellulases, which are important in the food, animal feed, and paper industries (Harman and Kubicek, 1998Go). Many, if not all, of these extracellular proteins are glycosylated. Stimulation of exoprotein secretion by addition of choline or peptone to the culture medium correlates well with an increased level of dolichol phosphate mannose (DPM) synthase (EC 2.4.1.83) activity in the fungus (Kruszewska et al., 1990Go). We have previously shown that in Trichoderma, DPM, which is synthesized by DPM synthase, donates the mannosyl residue that is transferred to the hydroxyl group of serine or threonine in protein O-mannosylation (Kruszewska et al., 1989Go). The obligatory requirement of DPM synthase for O-mannosylation was demonstrated in Saccharomyces cerevisiae with the finding that a temperature sensitive DPM-synthase mutant (dpm1) was completely blocked in O-mannosylation of the model protein chitinase (Orlean, 1990Go). Loss of dpm1 expression in yeast is lethal (Orlean, 1990Go). DPM synthase also participates in N-glycosylation of protein by supplying donor of the last four mannosyl residues during the assembly of the lipid-linked precursor oligosaccharide dolichylpyrophosphate GlcNAc2Man9Glc3, and the enzyme is required for the biosynthesis of glycosylphosphatidylinositol membrane anchors (Herscovics and Orlean, 1993Go). The protein and the gene from S.cerevisiae (Scdpm1) have been characterized (Orlean et al., 1988Go; Haselbeck and Tanner, 1989Go), and the isolation of DPM synthase genes from Ustilago maydis and Trypanosoma brucei has also been reported (Mazhari-Tabrizi et al., 1996Go; Zimmerman et al., 1996Go). More recently, Colussi et al. (1997)Go isolated human and Schizosaccharomyces pombe cDNAs encoding DPM synthases, and showed that these, together with C.briggsae DPM synthase, form a separate class of the enzyme whose members lack the COOH-terminal transmembrane domain present in the S.cerevisiae, U.maydis, and T.brucei enzymes. The overall identity between members of the two classes of DPM synthases is only about 30%, yet the proteins are functionally equivalent because members of both classes complement a lethal null mutation in the S.pombe dpm1+ gene (Colussi et al., 1997Go). However, the mammalian homologue of the S.cerevisiae Dpm1 protein does not correct the defect in DPM formation observed in Lec15 CHO cells, whereas the S.cerevisiae gene does (Tomita et al., 1998Go). These mammalian cells are defective in the Dpm2 protein, which regulates DPMS activity (Maeda et al., 1998Go). Our recent data indicate that overexpression of the S.cerevisiae DPM1 gene in T.reesei results in an increased level of protein secretion (Kruszewska et al., 1999Go). Moreover, Trichoderma DPM synthase, like its counterpart from rat parotid (Banerjee et al., 1987Go), was activated in vitro by cAMP dependent protein kinase (Kruszewska et al., 1991Go). This result suggests that DPMS activity may have a regulatory role in physiology of the fungus and in extracellular protein production. We therefore undertook to clone the T.reesei DPM-synthase gene in order to study the enzyme’s properties and regulation. Here we report that the T.reesei dpm1 gene encodes a 243 amino acid protein that lacks a COOH-terminal transmembrane domain, and which shows 67% and 65% identity, respectively, to the S.pombe and human DPM synthases. The T.reesei DPM synthase homologue is functional in S.pombe but not in S.cerevisiae.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Isolation of cDNA encoding a T.reesei DPM synthase homologue
DPM synthase has a key role in the assembly of glycoconjugates in eukaryotic cells, yet little is known about the structure and regulation of this enzyme. The availability of multiple DPM synthase sequences, together with the finding that there are two distinct classes of the enzyme (Colussi et al., 1997Go), made it possible to design degenerate primers for amplification of either class of DPM-synthase from T.reesei cDNA. Seven different combinations of primers were used for PCR amplification using Trichoderma genomic DNA and a Trichoderma cDNA library as templates. A PCR amplification product was obtained only with the primer pair 93u-94l and the cDNA library as template, and this DNA fragment was cloned into the plasmid pGEM and sequenced. The deduced amino acid sequence obtained was analyzed by the Blast program (Weilguny, 1991Go). The analysis showed that the amplified cDNA encoded a protein fragment with 62% identity to the sequences within the S.pombe and C.briggsiae DPM synthases and 51% identity to a stretch of amino acids in human Dpm1p. A full-length cDNA for Trichoderma DPM synthase was isolated by using a PCR-amplified fragment of the gene to screen a T.reesei RUT-C30 cDNA library. Positive clones were isolated, converted into the plasmid form, and their DNA insert was sequenced.

T.reesei DPM synthase belongs to the human class of the enzyme
Sequence analysis of the 729 bp open reading frame revealed it encoded a protein of 243 amino acids. The predicted T.reesei DPM synthase shows 65% identity and 82% similarity to the human protein (Figure 1), but only 28% and 30% identity to the S.cerevisiae and U.maydis Dpm1p sequences. Furthermore, hydropathy analysis indicates that, like the human protein, the T.reesei DPM synthase lacks a COOH-terminal transmembrane domain (Figure 2), a characteristic of the S.cerevisiae, U.maydis, T.brucei, and L.mexicana enzymes (Orlean et al., 1988Go; Mazhari-Tabrizi et al., 1996Go; Zimmerman et al., 1996Go; Colussi et al., 1997Go; Ilgoutz et al., 1999Go). The Trichoderma DPMS therefore is a member of the human and fission yeast class of DPM synthases. The deduced amino acid sequence of the Trichoderma DPM synthase homologue contains a potential site for phosphorylation by a cAMP-dependent protein kinase at Ser152 (Figure 1), a site also present in the S.cerevisiae protein at Ser141 (Orlean et al., 1988Go), and conserved in human and S.cerevisiae class DPM synthase sequences (Colussi et al., 1997Go).





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Fig. 1. The deduced amino acid sequence of the predicted Trichoderma protein and its alignment with the sequences of Dpm1 proteins of the S.cerevisiae and human classes. The DAD motif (amino acid residues 110–112) and the potential phosphorylation site (Ser152) are marked.

 


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Fig. 2. Hydropathy analysis of the S.cerevisiae, T.reesei, and chimeric TrDpm1p/ScDpm1p DPM synthases. Sequences were analyzed using the Tmpred program (Hoffmann et al., 1993).

 
Comparison of the amino acid sequences of all DPM synthase proteins listed in the database indicates the presence of the conserved DAD sequence (Figure 1). A similar conserved aspartate-containing sequence was found by hydrophobic cluster analysis in a range of ß-glycosyltransferases that transfer a single sugar residue (Saxena et al., 1995Go). More recently, it has been suggested that the DXD motif is a feature of the catalytic site or an element of a structural fold shared by many glycosyltransferases (Yanisch-Perron, 1985Go). The DAD motif is present at amino acid positions 110–112 of the Trichoderma Dpm1 protein (Figure 1).

Expression of the T.reesei dpm1 gene in S.pombe
If the Trdpm1 gene indeed encodes an DPM synthase of the human and fission yeast class, then it would be predicted to complement a lethal null mutation in the gene encoding its S.pombe counterpart, as is the case with human DPM1 (Colussi et al., 1997Go). To test whether this is so, the heterozygous S.pombe strain dpm1:: his7/dpm1+S 27 was transformed with plasmid pDW 232 containing the Trdpm1 gene. The specific DPM synthase activity measured in the membranes from the diploid transformants was 4-fold higher than that measured in membranes from the control S.pombe strain transformed with the plasmid without the Trdpm1 insert (Table I). However, when S.pombe was transformed with a chimeric Trdpm1 gene encoding TrDpm1p fused to the COOH terminus of ScDpm1p (see below), the specific DPMS activity of the transformants was only twice that of untransformed controls, suggesting that the COOH-terminal extension of TrDpm1p with the S.cerevisiae hydrophobic domain interferes with the function of the wild type TrDpm1p.


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Table I. DPM synthase activity in Schizosaccharomyces pombe
 
Heterozygous S.pombe dpm1::his7/dpm1+ diploids harboring the Trdpm1 gene were allowed to sporulate, and the meiotic progeny were submitted to random spore analysis to test whether haploid cells could grow in medium selective for both histidine and uracil prototrophy (Colussi et al., 1997Go). Because dpm1+ is essential for viability in S.pombe (Colussi et al., 1997Go), histidine prototrophic, haploid dpm1::his7+ are unable to grow on selective medium unless a functional DPM synthase is expressed in them. Indeed, there were no histidine prototrophic colonies among the meiotic progeny arising from dpm1::his7/dpm1+S 27 diploids that had been transformed with the empty pDW232 vector, whereas the diploids that had received pDW232 containing the Trdpm1 gene did give rise to colonies of histidine- and uracil-prototrophic haploids. We conclude that the product of the Trdpm1 gene is functional in S.pombe.

The T.reesei dpm1 gene does not function in S.cerevisiae
To determine whether the Trdpm1 gene’s product is functional in S.cerevisiae, we tested whether this gene could complement null or conditional mutations in the S.cerevisiae DPM1 gene. The yeast expression plasmid pAJ401 containing the Trdpm1 gene was introduced into the heterozygous S.cerevisiae dpm1::LEU2/DPM1, leu2/leu2 diploid and the transformants were allowed to sporulate and the resulting tetrads were dissected. All tetrads dissected yielded only two viable, leucine auxotrophic spores, indicating that Trdpm1 did not complement the lethal dpm1::LEU2 disruption. Moreover, transformation with pAJ401 did not rescue the temperature-sensitivity of the S.cerevisiae dpm1-6 mutant, which harbors a temperature-sensitive dpm1 allele on a plasmid (Orlean, 1990Go). The inability of Trdpm1 to complement mutations in its S.cerevisiae counterpart is consistent with its membership in the human and S.pombe class of DPMS: neither the human nor the fission yeast DPM1 genes encode proteins that are functional in S.cerevisiae (Colussi et al., 1997Go), and furthermore, Trdpm1, like its human and S.pombe counterparts, does not encode an DPM synthase that is active when expressed in E.coli (see Discussion).

A COOH-terminal transmembrane domain is not sufficient for T.reesei DPM synthase to function in S.cerevisiae or in E.coli
We considered the possibility that the failure of Trdpm1 to complement the yeast dpm1 defect might be due to the fact that the T.reesii DPMS lacks the COOH- terminal transmembrane domain that is present in the S.cerevisiae enzyme. We therefore tested whether addition of the S.cerevisiae Dpm1 protein’s transmembrane domain to the COOH-terminus of the TrDpm1 protein would render the Trichoderma enzyme functional in vivo when expressed in S.cerevisiae, and active in vitro when expressed in E.coli. A chimeric Trdpm1/ScDPM1 gene was constructed that contained the entire Trichoderma DPM synthase coding region in frame with DNA encoding the 27 amino acid COOH-terminal transmembrane domain of S.cerevisiae Dpm1p (Figure 2). This gene was cloned into plasmid pAJ401 and the resulting expression plasmid transformed into heterozygous S.cerevisiae dpm1::LEU2/DPM1 diploid JZY 251. The transformants were allowed to sporulate, and the resulting asci submitted to tetrad analysis. Out of 14 tetrads dissected, 12 yielded only 2 viable spores but 2 contained 3 viable spores which were analyzed further. Cells from the haploid colonies arising from the two tetrads with three viable spores were re-plated on YPD medium containing 5-fluororotic acid (5-FOA) to select against growth of cells harboring the URA3-marked plasmid expressing the chimeric Trdpm1/ScDPM1 gene. All three haploids from each tetrad all yielded colonies that could grow on 5-FOA-containing medium, indicating that growth of any dpm1::LEU2 haploid was plasmid-independent. Moreover, all six haploids were all uracil auxotrophs. We conclude that the presence of the COOH-terminal transmembrane domain is not sufficient for the Trichoderma DPM synthase to compensate for the S.cerevisiae DPM1 disruption. Similarly, the chimeric Trdpm1 gene did not complement the thermosensitive phenotype of the S.cerevisiae dpm1-6 mutant. Because the COOH-terminal domain of S.cerevisiae Dpm1p is likely to be a major determinant for S.cerevisiae Dpm1p’s localization to the yeast ER membrane, it seems less likely that the chimeric DPM synthase was inactive in S.cerevisiae on account of being mislocalized.

To test whether the chimeric protein was enzymatically active in E.coli, the gene encoding it was cloned into plasmid pRS316, which placed it under the control of the lacZ promoter, and the resulting construct was introduced into E.coli strain DH5{alpha}. In vitro DPM-synthase activity was then assayed in crude sonicates of the bacterial transformants, and radiolabeled mannolipids were extracted and separated by thin layer chromatography. The bacteria expressing the chimeric protein made no detectable [14C]DPM (Figure 3, lanes 3 and 4), whereas the control E.coli cells expressing yeast DPM1 did (Figure 3, lanes 1 and 2). Therefore, extension of the Trichoderma Dpm1 protein with the COOH-terminal hydrophobic domain of the yeast enzyme is not sufficient to obtain an active DPM synthase. To ensure that the TrDpm1 and chimeric TrDpm1/ScDpm1 proteins were expressed in E.coli, we exploited our observation that an anti-S.cerevisiae Dpm1p monoclonal antibody cross-reacts with the TrDpm1 protein. Samples of the sonicates of the E.coli transformants that had been assayed for DPM synthase activity were separated by SDS-polyacrylamide gel electrophoresis, then submitted to Western blotting using the anti-yeast Dpm1p antibody. A single immunoreactive band of about 30–32 kDa was detected in extracts from E.coli expressing Trdpm1 or S.cerevisiae DPM1 genes (Figure 4, lanes 1 and 2). The sizes of these proteins and the differences between them are consistent with the sizes predicted for the both proteins. The control strain harboring the vector alone contained no immunoreactive material (Figure 4, lane 3). The results of Western blot and immunostaining of the E.coli extracts transformed with the chimeric gene indicated that the hybrid protein of expected size has been also expressed (not shown). We conclude that the lack of activity of the bacterially expressed proteins is not due to lack of expression.



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Fig. 3. Expression of S.cerevisiae DPM1 and the chimeric Trdpm1/ScDPM1 genes in E.coli. E.coli strain DH5{alpha} was transformed with plasmid pRS316 containing the S.cerevisiae DPM1 gene (lanes 1 and 2) or with plasmid pRS316 containing the Trdpm1 reading frame extended at its 3' end with DNA encoding the COOH-terminal hydrophobic domain of S.cerevisiae Dpm1p (lanes 3 and 4). Expression of these genes was driven by the lacZ promoter. DPM synthase activity was determined in sonicates of the bacteria in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of exogenous C95-dolichol phosphate. Lipophilic reaction products were separated by thin layer chromatography using chloroform/methanol/water (65:25:4, by volume) as solvent, and [14C]-labeled lipids were detected by autoradiography. Lane 5 contains standard C95-dolichol phosphate mannose.

 


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Fig. 4. Expression of the TrDpm1 protein in E.coli. Samples of the bacterial sonicates prepared as in the experiments shown in Figure 3, and of harboring a plasmids containing Trdpm1and Sc.DPM! (150 µg of protein in each sample) were separated by SDS–polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, then analyzed by Western blotting using an anti-S.cerevisiae Dpm1p monoclonal antibody. Lanes 1 and 2, E.coli harboring the Trdpm1 and ScDPM1. Lane 3, E.coli harboring the empty vector pRS316.

 
Elevated expression of Trdpm1 is correlated with up-regulation of protein secretion by T.reesei QM9414.
Protein secretion by T.reesei is up-regulated when the fungus is grown on media containing urea or choline, and these events are correlated with an increase in DPM synthase activity measurable in the cells (Kruszewska et al., 1990Go). The availability of the Trdpm1 gene allowed us to test whether increased DPM synthase activity is correlated with an elevated level of expression of the Trdpm1 gene. Total RNA was isolated from cultures of T.reesei strain QM 9414 that had been grown for 140 h on minimal medium supplemented with choline, peptone, or urea. A 6-fold and almost a 9-fold increase in mRNA levels was observed in T.reesei cultivated in the presence of choline and urea, respectively, whereas inclusion of peptone in the medium had only a negligible effect (Figure 5). These findings, together with our previous observations on the effects of choline and urea on exoprotein production, support the suggestion that levels of DPM synthase activity are one of the factors regulating protein by T.reesei (Palamarczyk et al., 1998Go).



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Fig. 5. Elevated expression of the Trdpm1 gene in cultures grown in media that stimulate exoprotein production. Equal amounts of total RNA (20µg) were isolated from cultures of T.reesei strain QM9414 that had been grown on minimal medium supplemented with choline (2 mg/ml), urea (0.6 mg/ml), or peptone (2 mg/ml) and submitted to Northern blot analysis using a 1 kb EcoRI/XhoI fragment of the T.reesei dpm1 gene as a probe. (A) Autoradiogram of [32P]-labeled Trdpm1 mRNA from cells grown in media supplemented with choline (lane 1), peptone (lane 2), urea (lane 3), or from control cells (lane 4). (B) Levels of T.reesei act1 mRNA in the samples from (A), probed with a 1.9 kb KpnI fragment of the T.reesei act1 gene as a loading control for (A). (C) Quantification of levels of Trdpm1 mRNA, normalized against Tract1 mRNA levels. Quantification of the [32P] signals in (A) was performed using the Image Quant Program.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Our finding that increased protein secretion by the industrially important fungus Trichoderma reesei is correlated with elevated DPM synthase activity and stimulated by heterologous expression of the S.cerevisiae DPM1 gene led us to test whether expression of native Trdpm1 is elevated when cells are grown under the conditions that stimulate exoprotein production. To do this, we isolated a cDNA for T.reesei DPM-synthase. The Trdpm1 cDNA encodes a predicted protein of 243 amino acids and a molecular mass of about 36 kDa, and represents the first example of the cloning of an DPM synthase coding sequence from a filamentous fungus. Trichoderma DPM synthase is a member of the "human" class of DPM synthases. TrDpm1p has a potential site for phosphorylation by cAMP-dependent protein kinase at the same relative position in its amino acid sequence as all other DPM synthase sequences (Colussi et al., 1997Go). The presence of this site is consistent with our finding that cAMP stimulates DPM synthase activity in vitro (Kruszewska et al., 1991Go).

The observation that Trdpm1 mRNA levels rise 6- to 9-fold when the fungus is cultivated on media that stimulate exoprotein production supports our notion that DPM synthase is a regulator of this process. However, it is not the level of TrDpm1p alone, but rather, total DPM synthetic capacity, that must have the regulatory role. Thus, expression of the Trdpm1 cDNA in T.reesei does not result in a significant increase in in vitro DPM synthase activity or in increased protein secretion (J. Kruszewska, unpublished observations). Because "human" class DPM synthases consist of at least two subunits, Dpm1p and Dpm2p (Maeda et al., 1998Go), it is possible that in T.reesei, the levels of the Dpm2p subunit we would expect this fungus to have are limiting for DPM formation. We would therefore predict that, if present in T.reesei, Trdpm2, like Trdpm1, would also be expressed at elevated levels when exocellular protein secretion is stimulated. Our finding that overexpression of Trdpm1 cDNA in a wild type S.pombe strain results in a 4-fold increase in in vitro DPM synthase activity suggests that S.pombe Dpm2p is not limiting for Trichoderma DPM synthase activity in fission yeast.

The Trichoderma DPM synthase is functional in vivo because the Trdpm1 gene complements a lethal null mutation in the Schizosaccharomyces pombe dpm1+ gene. Further, as expected from TrDpm1p’s membership in the "human" class of DPM synthases and the likely requirement for an auxiliary subunit for activity (Colussi et al., 1997Go; Maeda et al., 1998Go), the Trdpm1 gene neither rescues an S.cerevisiae dpm1::LEU2 disruptant nor confers DPM synthase activity on E.coli, despite the observation that the full-sized TrDpm1 and chimeric TrDpm1/ScDpm1 proteins are made in the bacteria.

However, TrDpm1p’s lack of in vivo and in vitro activity when expressed in S.cerevisiae and E.coli respectively is not simply due to the absence of a COOH terminal transmembrane domain from this "human" class synthase. Thus, addition of the COOH-terminal hydrophobic domain of S.cerevisiae Dpm1p at the COOH terminus of TrDpm1p does not generate a functional DPM synthase. The S.cerevisiae COOH-terminal domain therefore does not mimic the auxiliary subunit required by TrDpm1p. Indeed, most of the COOH terminal hydrophobic domain of S.cerevisiae Dpm1p is dispensable for in vivo function in S.cerevisiae, for a truncated protein retaining only three COOH terminal hydrophobic residues still rescues the dpm1::LEU2 disruption (Zimmerman, 1996Go).

The finding that T.reesei has a "human" class DPM synthase is an important step in identifying the mechanism of DPM synthesis in this filamentous fungus and manipulating the process. Thus, we have established that auxiliary proteins are likely to be required for the stable overexpression of the homologous DPM synthase, and, with the availability of the Trdpm1 gene, we can test the significance of TrDpm1p’s phosphorylation site for regulation of enzymatic activity. The studies now made possible will help us toward our goal to develop T.reesei strains that constitutively overexpress DPM synthase activity and exhibit hyper-secretory capacity.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Strains, Trichoderma c-DNA library, and growth media
The yeast strains used in this study were S.cerevisiae diploid JZY251 (dpm1::LEU2/ DPM1, ura3-52/ura3-52, leu2-3,112/leu2-3,112 (Zimmerman, 1996Go), S.cerevisiae strain dpm1-6 (MAT{alpha}, dpm1::LEU2, leu2-3, lys2-801, trp1-1, ura3-52) harboring plasmid pDM8-6, which carries the temperature sensitive dpm1-6 (Pro157 to Leu) allele and the TRP1 marker (Orlean, 1990Go), and S.pombe diploid strain S27 (dpm1+::his7, ade6, leu1-32, ura4-d18 (Colussi et al., 1997Go).

E.coli strains DH5{alpha} and XL1 Blue MRF' were from Bethesda Research Laboratories and T.reesei strains QM9414 and RutC-30 were from American Type Culture Collection (ATTC 26921 and ATTC 56765). The T.reesei RutC-30 cDNA libraries were constructed in the Lambda ZAP II vector (Stratagene) and in the yeast expression vector pAJ401 (Stalbrand et al., 1995Go). Yeast strains were grown in SD medium (Sherman, 1991Go) containing the supplements necessary to complement strain auxotrophies. The strains from which DNA was extracted were grown in YPG medium (Sherman, 1991Go). Standard media and procedures were used for crossing, sporulation, and tetrad analysis of yeast (Rose and Hieter, 1990Go). The S.pombe diploid was sporulated on medium containing 1% (w/v) glucose, 0.3% (w/v) maltose, 0.5% (w/v) peptone, and 0.3% (w/v) yeast extract. For yeast transformation, a "one-step" method was used (Chen et al., 1992Go). For analysis of Trdpm1 expression, T.reesei QM 9414 was cultivated on minimal medium and RNA was isolated from it as described (Kruszewska et al., 1999Go).

Isolation of the T.reesei dpm1 gene
A fragment of DNA encoding a portion of a putative Trichoderma DPM synthase was amplified by PCR using a Trichoderma cDNA library as template. The DNA fragment was then used to screen a Trichoderma lambda ZAP cDNA library by plaque hybridization. The degenerate primers used were designed using human, S.pombe and Caenorhabditis briggsae Dpm1 protein sequences as guides (Table II). PCR amplification reactions were carried out using Dynazyme (Finnzymes, Finland) in incubation mixtures (total volume, 100 µl) containing 300 pmol of primers (93u with 94l, 95u with 94l, and 93u, 95u with oligo dT), 200 mM dNTPs, reaction buffer, 3 mM MgCl2, and 3 µg of Trichoderma cDNA library in the yeast pAJ401 vector as a template. Amplification conditions consisted of a 3 min denaturation step at 94°C, and then 7 cycles of incubation at 94°C for 45 s, 37°C for 45 s, and 72°C for 1 min. The temperature was increased from 37°C to 72°C by 0.2°C per second. Amplification was then continued for 30 further cycles with an annealing temperature of 50°C and concluded with a 5 min incubation at 72°C. The PCR products were analyzed on a 1.2% agarose gel. The DNA fragment of about 550 bp that was obtained with the 93u and 94l primers was isolated, cloned into the pGEM-T vector (Promega pGEM-T Vector System), and sequenced. The Trichoderma DNA fragment, which encoded an amino acid sequence 62% identical to that of a portion of the S.pombe Dpm1 protein, was used for probe preparation using radioactive d{alpha}ATP[32P] and the Amersham Megaprime DNA labeling system according to the standard Amersham protocol. E.coli XL1-Blue MRF' cells were infected with the lambda ZAP II phage T.reesei RutC-30 cDNA library using standard methods (Sambrook, et al., 1989Go). Of the 500,000 bacteriophage plaques analyzed, 6 gave a radioactive signal. Bluescript plasmids, containing the ~950 bp cDNA inserts (pJSK2-7), were isolated from the bacteria (Zimmerman and Robbins , 1993Go; Short et al., 1988Go; Sambrook, et al., 1989Go). The inserts in three of the plasmids were sequenced using the Pharmacia automatic sequencer (ALF), either with the Pharmacia fluorescent primer sequencing kit or with custom-synthesized oligonucleotide primers and internal labeling with a fluorescent derivative of dATP (Pharmacia).


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Table II. title
 
Construction of a chimeric Trdpm1 gene by plasmid gap-repair method using PCR generated cassette
The plasmid gap-repair method, which uses a PCR-generated cassette to create gene fusions, is based on the technique described by Orr-Weaver et al. (1983)Go. This method was used to create a chimeric dpm1 gene consisting of the Trdpm1 coding sequence without its stop codon and a fragment of the S.cerevisiae DPM1 gene coding for the transmembrane domain of DPM synthase. The S.cerevisiae DPM1 gene fragment was generated by PCR with 75-base oligonucleotide primers. The upper strand primer, 5'- GAC GAG ATT GTC GAG TAC GCC AAG GGC GTG TTT TCC CTG TGG CTC AAG GTC GCC AAT AAC CTT ATC CTT TTC ATT contained Trdpm1 sequences followed by the S.cerevisiae sequence coding for the transmembrane domain, and the lower strand primer, 5'- CTC GGA ATT AAC CCT CAC TAA AGG GAA CAA AAG CTG GGT ACC GGG CCC CCC CTA TGT GTG GGG GTG GGG CAG AAT, encoded 27 amino acids of the S.cerevisiae Dpm1 protein’s transmembrame domain and also contained DNA complementary to sequence of plasmid pRS316, a shuttle vector containing the inducible LacZ promoter and the yeast URA3 and ampicillin resistance genes as selectable markers. Yeast cells were cotransformed with 100 ng of XhoI-linearized plasmid pRS316 containing the native Trdpm1 gene ligated into to its EcoRI/XhoI sites, and a 20-fold molar excess of the PCR-generated DNA fragment which was flanked by 50 nucleotides identical to the plasmid’s DNA at one end, and by 50 nucleotides identical to Trdpm1 sequence at the other end. Plasmids from Ura+ transformants were isolated as described (Hoffmann and Winston, 1987Go) and used in turn to transform E.coli DH5{alpha} cells. The resulting plasmids were digested with KpnI/EcoRI to analyze the insert. Standard molecular cloning techniques (Sambrook, et al., 1989Go) were used for isolating and analyzing plasmid DNA. The resulting hybrid insert was sequenced to check the PCR product and ascertain its correct ligation in the vector.

Construction of expression plasmids for Trdpm1
For expression of Trdpm1 in S.cerevisiae, a cDNA fragment of about 950 bp, containing the Tr.d.p.m.1 reading frame or the chimeric Trdpm1/ScDPM1 gene were cloned into the XhoI/EcoRI sites of plasmid pAJ401 (Stalbrand et al., 1995Go). For expression in S.pombe, the fragments of Trichoderma cDNA containing the Trdpm1 reading frame or the chimeric Trdpm1/ScDPM1 gene were cloned into the KpnI and BamHI sites of plasmid pDW232, which contains the S.pombe ura4+ gene as marker (Weilguny et al., 1991Go). For expression in E.coli, the Trdpm1 gene, the S.cerevisiae DPM1 gene, and the chimeric Trdpm1/ScDPM1 genes were each cloned into the pRS316 vector at its EcoRI and XhoI sites, which placed them under the control of the LacZ promoter.

DPM synthase assay
Control and transformed S.pombe strains were grown at 30°C in 1 l of SD medium to an OD600 of 1, then harvested by centrifugation and resuspended in 25 ml of 150 mM Tris–HCl buffer pH 7.4 containing 15 mM MgCl2 and 9 mM 2-mercaptoethanol. The cells were homogenized in a Beadbeater with 0.5 mm diameter glass beads, and the homogenate then centrifuged at 4000 x g for 10 min to remove unbroken cells and cell debris. The supernatant liquid was centrifuged for 1 h at 50,000 x g. DPM synthase activity was measured in the pelleted membrane fraction by incubating it with GDP-[14C]Mannose (sp. act.: 288 Ci/mol, Amersham), according to Kruszewska et al. (1989)Go. To assay for DPM synthase activity in bacteria, E.coli DH5{alpha} transformants expressing DPM1 genes were grown overnight in LB medium containing 100 µg ampicillin/ml. One ml of the overnight culture was then transferred to 100 ml of fresh LB-ampicillin medium supplemented with 0.4 mM isopropyl-ß-D-thiogalactopyranoside and grown to an OD600 of 0.5–0.8 at 37°C. DPM synthase activity was determined in crude lysates of E.coli prepared by cell sonication as described by Orlean et al. (1988)Go, and the chloroform/methanol extractable, [14C]-labeled products were analyzed on Silica Gel 60 TLC plates, which were developed in the solvent system chloroform/methanol/water (65:25:4 by volume). A standard of [14C]-labeled yeast DPM was run in parallel as a standard.

Immunoblotting
Samples of sonicates of E.coli transformants containing 150 µg protein were separated by SDS–polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, then analyzed by Western blotting using an anti-S.cerevisiae Dpm1p monoclonal antibody (Molecular Probes, Eugene, OR, USA) using the conditions recommended by the supplier. Immunoreactive material was detected using an anti-mouse IgG secondary antibody conjugated to alkaline phosphatase (Promega).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Ms. Riitta Nurmi (VTT Biotechnology and Food Research, Espoo, Finland) for technical assistance, and Dr. Irene Jensen for helpful suggestions. The work was partially financed by The Committee for Scientific Research (KBN, Poland, Grant 6P04B 01712) and the exchange program between the Finnish and Polish Academies of Science.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
DPM, dolichol phosphate mannose; ScDpm1p and TrDpm1p, the proteins encoded by the ScDPM1 and Trdpm1 genes respectively; dNTP, deoxyribonucleoside triphosphate; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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