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.
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Abstract |
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Key words: dpm1 gene/dolichol phosphate mannose synthase/Trichoderma reesei
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Introduction |
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Results |
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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., 1988; Mazhari-Tabrizi et al., 1996
; Zimmerman et al., 1996
; Colussi et al., 1997
; Ilgoutz et al., 1999
). 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., 1988
), and conserved in human and S.cerevisiae class DPM synthase sequences (Colussi et al., 1997
).
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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., 1997). 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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The T.reesei dpm1 gene does not function in S.cerevisiae
To determine whether the Trdpm1 genes 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, 1990). 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., 1997
), 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 proteins 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 Dpm1ps 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. 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 3032 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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Discussion |
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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., 1998), 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 TrDpm1ps membership in the "human" class of DPM synthases and the likely requirement for an auxiliary subunit for activity (Colussi et al., 1997; Maeda et al., 1998
), 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, TrDpm1ps 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, 1996).
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 TrDpm1ps 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.
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Materials and methods |
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E.coli strains DH5 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., 1995
). Yeast strains were grown in SD medium (Sherman, 1991
) containing the supplements necessary to complement strain auxotrophies. The strains from which DNA was extracted were grown in YPG medium (Sherman, 1991
). Standard media and procedures were used for crossing, sporulation, and tetrad analysis of yeast (Rose and Hieter, 1990
). 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., 1992
). 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., 1999
).
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 dATP[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., 1989
). 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 , 1993
; Short et al., 1988
; Sambrook, et al., 1989
). 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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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., 1995). 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., 1991
). 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 TrisHCl 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). To assay for DPM synthase activity in bacteria, E.coli DH5
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 0.50.8 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)
, 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 SDSpolyacrylamide 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).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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