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Cloning and functional analysis of dpm2 and dpm3 genes from Trichoderma reesei expressed
in Saccharomyces cerevisiae dpm1∆ mutant
Patrycja Zembek , Urszula Perlińska-Lenart , Katarzyna Rawa , Wioletta Górka-Nieć ,
Grażyna Palamarczyk , Joanna S. Kruszewska*
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02106 Warsaw, Poland
Address for correspondence: Dr. Joanna S. Kruszewska, Institute of Biochemistry and
Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland. Phone (+
48 22) 592 12 09 Fax: (+48 22) 658 46 36
E-mail [email protected]
RUNNING TITLE: Functional analysis of T.reesei DPM synthase subunits
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SUMMARY
In Trichoderma, dolichyl phosphate mannose synthase, a key enzyme in the O-glycosylation
process, needs three proteins for full activity. In this study the dpm2 and dpm3 genes coding
for the DPMII and DPMIII subunits of Trichoderma DPM synthase were cloned and analyzed
functionally after expression in the S.cerevisiae dpm1∆ mutant. It was found that apart from
the catalytic subunit DPMI, also the DPMIII subunit was essential to form an active DPM
synthase in yeast. Additional expression of the DPMII protein, considered to be a regulatory
subunit of DPM synthase, decreased the enzyme activity. We also characterized S.cerevisiae
strains expressing dpm1,2,3 or dpm1,3 genes and analyzed the consequences of dpm
expression on protein O- glycosylation in vivo and on the cell wall composition.
KEY WORDS: Trichoderma; DPM synthase; O-glycosylation; cell wall composition.
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INTRODUCTION
Dolichyl phosphate mannose synthase (DPM synthase) catalyzes the transfer of mannosyl
residue from GDP mannose to dolichyl phosphate (DolP) to form dolichyl phosphate
mannose (DPM). DPM serves as a donor for protein O-mannosyltransferases in Oglycosylation and for mannosyltransferases elongating the DolPP(GlcNAc)2Man5
oligosaccharide in the N-glycosylation pathway (Orlean 1992; Herscovics and Orlean 1993;
Strahl-Bolsinger et al., 1999, Maeda and Kinoshita 2008). DPM also takes part in Cmannosylation and in the synthesis of glycosylphosphatidylinosytol anchor (GPI), and cells
devoid of DPM synthase are non viable (Doucey et al., 1998; Orlean 1990). Moreover,
limitation in the synthesis of DPM in human cells causes a congenital disorder of
glycosylation (CDG) called carbohydrate-deficient glycoprotein syndrome (CDGS); show
developmental delay, hypotonia, seizures, and acquired microcephaly (Imbach et al., 2000;
Kim et al., 2000, Lefeber et al. 2009).
The DPM synthases cloned up to now are divided into two groups. The first group contains
Dpm1 proteins similar in structure to the Saccharomyces cerevisiae DPM synthase which is
encoded by the DPM1 gene and characterized by a C-terminal transmembrane domain which
is absent in the second group of the Dpm1 proteins called the human group. The human group
contains synthases from human, rat, Caenorhabditis elegans, Schizosaccharomyces pombe
and Trichoderma (Orlean et al., 1988; Colussi et al., 1997; Kruszewska et al., 2000). In
human cells DPM synthase is a complex of three proteins, where DPM3 stabilizes the DPM1
protein and DPM3 itself is stabilized by DPM2 (Maeda et al., 2000, Ashida et al. 2006).
DPM2 is a hydrophobic protein associated with the hydrophobic N-terminal part of DPM3
(about 60 amino acids). In turn, a hydrophobic C-terminal part of DPM3 (about 30 amino
acids) associates with DPM1. Maeda et al. (1998) proposed a model of the DPM synthase
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complex and described it in detail, showing that for association of DPM1 with DPM2 24 Cterminal amino acids of DPM1and Phe 21 and Tyr 23 from DPM2 were important.
Furthermore, DPM3 alone was able to stabilize DPM1 and DPM synthase had an enzymatic
activity without the DPM2 subunit, however, the activity was ten-fold higher when DPM2
was present as well. It is not known why in some organisms the structure of DPM synthase is
so complex; it has been suggested that a multicomponent system could be more suitable for
regulation (Maeda et al., 2000), but a similar system of regulation has been proposed for DPM
synthases from both groups. DPM synthase from Trichoderma, like its counterparts from the
rat, Aspergillus nidulans, S.cerevisiae or Candida albicans has been shown to be activated in
vitro by cAMP-dependent phosphorylation (Kruszewska et al., 1991; Banerjee et al., 1987,
2005; Perlińska-Lenart et al., 2005; Arroyo-Flores et al., 2005). The predicted site of
phosphorylation, Ser 152 in Trichoderma, was also found in S.cerevisiae Dpm1 at Ser 141
and is generally conserved in the human and S.cerevisiae classes of DPM synthases (Orlean et
al., 1988; Colussi et al., 1997; Kruszewska et al., 2000; Banerjee et al., 2005).
A comparison of sequences of Dpm1 proteins has revealed 30% identity between members
from different groups and more than 60% identity between Dpm1 proteins from the same
group (Kruszewska et al., 2000). Moreover, our interest was stirred up by the presence of the
C-terminal hydrophobic domain distinguishing the two groups of Dpm1 proteins. The domain
is likely to be a major determinant for the localization of the yeast-type Dpm1 proteins in the
endoplasmic reticulum membrane. From our earlier study we know that addition of the Cterminal transmembrane domain from S.cerevisiae Dpm1to the T.reesei DPMI protein creates
a chimeric protein which is not functional in S.cerevisiae or bacteria and has a decreased
activity when expressed in an S.pombe dpm1 mutant (Kruszewska et al., 2000). This result
has led to the conclusion that the transmembrane domain cannot replace the DPMII and
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DPMIII subunits of Trichoderma DPM synthase and suggested that the enzyme is inactive in
vivo on account of being mislocalized. Since organisms belonging to the S.cerevisiae group
lack the Dpm2 and Dpm3 subunits of DPM synthase, the Dpm1 protein from the human
group cannot replace the Dpm1 protein from the S.cerevisiae group (Tomita et al., 1998;
Maeda et al., 1998).
Here we expressed DPM proteins from Trichoderma in different combinations in the
S.cerevisiae dpm1∆ mutant. We report that apart from the DPMI catalytic subunit also the
DPMIII protein is essential for survival of the mutant. Additional expression of the DPMII
regulatory protein resulted in the inhibition of DPM synthase activity. As a consequence of
the decreased activity of the enzyme, chitinase, a model O-glycosylated protein ,was partially
under glycosylated. In addition, the lower-than-normal activity of DPM synthase in yeast
strains dpm1,2,3 or dpm1,3 resulted in changes in the cell wall composition which were more
pronounced in the triple transformant than in both dpm1,3 and dpm1*3(GAL)-expressing
strains.
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RESULTS
Cloning of the dpm2 and dpm3 genes from T.reesei
Cloning of the dpm1 gene from Trichoderma (Kruszewska et al., 2000) showed that the
encoded DPM synthase belongs to the human group of the enzymes and probably works as a
complex of three proteins DPMI, DPMII and DPMIII. Fragments of DNA encoding putative
DPMII and DPMIII proteins were synthesized as described in the Methods and sequenced.
Sequence analysis of the 240 bp open reading frame (ORF) of dpm2 revealed it encoded a
protein of 80 amino acids, a predicted T.reesei DPMII. This protein shows 86% identity and
93% similarity to the Gibberella zeae hypothetical Dpm2 protein and only 54% identity and
78% similarity to the S.pombe Dpm2p (Figure 2). The second ORF of 276 bp encoded a
protein of 92 amino acids a predicted T.reesei DPMIII. Analysis of the protein sequence
showed 79% identity and 90% similarity to the Dpm3 protein from Gibberella zeae, 64 %
identity and 76 % similarity to the Neurospora crassa Dpm3 and only 30% identity and 63%
similarity to S.pombe Dpm3p (Figure 2) (Altschul et al., 1997).
Phylogenetic trees for the Dpm2 (Figure 3) and Dpm3 (Figure 4) proteins supported the
results from protein sequence analysis (Figure 2) showing the closest relationship of
Trichoderma with other filamentous fungi.
The putative DPMII and DPMIII proteins were analyzed by the TmPred program (Hoffmann
et al., 1993) to find potential membrane spanning domains.
Hydropathy analysis indicated that, like the human DPM2 protein (Maeda et al., 1998),
Trichoderma DPMII is a hydrophobic protein with two predicted transmembrane domains
(Figure 2), and analysis by the PSORT II program (http://psort.nibb.ac.jp) revealed a putative
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ER membrane retention signal, a KKXX-like motif (AKKK) at the C-terminus. Hydropathy
profile of the predicted T.reesei DPMIII showed a structure very similar to that of the human
DPM3 protein (Maeda et al., 2000) with two membrane spanning domains (Figure 2), a
hydrophobic domain at the C-terminus and a putative ER retention signal TRAQ at the Nterminus.
In the cloned genomic copies of the two genes we found one intron (82 bp) in the 322 bp
sequence of dpm2 and two introns (104 bp and 97 bp) in the 477 bp sequence of dpm3.
Expression of the dpm2 and dpm3 genes in S.cerevisiae
A yeast diploid strain carrying deletion of one copy of the DPM1gene was transformed with
Trichoderma dpm1, dpm2 and dpm3 or dpm1and dpm2 or dpm1and dpm3 genes and selected
on -Leu -Ura SC medium. Eleven different combinations of plasmids were used for
transformation (Table 2).
Transformants were then cultivated on sporulation medium and tetrads dissected. Four viable
spores were obtained for S.cerevisiae transformed with non-flagged dpm1,2,3 or dpm1,3
genes. These transformants contained the dpm1 gene under the constitutive GPD promoter.
We also isolated one transformant (dpm1*3(GAL)) containing FLAG-dpm1 gene under the
GAL1 promoter in combination with non-tagged dpm3 gene. S.cerevisiae dpm1∆ diploid
transformed with this combination of genes gave only two or three viable spores. The
combination of dpm1and dpm2 genes could not complement the DPM1 deletion, giving only
two viable spores containing the original yeast DPM1 gene.
Next, the spore clones were analyzed as described in the Methods to confirm the presence of
the appropriate dpm genes and finally plasmids isolated from the strains were sequenced. For
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the subsequent experiments we took the dpm1,2,3, dpm1,3 and dpm1*3(GAL) transformants
and a control haploid S.cerevisiae strain containing the native DPM1 gene.
Influence of heterologously expressed dpm genes on strain growth and activity of DPM
synthase, protein O-mannosyltransferases and mannosyltransferases elongating Olinked sugar
The transformants and control strain were cultivated in SC medium with galactose and every
1.5 h absorbtion of the culture was measured (Figure 5). The dpm1,2,3-transformed strain
grew more slowly than did the control and dpm1,3 or dpm1*3(GAL) strains. Both dpm1,3, and
dpm1*3(GAL)-transformed strains and the control reached stationary phase after 9 h while the
dpm1,2,3 transformant required 20 h of cultivation.
Next, the strains were cultivated to mid-exponential phase (OD600=1), membrane fractions
were isolated and activity of DPM synthase was measured. The membrane fractions were also
used for Western analysis of DPMI protein content in the ER of the transformants. The
activity of DPM synthase in the membrane fraction from the dpm1,2,3-transformed strain was
by 35% lower than the activity from both dpm1,3-transformed strains and the control (Table
3). The decreased activity of DPM synthase in the membrane fraction from dpm1,2,3transformed strain was in agreement with the low amount of DPMI protein detected by
Western blot (Figure 6).
Since the DPM synthase activity in the dpm1,2,3 transformant was decreased one could
expect that this strain produced lower amounts of dolichyl phosphate mannose a crucial
substrate for protein O-mannosyltransferases; to check the validity of this assumption activity
of protein O-mannosyltransferases was measured. Unexpectedly, despite the lower activity of
DPM synthase in the dpm1,2,3 transformant, the activity of protein O-mannosyltransferases
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was in fact higher by 75% than that in the three other strains (Table 3). On the other hand, a
decrease of substrate (DPM) concentration might have been compensated by protein Omannosyltransferases activity or their expression rates. Protein O-mannosyltransferases were
only slightly more active (about 12%) in both dpm1,3 transformed strains than in the control
and this increase was not statistically significant according to t-Student test.
At the same time the activity of mannosyltransferases catalyzing elongation of the O-linked
sugar chain was lower by 70 and 26% in the dpm1,2,3 and dpm1,3 strains, respectively, than
in the control strain (Table 3). The 9.2% decrease of mannosyltransferases activity in the
dpm1*3(GAL) was not statistically significant.
Influence of heterologously expressed dpm genes on protein O- glycosylation in vivo
The decreased activities of DPM synthase and mannosyltransferases elongating O-linked
carbohydrate chain in the dpm1,2,3 transformant suggested that O-glycosylation of some
proteins could be altered in this strain. Chitinase, a model O-glycosylated protein, was chosen
to examine its O-glycosylation in vivo. We found that chitinase produced by the dpm1,2,3
transformant had a different pattern of electrophoretic mobility compared to those in the
dpm1,3- or dpm1*3 (GAL) transformed strains and the control (Figure 7), apparently due to the
presence of under-O-glycosylated forms of the enzyme.
Influence of heterologously expressed dpm genes on cell wall formation
Our previous experience with the impact of DPM1 expression in Trichoderma (PerlińskaLenart et al., 2006) on the cell wall composition prompted us to investigate whether the cell
wall composition of the dpm-transformed yeast strains would also be altered. For a simple
screen of the cell wall status we cultivated our transformants and the control strain in the
presence of Calcofluor white (40 g/ml). Cultivation with Calcofluor white, a fluorochrome
dye that binds to chitin in cell walls of fungi, should give proportional effect to the chitin
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concentration in the cell wall of examined strains. The experiment showed significant
differences between the examined strains in their sensitivity to this antifungal agent. The
control strain exhibited no sensitivity to Calcofluor white, while the dpm1,2,3-transformed
stain was unable to grow in the presence of the drug (Figure 8). Growth of both dpm1,3 and
dpm1*3 (GAL) expressing transformants was also not inhibited in the presence of Calcofluor
white. These results led us to propose that expression of three Trichoderma dpm genes altered
the cell wall composition of the transformant. To investigate these alterations, we measured
the content of the cell wall glucans and chitin. The analysis of carbohydrates showed that the
amount of β-1,3 glucan in the cell wall of the dpm1,2,3, dpm1,3 and dpm1*3 (GAL)
transformants was increased by 17, 12 and 20%, respectively, compared to the control, while
the amount of alkali-insoluble β-1,6 glucan was slightly decreased in the transformants. At the
same time, the content of chitin was 3.8 and 1.3-fold higher in the dpm1,2,3 and dpm1,3transformed strain, respectively, compared to the control and was unaffected in the dpm1*3
(GAL) transformant (Table 4).
DISCUSSION
Our earlier studies have shown that DPM synthase from Trichoderma belongs to the human
type of the enzymes, where the catalytic DPM1 subunit forms an active complex with two
other DPM proteins, DPM2 and DPM3 (Kruszewska et al., 2000; Colussi et al., 1997).
In this study we have cloned the dpm2 and dpm3 genes from Trichoderma and analyzed their
function by expression in an S.cerevisiae dpm1∆ mutant. Basing on the finding that
expression of Trichoderma DPMI protein alone in the S.cerevisiae dpm1∆ mutant could not
suppress the mutation (Kruszewska et al., 2000), we transformed a diploid dpm1∆ yeast
mutant with different combinations of Trichoderma dpm genes: dpm1 and 2, dpm1 and 3, and
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dpm1, 2 and 3. Both dpm1 and dpm2 genes were expressed under the GAL1 promoter with the
FLAG epitope inserted upstream of the genes, while the dpm3 gene was cloned under the
GAL10 promoter and tagged with the myc epitope. Yeast sporulation and tetrad dissection
gave only two viable spores containing yeast DPM1 gene showing that expression of the
tagged dpm genes did not lead to suppression of the dpm1∆ mutation.
The DPM3 protein from the human DPM synthase complex was shown to be associated by its
C- and N- terminus with the DPM1and DPM2 subunits, respectively(Maeda et al., 2000). It
seemed possible that the FLAG or myc epitopes fused with the DPMI or DPMIII proteins
could disturb formation of the DPM synthase complex. To overcome this possible obstacle we
transformed yeast dpm1∆ mutant with combinations of tagged and non- tagged genes
(Table1). Moreover, dpm1 gene was expressed under two different promoters (see Material
and Methods). Viable spores expressing Trichoderma genes were obtained only when
expression of dpm1 was driven by the constitutive GPD promoter and when formation of the
DPM synthase complex was not disturbed by the FLAG or myc epitop. We observed only one
exception when the dpm1∆ mutation was suppressed by the flagged dpm1* gene expressed
under the GAL1 promoter in combination with non-flagged dpm3 gene. Western analysis
showed that in the dpm1*3(GAL) transformant the amount of DPMI protein was elevated
compared to the dpm1,3 transformant where the dpm1 gene was expressed under the GPD
promoter. We thus concluded that successful suppression of the dpm1∆ mutation was
obtained by expression of non- flagged genes and when the dpm1 gene was expressed under
the GPD promoter. The high expression of the flagged dpm1* gene under the GAL1 promoter
(dpm1*3(GAL) strain), failed to increase the DPM synthase activity over the control level,
despite producing an elevated amount of DPMI protein.
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Furthermore, our study confirmed that the DPMII subunit was not essential for the
functioning of DPM synthase, in contrast to the DPMIII subunit. We did not investigate the
possibility of obtaining an active DPM synthase in the absence of DPMI, as the latter is
obviously the only catalytically active one.
In human cells the DPM1protein stabilized by DPM3 had an enzymatic activity, although in
the presence of DPM2 the activity was 10-fold higher (Maeda et al., 2000). Since the DPM2
protein is very hydrophobic the authors suggested that it could facilitate the use of dolichyl
phosphate or, being closely associated with DPM3, it could cause a steric change in DPM3
enhancing the enzymatic activity.
In our case, expression of Trichoderma DPMI and DPMIII proteins in yeast cells resulted in a
higher activity of DPM synthase than expression of all three subunits. This result suggests that
the heterologous regulatory signal coming from the DPMII protein was recognized by the
yeast cells and somehow limited the activity of the enzyme. Moreover, expression of the
DPMII protein in the control strain did not affect the activity of yeast Dpm1 protein (data not
shown). This experiment suggested that the DPMII regulatory activity was connected with a
direct physical interaction between the Trichoderma DPM synthase subunits.
Although the activity of DPM synthase was lower in the dpm1,2,3-transformed strain, the
activity of the next enzymes in the O-glycosylation pathway, protein O-mannosyltransferases,
was elevated. Paradoxically, Western blotting analysis revealed that the model O-glycosylated
protein chitinase was under- O-glycosylated in this strain. This could be a result of the
significantly decreased activity of mannosyltransferases elongating O-linked carbohydrate
chains in the dpm1,2,3 transformant.
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Changes in the O-glycosylation of chitinase were often observed as a consequence of limited
activity of the O-glycosylation pathway (Agaphonov et al., 2001; 2005; Janik et al., 2003;
Kim et al., 2006; Strahl-Bolsinger et al., 1999; Zakrzewska et al., 2003). Shortage of GDPmannose production (Agaphonov et al., 2001) and limited activity of protein Omannosyltransferases (Agaphonov et al., 2005; Strahl-Bolsinger et al., 1999) as well as
blocked activity of DPM synthase (Janik et al., 2003) resulted in the production of under-Oglycosylated chitinase or, for an S.cerevisiae dpm1-6 mutant, a totally un-O-glycosylated
form.
The lower activity of DPM synthase in the yeast transformants studied here affected also the
cell wall composition. On the other hand, a higher activity of DPM synthase in Trichoderma
also influenced the cell wall composition (Kruszewska et al., 1999; Perlińska-Lenart et al.,
2006). These findings combined show that proper activity of DPM synthase is crucial to the
cell wall status. Here we found that the decreased activity of this enzyme caused small
changes in the glucan content and a significant elevation of chitin level. Similarly the cell wall
of the yeast dpm1-6 mutant had the content of chitin increased by 64% and an unchanged
content of glucans (Orłowski et al., 2007). Variations in glycosylation often correlated with
activation of the cell wall integrity pathway manifested by increased synthesis of chitin
(Strahl-Bolsinger et al., 1999; Lommel et al., 2004; Orłowski et al., 2007).
In this study we cloned and characterized functions of the dpm2 and dpm3 genes from
Trichoderma. Characterization of the dpm1,2,3, dpm1,3 and dpm1*3 (GAL) transformed yeast
strains proved that we did clone subunits of Trichoderma DPM synthase. Furthermore, we
showed that the DPMII regulatory subunit decreased the activity of Trichoderma DPM
synthase expressed in yeast. Moreover, we showed that the decreased activity of DPM
13
synthase in the dpm1,2,3-transformed strain resulted in specific changes in the Oglycosylation of chitinase and influenced the formation of the cell wall components. We
believe that our observations might be useful in better understanding of the O-glycosylation
process in yeast and Trichoderma.
MATERIAL AND METHODS
Strains and growth conditions
T.reesei QM 9414 (ATCC 26921) was used for DNA isolation. Escherichia coli strain
DF’ was used for plasmid propagation (Yanish-Perron et al., 1985). T.reesei was
cultivated at 30°C on a rotary shaker (250 rpm) in 2 l shake flasks containing 1 l of minimal
medium (MM): 1 g MgSO4 x 7H2O, 6 g (NH4)2SO4, 10 g KH2PO4, 3 g sodium citrate x 2H2O
and trace elements (25 mg FeSO4 x 7H2O, 2.7 mg MnCl2 x 4H2O, 6.2 mg ZnSO4 x 7H2O, 14
mg CaCl2 x 2H2O) per liter and 1% (w/v) glucose as a carbon source.
S.cerevisiae BY4743 diploid strain carrying deletion of DPM1 (YPR183w) gene was used for
expression of dpm1, dpm2 and dpm3 genes from Trichoderma (NCBI Acc. No.: dpm1 AF102883; dpm2 - GQ200738; dpm3 - GQ200739).
The yeast strain was bought in Euroscarf (Y25598; genotype BY4743; Mat a/;
his31/his31; leu20/leu20; lys20/LYS2; MET15/met150; ura30/ura30;
YPR183w::kanMX4/YPR183w).
Yeast strains were grown in SC medium (Sherman 1991) with the necessary supplements and
for sporulation 1% potassium acetate pH 7 and 2% bacto-agar was used. Galactose was used
at 2% as a carbon sources.
Cloning of dpm2 and dpm3 genes from T. reesei
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Sequences of putative dpm2 and dpm3 genes were found in the genomic data base of
Trichoderma (http://genome.jgi-psf.org/Trire2/Trire2.home.html) using sequences of dpm2
and dpm3 genes from Botryotinia fuckeliana (NCBI Acc. No.: dpm2 - XM 001556879; dpm3
- XM 001561034), Gibberella zeae (XM 387389; XM 388453) and Neurospora crassa (XM
951284; XM955413).
DNA fragments encoding putative Trichoderma DPMII and DPMIII proteins were amplified
by PCR using T. reesei QM9414 genomic DNA or cDNA as templates and specific primers
(Table 1). The amplified DNA was cloned into the pGEM T-Easy vector (Promega) and
sequenced.
Expression plasmids
cDNA fragments of 240 bp and 276 bp coding for DPMII and DPMIII, respectively,
amplified as above using Trichoderma cDNA library as template were ligated together or
separately into the multiple cloning vector pESC-URA (Stratagene). Putative Trichoderma
dpm2 and dpm3-encoding cDNAs were amplified primers using dpm2U2 and dpm2F2 or
dpm3U2 and dpm3F2, respectively (Table 1). The genes were cloned under the GAL1/GAL10
(galactokinase/ UDP glucose 4-epimerase) divergent promoters in cloning sites 1 (dpm2) and
2 (dpm3) using restriction sites BglII/PacI for dpm2 gene and XhoI/KpnI for dpm3. This way
of cloning allowed us to fuse DPMII and DPMIII proteins with the FLAG and myc-tag,
respectively. To clone the genes without the tags under the same promoters, BamHI/HindIII
and EcoRI restriction sites were used for dpm2 and dpm3 genes, respectively. The respective
cDNA was amplified using dpm2U and dpm2F or dpm3U and dpm3F primers (Table 1).
For expression of T.reesei dpm1gene in S.cerevisiae, a cDNA fragment of about 810 bp
containing the dpm1 open reading frame was amplified by PCR using dpm1U2 and dpm1F2
primers and cloned into the pESC-LEU vector (Stratagene) under the GAL1promoter and the
15
CYC1 (cytochrome-c-oxidase) terminator in cloning site 2 using XhoI/NheI restriction sites
and fused with the myc-tag. The dpm1 gene amplified with the dpm1U3 and dpm1F2 primers
was also cloned under the GAL1 promoter in cloning site 2 using digestion with BamHI/NheI
to remove the myc-tag. Next, dpm1 c DNA was amplified using dpm1U and dpm1F primers
(Table 1) and cloned into the p415 GPD LEU plasmid under the GPD (glyceraldehyde-3phosphate dehydrogenase) promoter and the CYC1 (cytochrome-c-oxidase) terminator
(Munberg et al., 1995).
For yeast transformation, the one-step transformation method of Chen et al. (1992) was used.
The diploid yeast strain carrying deletion of one copy of the DPM1gene was transformed with
Trichoderma dpm1 and dpm2, dpm1, dpm2, and dpm3, or dpm1 and dpm3 genes and selected
on -Leu -Ura minimal medium. Transformants were then cultivated on the sporulation
medium and tetrads dissected. Spore clones carrying disruption of the yeast DPM1 gene were
selected on plates containing Geneticin (200 g/ml).
From the selected strains plasmids were isolated as described (Hoffman and Winston 1987)
and used to transform E. coli DH5F. The plasmids were digested with BglII/PacI,
XhoI/KpnI, XhoI/NheI, BamHI/HindIII, BamHI/NheI or EcoRI to analyze the inserts of
dpm2, dpm3 and dpm1genes, respectively. The restriction analysis was confirmed by
sequencing. Standard techniques (Sambrook et al., 1989) were used for isolating and
analyzing plasmid DNA.
A control haploid strain was obtained from non-transformed diploid BY4743dpm1∆ by
sporulation and taking a viable spore clone containing native DPM1 gene.
Molecular biology methods
16
Chromosomal DNA was isolated from T. reesei using the Promega Wizard Genomic DNA
Purification kit. Total RNA was isolated using the single-step method described by
Chomczynski and Sacchi (1987). Other molecular biology procedures were performed
according to standard protocols (Sambrook et al., 1989).
For Northern analysis, 10 g of total RNA was loaded onto agarose gel, blotted and
hybridized with the whole T.reesei dpm1, dpm2 or dpm3 genes. A control hybridization was
performed with a 0.6 kb fragment of the yeast ACT1(actin encoding) gene. The radioactive
probes were prepared using [32P] dATP and the Fermentas HexaLabel Plus DNA labeling
system according to the standard Fermentas protocol (Figure 1).
Biochemical methods
Protein assay
Protein concentrations were estimated according to Lowry et al. (1951).
Enzyme activity assays
Membrane preparation and DPM synthase activity
S.cerevisiae strains were cultured at 30oC in 1 l of SC-Leu -Ura medium to OD600=1; the cells
were then harvested by centrifugation and resuspended in 25 ml of 150 mM Tris buffer pH
7.4 containing 15 mM MgCl2 and 9 mM 2-mercaptoethanol. The suspension was
homogenized with 0.5 mm glass beads in a bead beater and the homogenate was centrifuged
at 4 000xg for 10 min to remove unbroken cells and cell debris. The supernatant was
centrifuged for 1 h at 50 000xg and DPM synthase activity was assayed in the pelleted
membrane fraction (about 100g protein) by 5 min incubation at 30oC with GDP[14C]-
17
mannose (sp.act. 288 Ci/mol, Amersham) and 5 ng of dolichyl phosphate (Dol-P), according
to Kruszewska et al. (1989, 1999).
Enzymatic activity of protein O-mannosyltransferases
S.cerevisiae strains were cultured and membrane fraction was prepared as described above.
Protein O-mannosyltransferase activity was assayed in the pelleted membrane fraction by 1 h
incubation at 30oC with GDP[14C]-mannose (sp.act. 288 Ci/mol, Amersham) and 5 ng of
dolichyl phosphate (Dol-P), according to Kruszewska et al. (1989). Total membrane proteins
(about 300g) were used as the sugar acceptor. The first mannosyl residue was transferred
from GDP[14C]Man via DPM (dolichyl phosphate mannose) in the presence of 10 mM MgCl2
for 1 h (Sharma et al., 1974). To activate the elongation of the O-linked sugar chain the
reaction mixture was supplemented with 10 mM MnCl2 and the reaction was carried out for 1
h. Radioactivity was measured in the protein fraction. The elongation of the sugar chain was
calculated as the difference between the amount of radioactive mannose bound to the protein
in the presence of MnCl2 (the first mannosyl residue and the elongation) and of the mannose
transferred in the presence of MgCl2 (only the first mannosyl residue transferred) (Sharma et
al., 1974) .
Immunodetection of DPMI protein
Total membrane fraction use for activity assay was also examined for the concentration of the
DPMI protein by immunostaining with monoclonal antibody against S.cerevisiae Dpm1p
(Molecular Probes, Eugene, OR, USA). Membrane proteins (200 g) were subjected to the
10% SDS PAGE then transferred to the Immobilon P membrane (Milipore) and Trichoderma
DPMI protein detected by immunological reaction with S.cerevisiae Dpm1 antibody.
18
Immunoreactive material was detected using an anti-mouse IgG secondary antibody
conjugated to alkaline phosphatase (Sigma).
Immunodetection of chitinase
Yeast cultures were grown to the logarithmic phase, washed with 50 mM Tris-HCl, pH 7.4,
50 mM MgCl2 and broken with glass beads in the same buffer. Cell walls were centrifuged
down at 3000xg and the pellet was resuspended in the same buffer. Cell wall proteins (about
100 g per lane) from the resulting pellet were separated by SDS-PAGE on a 6% gel.
Chitinase was detected by immunostaining with an antibody against the deglycosylated
protein EC 3.2.1.14 (a gift from Prof. Widmar Tanner, Lehrstuhl für Zellbiologie und
Pflanzenphysiologie, Universität Regensburg, Germany) ( Kuranda and Robins 1991;
Immervoll et al., 1995). Immunoreactive material was detected using an anti-rabbit IgG
secondary antibody conjugated to horseradish peroxidase (HRP) (DAKO).
Isolation of the cell wall
Cells were grown in medium containing 2% galactose (supplemented with appropriate amino
acids) at 28oC. Logarithmic cell cultures (OD600=1)
(100 ml) were harvested by centrifugation, washed with water and resuspended in 400 μl
water and broken with glass beads. The glass beads were removed and the cell free extracts
were centrifuged (1000x g for 15 min). The supernatants were discarded and the pellets,
containing the cell wall, were lyophilized.
Analysis of the cell wall components
For alkali-insoluble β-glucan determination, isolated cell walls were treated with 3% NaOH
three times for 1 h at 75oC to remove mannoproteins and alkali soluble glucan. The cell walls
were then neutralized by washing twice with 0.1 M Tris–HCl, pH 7.4, followed by 10 mM
19
Tris–HCl, pH 7.4. The alkali-insoluble fraction was digested overnight in 10 mM Tris–HCl,
pH 7.4, containing 5 mg/ml zymolyase 20T (ICN Biomedicals Inc.). Subsequently, insoluble
material was removed by centrifugation at 18 000 × g for 15 min). After dialysis of the
zymolyase-solubilized material, β1,6-glucan was collected and quantified by the method of
Miller et al. (1960). Total alkali-insoluble glucan was determined as reducing sugars content
before dialysis by the method cited above. The alkali-insoluble β1,3-glucan level was
calculated by substraction of the β1,6-glucan content from that of total glucan.
For chitin measurements alkaline hydrolysis of cell walls was performed in 6% KOH for 90
min at 80oC in order to release cell wall proteins. After neutralization with acetic acid, the cell
walls were washed with phosphate-buffered saline and chitinase buffer, pH 6.0, containing 18
mM citric acid and 60 mM dibasic sodium phosphate. Subsequently, the cell walls were
treated with chitinase C (InterSpex Products) for 3 h at 37o C. The level of chitin was
measured with Ehrlich’s reagent as described (Reissig et al., 1955).
20
ACKNOWLEDGMENTS
We wish to thank Prof. Markku Saloheimo from VTT, Finland for the cDNA bank from
Trichoderma and Prof. Robert Mach and Dr. Kurt Bunner from Vienna University of
Technology, Austria for the analysis of genomic data bases and designing primers. We also
thank Dr. Jacek Lenart from The Museum and Institute of Zoology, PAS, Warsaw for
preparation of phylogenetic trees.
This work was partially supported by the State Committee for Scientific Research (KBN),
Warsaw, Poland, grant No. 1307/P01/2006/31 to J.S. Kruszewska.
21
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28
FIGURE LEGENDS
Figure 1
A. Transcription of dpm genes in the dpm1,2,3 (1,2,3), dpm1,3 (1,3) and dpm1*3(GAL)
(1*3(GAL)) transformed strains compared to the control (C) after cultivation in galactose based medium. Gene expression was compared with the expression of actin gene (ACT1)
analyzed under the same conditions.
B. The columns show the signal intensity in arbitrary units after normalization for the actin
band.
Figure 2
Deduced amino acid sequences of Trichoderma DPMII (A) and DPMIII (B) proteins and their
alignment with sequences of DPM2 and DPM3 proteins of Gibberella zeae , Botryotinia
fuckeliana, Neurospora crassa, S.pombe, and human. The potential membrane spanning
regions of Trichoderma proteins are indicated by black line above the sequence. Hydropathy
analysis was done using the Tmpred program (Hoffmann et al., 1993).
Figure 3
Phylogenetic tree of putative DPMII proteins. Phylogenetic analysis was performed using the
Geneious Pro 4.5.5 program and the UPGMA tree building method. Numbers indicate genetic
distances calculated based on Jukes-Cantor method.
Figure 4
Phylogenetic tree of putative DPMIII proteins. For details see Figure 3.
Figure 5
Growth curves of dpm1,2,3, dpm1,3 and dpm1*3 (GAL) transformants and the control strain
cultivated in minimal media containing 2% galactose as a carbon source. Presented data are
the mean  SE of three independent experiments.
29
Figure 6
Immunodetection of DPMI protein from dpm1,2,3, dpm1,3 and dpm1*3 (GAL) transformants
with S.cerevisiae anti-Dpm1 protein antibody. Membrane fraction from S.cerevisiae
containing yeast Dpm1 protein was used as a positive control (st).
Figure 7
Immunodetection of chitinase synthesized by dpm1,2,3, (1,2,3) dpm1,3 (1,3) and dpm1*3
(GAL) (1*3(GAL)) transformants and the control strain (C) containing yeast DPM1 gene. st –
molecular standards.
Figure 8
Calcofluor white sensitivity of yeast strains. Serial dilutions of the dpm1,2,3 dpm1,3 and
dpm1*3 (GAL) transformants and the control strain were plated on SC medium containing 40
μg/ml Calcofluor white or without Calcofluor white and cultivated at 28 oC.
30