Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
Correspondence
Kensuke Furukawa
kfurukaw{at}agr.kyushu-u.ac.jp
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ABSTRACT |
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The GenBank accession number for the sequence reported in this paper is AF225551.
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INTRODUCTION |
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Aspergillus species belong to the filamentous fungi; they include many industrially useable strains such as Aspergillus oryzae, Aspergillus awamori and Aspergillus niger. These fungi produce large amounts of extracellular hydrolases and organic acids, and thus have been used for the production of fermented foods (van den Hondel et al., 1992). Besides these useful Aspergillus strains, opportunistic pathogens such as Aspergillus fumigatus, aflatoxin B1-productive Aspergillus flavus and Aspergillus parasiticus are also known. Therefore, Aspergillus species have been widely investigated in the industrial and medical fields. However, little is known about O-glycosylation in Aspergillus species, although it has been suggested that it is closely associated with secretion (Zakrzewska et al., 2003
), stability (Harty et al., 2001
; Goto et al., 1999
) and localization of proteins (Bourdineaud et al., 1998
). Structural analyses of O-linked oligosaccharides of Aspergillus glycoproteins have revealed that O-linked oligosaccharides are predominantly composed of mannose moieties ranging in size from one to three mannose units with linkages of
-1,2,
-1,3 and
-1,6, including not only a linear form but also a branched structure (Pazur et al., 1980
; Gunnarsson et al., 1984
). Moreover, the O-linked oligosaccharides contain glucose and galactose moieties, in addition to mannose moieties, in the same protein (Gunnarsson et al., 1984
; Wallis et al., 1999
). Thus, O-linked oligosaccharides in proteins from Aspergillus species are different from those of S. cerevisiae. Recently, Shaw & Momany reported that the swoA mutation, which causes abnormal hyphae development, is identical to the pmtA mutation in Aspergillus nidulans (Momany et al., 1999
; Shaw & Momany, 2002
). In the present paper, we demonstrate the principal functions of the pmtA gene, and also discuss its role, by providing instances of abnormal cell phenotypes in a pmtA disruptant.
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METHODS |
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Isolation of the pmtA gene.
Two types of partial genomic DNA library of A. nidulans FGSC26 were constructed. Genomic DNA was digested with SalI and EcoRI, and 3·8 kb DNA fragments were obtained. Genomic DNA was also digested with SalI and ClaI, and 3·7 kb DNA fragments were obtained. The DNA fragments thus obtained were inserted into the corresponding sites of pBluescript-II KS+. E. coli XL-1 Blue transformants carrying two types of genomic DNA library were analysed by colony hybridization. A partial region of the pmtA gene was amplified by PCR with primers pmtA-F (5'-CAGGCCGTAACCTTCATTCCCA-3') and pmtA-R (5'-CCGGCGCGTAGATAACAACCC-3') and was used as a hybridization probe. Two positive clones carrying pBS-C-pmtA and pBS-N-pmtA were isolated from the two types of genomic library. The 1·86 kb SalISphI fragment from pBS-N-pmtA and the 2·69 kb SphIBamHI fragment from pBS-C-pmtA were co-inserted into the SalI and BamHI sites of pBluescript-II KS+, to yield pBS-pmtA, which carries a 4·55 kb SalIBamHI fragment containing the entire pmtA gene. In order to isolate the cDNA of pmtA, RT-PCR was performed with a High Fidelity RNA PCR kit (Takara), using the primers pmtA-RT-F (5'-TTGGATCCGAAATGGCTGAAATTGGCTTTG-3') and pmtA-RT-R (5'-GTTCCTCGAGGTTAGCGATTCGCCAAC-3').
Analysis of pmtA transcription.
Total RNA was isolated by Trizol reagent (Gibco-BRL). An 18 µg sample of total RNA was separated by electrophoresis in a 1·0 % agarose gel with 15 % (v/v) formaldehyde and transferred to a Hybond-N membrane (Amersham Biosciences). Hybridization with DIG-labelled probes (Roche) and subsequent detection with CDP-star (Roche) were done according to the supplier's manual. Primer-extension experiments using the mRNA of strain FGSC26 were done with the Primer Extension System-AMV Reverse Transcriptase (Promega) and the primer PE-pmtA (5'-IRD800-GGTCGATGCAAAGCCAATTTCAGCCATG-3'). A sequencing reaction was performed using a Thermo Sequenase Cycle Sequencing Kit (USB) with a 1·5 µM ddNTP (Amersham Biosciences) and 150 µM dNTP (Promega) mixture. Analysis of reaction products was done on a model LIC4200L-2G DNA sequencer (LI-COR).
Construction of the pmtA disruptant.
A plasmid, pBS-pmtA : : argB, for disruption of the pmtA gene, was constructed as follows. The 1·7 kb EcoRI fragment of pDC1 (Aramayo et al., 1989
), containing the argB gene of A. nidulans, was inserted into the middle of the pmtA gene at the EcoRI site of pBS-pmtA. The 6·0 kb DNA fragment containing
pmtA : : argB that had been prepared by digestion of pBS-
pmtA : : argB with BssHII was used for transformation of A. nidulans FGSC89. The disruption of the pmtA gene in the argB+ transformants was confirmed by Southern blot analysis. The 0·9 kb terminator region of pmtA was amplified by PCR with the primers pmtA-TF (5'-AAAAGGGCCCGAACCGTGATAGAGCG-3') and pmtA-TR (5'-AAAAGGGCCCGGATCCCGCATTGTCC-3') and used as a probe.
Protein O-mannosylation activity in vitro.
A. nidulans was grown on MM medium for 24 h at 30 °C. Dol-P-[14C]Man was synthesized according to the methods of Sharma et al. (1974). The in vitro peptide assay for O-mannosylation activity was performed according to Gentzsch & Tanner (1996)
. Dol-P-[14C]Man as a sugar donor and Ac-YATAV-NH2 as an acceptor peptide were used as substrates. The reaction mixture contained 5000 c.p.m. Dol-P-[14C]Man, 1·5 mM acceptor peptide, 7 mM Tris/HCl, pH 7·5, 7 mM MgCl2, 0·14 % (w/v) Triton X-100 and 0·20·3 mg ml1 microsomal membranes of A. nidulans. The mixtures were incubated at 30 °C for 20 min. The amount of [14C]mannosylated peptide was measured by a liquid scintillation counter. The assay was done three times independently.
Expression of pmtA and glaA.
The plasmids for expression of pmtA and the glucoamylase-encoding glaA were constructed as follows. The 4·55 kb SalIBamHI fragment carrying the pmtA from pBS-pmtA was inserted into the corresponding sites of pUC19, to yield pUC-pmtA. pUC-pmtA was digested by PstI and SmaI, and the resultant pmtA fragment was inserted into the PstI and SmaI sites of pPTR-I, carrying the ptrA gene as a selection marker (Takara), to yield pPTR-pmtA. The glaA gene of A. awamori, from pBR-glaA, (Goto et al., 1997) was inserted into the SmaI site of pPTR-I, to generate pPTR-glaA. The pPTR-pmtA and pPTR-glaA were linearized by digestion with PstI and HindIII, respectively. Transformants showing pyrithiamine resistance were selected on MM medium supplemented with 0·1 µg pyrithiamine ml1 and 0·8 M NaCl. Transformants carrying the glaA gene were cultured in 100 ml MM medium with 1 % (w/v) maltose, to induce glaA expression, and with 20 mM tunicamycin as an inhibitor for N-glycosylation of protein, for 24 h at 30 °C.
Colony growth rate.
Colony growth rates were measured as described by Kellner & Adams (2002). Briefly, conidia from each of the strains were point-inoculated into the centre of agar medium plates and incubated at 37 °C. Colony diameters were measured at 29, 49, 66 and 91 h. The growth rates were observed for each colony in mm h1 for each of the above three intervals, and the values were averaged across the entire time interval. The growth rates were expressed as a percentage of the growth rate of the wild-type (FGSC26) on YG medium, which was 0·59 (±0·085) mm h1 (n=15). Measurements of independent growth rates for all strains were done 15 times.
Analysis of efficiency of conidiation.
Efficiency of conidiation was analysed as described by Motoyama et al. (1997). Briefly, approximately 105 conidia were spread onto an 84 mm MM medium plate. After 5 days of incubation at 30 °C, the conidia were suspended in 5 ml 0·01 % (w/v) Tween 20 solution and counted using a haemocytometer.
Microscopy.
The germlings of A. nidulans were observed according to the protocol described by Harris et al. (1994). Conidia were inoculated into YG liquid medium, and the culture was poured into a Petri dish containing a glass coverslip. After incubation at 30 °C for 614 h, the germlings adhering to the coverslip were fixed in 3·7 % (v/v) formaldehyde, 50 mM phosphate buffer (pH 7·0) and 0·2 % (w/v) Triton X-100 for 30 min. Coverslips were then washed with water and incubated with 10 ng ml1 of fluorescent brightener 28 (Calcofluor white; Sigma) for 5 min. The coverslips were washed again and mounted on a slide glass. Germlings were observed using the Eclipse E600 (Nikon).
Preparation of microsomal membrane and cell-wall fractions.
A. nidulans was grown on MM or YG medium for 24 h, harvested, washed with ice-cold TM buffer (50 mM Tris/HCl, pH 7·5, 0·3 mM MgCl2) three times and broken twice in a French press (Ohtake) in the same buffer. The lysed cells were centrifuged at 3000 g for 20 min. The resultant pellet was washed ten times with 250 mM phosphate buffer (pH 7·0), washed with distilled water five times, lyophilized, and designated a cell-wall fraction. The supernatant was centrifuged at 100 000 g for 90 min, suspended in TM buffer, and designated a membrane fraction.
Analysis of the contents of glucan and chitin.
Analysis of the glucan and chitin contents was done by the methods described by Borgia & Dodge (1992), with a slight modification. Cells were cultured in YG medium for 24 h at 30 °C. For the quantification of total glucan, 10 mg of cell walls was suspended in 1·0 ml 98 % formic acid, heated at 100 °C for 20 min and centrifuged. The supernatant was dried in a vacuum desiccator, and the amount of neutral carbohydrate was estimated by the phenol-sulfuric acid method (Dubois et al., 1956
). For the quantification of alkali-soluble and alkali-insoluble fractions, 10 mg of cell walls was suspended in 1·0 ml distilled water, heated at 100 °C for 30 min and centrifuged. The pellet was suspended in 1·0 ml 1·0 M KOH solution and heated at 70 °C for 30 min prior to centrifugation. The supernatant was dried and used to estimate the amount of alkali-soluble glucan, as above. The alkali-insoluble pellet was suspended in 1·0 ml 98 % formic acid and heated at 100 °C for 20 min, dried and used to estimate the amount of alkali-insoluble glucan. For the quantification of total GlcNAc, 10 mg of cell walls was digested with 2 mg Yatalase ml1 (Takara) in 250 mM phosphate buffer (pH 7·0) for 16 h. After digestion, the suspension was centrifuged, dried and used to estimate the amount of total GlcNAc by the MorganElson method (Reissig et al., 1955
). The analyses were done three times independently.
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RESULTS |
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Transcription of the pmtA gene
Northern blot analysis of pmtA was carried out at various growth stages, using the pmtA cDNA as a probe. The amounts of the pmtA transcripts under incubation in MM medium were almost comparable with the -actin gene from 16 h to 48 h (Fig. 1
a). This result indicates that pmtA is transcribed throughout hyphal growth. The transcriptional start points of the pmtA gene were determined by primer-extension analysis, and indicated that pmtA has at least five transcriptional initiation sites, at 27, 46, 75, 127 and 226 bp upstream from the translational start site (Fig. 1b
). Analysis of the 5'-noncoding region revealed the presence of putative promoter-proximal elements of a TATA-like sequence involved in the expression of the pmtA gene. The promoter contains a putative binding sequence of the pH signalling transcription factor PacC (Espeso et al., 1997
) 373 bp upstream from the translation start codon (Fig. 1b
).
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The pmtA disruptant formed a colony approximately 90 % of the diameter of that of the wild-type strain, when cultured on MM medium at 30 °C for 3 days (Fig. 4). However, in the case of growth on YG medium at 37 °C, the pmtA disruptant P3 showed remarkable inferiority in its hyphal extension ability: its growth rate was reduced to 41·8 % compared to the wild-type (Table 1
). We attempted to obtain transformants whose pmtA mutation was complemented, where one to three copies of the pmtA gene were integrated via introduced plasmid pPTR-pmtA. The colony growth rate of the transformants (strain P3 carrying wild-type pmtA) was fully restored (Table 1
), indicating that the abnormal hyphal extension is due to the pmtA mutation. The growth rate was fully restored on YG medium plates in the presence of 0·6 M KCl. The growth-rate recovery was also observed at the same level when 0·8 M NaCl and 1·2 M sorbitol were used, instead of 0·6 M KCl, as an osmotic stabilizer (data not shown). In addition, we often observed lysis of the hyphal tip in the MM liquid medium without the stabilizer. However, the hyphal lysis was repressed when the stabilizer was incorporated. Morphology of the germlings in the early growth stage was observed by microscopy. On YG and MM media, swollen hyphal formation (balloon formation) appeared at the apical or umbilical regions of the hyphae (Fig. 3
a). This morphology was observed in moribund regions, in particular.
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pmtA disruption causes hypersensitivity to antifungal reagents
Congo red and Calcofluor white are known to be adsorbed on cell walls composed of polysaccharides, and therefore exhibit antifungal effects. In the presence of Congo red and Calcofluor white, the hyphal growth of the pmtA disruptant on MM medium was markedly inhibited compared with the wild-type strain (Fig. 4).
Cell-wall chitin and -glucan content of wild-type and pmtA-disruptant strains
The alkali-soluble fraction contains -glucan and soluble
-1,3-/1,6-glucan. The alkali-insoluble fraction contains
-1,3-/1,6-glucan covalently linked to chitin (Borgia & Dodge, 1992
; Fontaine et al., 2002
; Lee et al., 2002
). The pmtA disruptant showed a 20 % decrease in the alkali-insoluble fraction. The alkali-soluble fraction and the GlcNAc content of the cell walls of the pmtA disruptant were increased by 44 % and 33 %, respectively, compared with the wild-type strain. The total sugar content in the cell walls of the pmtA disruptant was comparable to that of the wild-type strain (Table 2
). The alkali-insoluble fraction is believed to be responsible for fungal cell-wall rigidity (Fontaine et al., 2002
). The decrease in the alkali-insoluble fraction indicates that the cell wall was weakened.
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DISCUSSION |
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The pmtA disruption gave a phenotype of A. nidulans identical to the temperature-sensitive swoA mutant reported by Momany et al. (1999) and Shaw & Momany (2002)
. Inhibition of hyphal extension, multinucleation of the cell, and enhanced susceptibility to heat and Calcofluor white were observed. In addition to the properties of the swoA mutant, we observed hypersensitivity to Congo red (Fig. 4
) and a decrease in conidia formation. Disruption of the pmtA gene revealed that it encodes the functional protein O-D-mannosyltransferase. The pmtA-disrupted strain exhibited greatly reduced protein O-D-mannosyltransferase activity (6 %), compared to the wild-type. The remnant activity would be derived from Pmt isozymes. Thus, A. nidulans is assumed to possess several pmt genes, as in other organisms (Gentzsch & Tanner, 1996
; Timpel et al., 2000
). In fact, we cloned two genes homologous to pmtA and confirmed their transcripts in A. nidulans (unpublished data).
In order to confirm in vivo mannosylation activity toward a secretory protein, GAI of A. awamori was heterologously expressed in A. nidulans. The Ser/Thr-rich region of GAI, which could be the target for O-glycosylation, contains 27 hydroxyamino acids among 38 amino-acid residues (Hayashida et al., 1989). Unexpectedly, only a 2 kDa difference in the molecular mass of GAI was observed between the wild-type strain and the pmtA disruptant, suggesting that approximately 11 mannose moieties are absent in the pmtA disruptant. This result supports the fact that PmtA protein catalyses O-mannosylation with an inherent substrate specificity for certain proteins. Gentzsch & Tanner reported that, in S. cerevisiae, Pmt proteins have distinct substrate specificities: for proteins that contain Ser or Thr (Gentzsch & Tanner, 1997
). Therefore, the slight difference in glycosylation in GAI would be due to a lower preference of A. nidulans PmtA for the foreign GAI polypeptide as the substrate.
It is well documented that some triple mutants of PMT14 show a lethal phenotype in S. cerevisiae. On the other hand, all single and double mutants of PMT were viable (Gentzsch & Tanner, 1996). The pmtA disruptant of A. nidulans is also viable. However, a defect in the PmtA function resulted in repression of normal hyphal growth and normal conidial development, particularly under conditions of low osmotic potential. pmtA is transcribed at abundant levels through the early to late growth-stages in the wild-type strain (Fig. 1a
). Because hyphal extension is in concert with cell-wall formation in filamentous fungi, the pmtA transcribed abundantly in conventional growth would be required for cell-wall formation. The pmtA disruptant of A. nidulans showed higher sensitivity to antifungal agents than the wild-type strain (Fig. 4
). A similar response was observed in C. albicans pmt1 and pmt1pmt6 mutants (Timpel et al., 2000
). Therefore, it is not surprising that the same effects are observed in the pmtA disruptant of A. nidulans. The addition of osmotic stabilizers to the growth medium resulted in recovery of the defect in hyphal growth of the pmtA disruptant. These results indicated that the structure and mechanical rigidity of the cell walls are drastically altered by the pmtA disruption. Unlike the pmt mutants of C. albicans, the pmtA disruption also caused swollen hyphae locally in the mycelia, although, overall, mycelial development did take place (Fig. 3a
). In Aspergillus species, the formation of swollen structures in the hyphae was reported in mutants of chitin synthase or NAD-dependent glycerol-3-phosphate dehydrogenase (Borgia et al., 1996
; Horiuchi et al., 1999
; Fillinger et al., 2001
). The cell-wall rigidity of those mutants was defective because the biosynthesis of chitin or glycerol was significantly reduced. These morphological rigidity defects were restored by the addition of an osmotic stabilizer.
Cell-wall components are functionally classified into two groups: the skeletal polysaccharides and the polysaccharides of the wall matrix (Farkas, 1985). The skeletal polysaccharides include
-1,3-glucan interconnected covalently with
-1,6-glucan and chitin, which are highly crystalline and provide mechanical rigidity to the wall. These polysaccharides were recovered as an alkali-insoluble fraction (Borgia & Dodge, 1992
; Fontaine et al., 2002
; Lee et al., 2002
). The alkali-soluble fraction contains
-glucan and soluble
-1,3/1,6-glucan, which fills the space between the skeletal polysaccharides and serves as a cementing substance (Borgia & Dodge, 1992
; Farkas, 1985
). The pmtA disruptant exhibited a significant alteration in the carbohydrate composition of the cell-wall fractions (Table 2
). The cell walls in the pmtA disruptant changed to a defective structure, due to the reduction of the skeletal polysaccharides of the
-glucans and the increase in chitin content. The polysaccharides of the wall matrix increased in the pmtA disruptant. Therefore, the pmtA disruptant gives rise to critical damage to the skeletal structure and wall matrix of the cell wall. Accordingly, mycelial extension can be repressed in the pmtA disruptant.
In S. cerevisiae, it is known that the KRE1 and KRE9 genes encode O-glycosylated proteins involved in the synthesis of -1,6-glucan. KRE1 and KRE9 mutants exhibit fragile cell-wall phenotypes, due to the reduction of the amount of
-1,6-glucan in the cell wall (Brown et al., 1993
; Brown & Bussey, 1993
). Therefore, the underglycosylation caused by the pmtA disruption may affect the synthesis of
-1,6-glucan. It has also been reported that disruption of the
-glucan synthase gene results in an increase in cell-wall chitin content in S. cerevisiae. (Garcia-Rodriguez et al., 2000
). The increase in chitin content in the pmtA disruptant of A. nidulans may be due to a similar mechanism, through a kind of salvage mechanism for covering cell-wall weakening. Philip & Levin (2001)
reported that Wsc1 and Mid2, which are cell-surface sensors responsible for cell-wall integrity through the MAP kinase pathway, are modified by ScPmt2p. Thus, one cannot exclude the possibility, in A. nidulans, that the impairment in O-mannosylation triggers the cascade of events that leads first to damage to cell-wall integrity and then to the cell response involving its repair through cell-wall compensatory mechanisms. Alternatively, PmtA could be directly involved in the mannosylation of proteins covalently attached to the polysaccharides of the wall matrix, whereby the pmtA disruption may cause alteration of the cell-wall structure.
A number of proteins secreted into the culture and localized in the cell wall are O-glycosylated (Mrsa & Tanner, 1999; Woo et al., 2003
). However, in Aspergillus, most of the O-glycosylated proteins localized in the cell walls or plasma membrane have not yet been identified. We are currently involved in an investigation using the pmtA disruptant to identify the target proteins that PmtA modulates in structure and function.
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REFERENCES |
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Aramayo, R., Adams, T. H. & Timberlake, W. E. (1989). A large cluster of highly expressed genes is dispensable for growth and development in Aspergillus nidulans. Genetics 122, 6571.
Barratt, R. W., Johnson, G. B. & Ogata, W. N. (1965). Wild-type and mutant stocks of Aspergillus nidulans. Genetics 52, 233246.
Borgia, P. T. & Dodge, C. L. (1992). Characterization of Aspergillus nidulans mutants deficient in cell wall chitin or glucan. J Bacteriol 174, 377383.[Abstract]
Borgia, P. T., Iartchouk, N., Riggle, P. J., Winter, K. R., Koltin, Y. & Bulawa, C. E. (1996). The chsB gene of Aspergillus nidulans is necessary for normal hyphal growth and development. Fungal Genet Biol 20, 193203.[CrossRef][Medline]
Bourdineaud, J. P., van der Vaart, J. M., Donzeau, M., de Sampaio, G., Verrips, C. T. & Lauquin, G. J. (1998). Pmt1 mannosyl transferase is involved in cell wall incorporation of several proteins in Saccharomyces cerevisiae. Mol Microbiol 27, 8598.[CrossRef][Medline]
Brown, J. L. & Bussey, H. (1993). The yeast KRE9 gene encodes an O glycoprotein involved in cell surface beta-glucan assembly. Mol Cell Biol 13, 63466356.[Abstract]
Brown, J. L., Kossaczka, Z., Jiang, B. & Bussey, H. (1993). A mutational analysis of killer toxin resistance in Saccharomyces cerevisiae identifies new genes involved in cell wall (1-6)-beta-glucan synthesis. Genetics 133, 837849.
Burda, P. & Aebi, M. (1999). The dolichol pathway of N-linked glycosylation. Biochim Biophys Acta 1426, 239257.[Medline]
Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Anal Chem 28, 350356.
Espeso, E. A., Tilburn, J., Sanchez-Pulido, L., Brown, C. V., Valencia, A., Jr, Arst, H. N. & Penalva, M. A. (1997). Specific DNA recognition by the Aspergillus nidulans three zinc finger transcription factor PacC. J Mol Biol 274, 466480.[CrossRef][Medline]
Farkas, V. (1985). The fungal cell wall. In Fungal Protoplasts, pp. 329. Edited by J. F. Peberdy & L. Ferenczy. New York: Marcel Dekker.
Fillinger, S., Ruijter, G., Tamas, M. K., Visser, J., Thevelein, J. M. & d'Enfert, C. (2001). Molecular and physiological characterization of the NAD-dependent glycerol 3-phosphate dehydrogenase in the filamentous fungus Aspergillus nidulans. Mol Microbiol 39, 145157.[CrossRef][Medline]
Fontaine, T., Simenel, C., Dubreucq, G., Adam, O., Delepierre, M., Lemoine, J., Vorgias, C. E., Diaquin, M. & Latge, J. P. (2002). Molecular organization of the alkali-insoluble fraction of Aspergillus fumigatus cell wall. J Biol Chem 275, 2759427607.
Garcia-Rodriguez, L. J., Trilla, J. A., Castro, C., Valdivieso, M. H., Duran, A. & Roncero, C. (2000). Characterization of the chitin biosynthesis process as a compensatory mechanism in the fks1 mutant of Saccharomyces cerevisiae. FEBS Lett 478, 8488.[CrossRef][Medline]
Gemmill, T. R. & Trimble, R. B. (1999). Overview of N- and O-linked oligosaccharide structures found in various yeast species. Biochim Biophys Acta 1426, 227237.[Medline]
Gentzsch, M. & Tanner, W. (1996). The PMT gene family: protein O-glycosylation in Saccharomyces cerevisiae is vital. EMBO J 15, 57525759.[Abstract]
Gentzsch, M. & Tanner, W. (1997). Protein-O-glycosylation in yeast: protein-specific mannosyltransferases. Glycobiology 7, 481486.[Abstract]
Goto, M., Ekino, K. & Furukawa, K. (1997). Expression and functional analysis of a hyperglycosylated glucoamylase in a parental host, Aspergillus awamori var. kawachi. Appl Environ Microbiol 63, 29402943.[Abstract]
Goto, M., Tsukamoto, M., Kwon, I., Ekino, K. & Furukawa, K. (1999). Functional analysis of O-linked oligosaccharides in threonine/serine-rich region of Aspergillus glucoamylase by expression in mannosyltransferase-disruptants of yeast. Eur J Biochem 260, 596602.
Gunnarsson, A., Svensson, B., Nilsson, B. & Svensson, S. (1984). Structural studies on the O-glycosidically linked carbohydrate chains of glucoamylase G1 from Aspergillus niger. Eur J Biochem 145, 463467.[Abstract]
Harris, S. D., Morrell, J. L. & Hamer, J. E. (1994). Identification and characterization of Aspergillus nidulans mutants defective in cytokinesis. Genetics 136, 517532.
Harty, C., Strahl, S. & Romisch, K. (2001). O-Mannosylation protects mutant alpha-factor precursor from endoplasmic reticulum-associated degradation. Mol Biol Cell 12, 10931101.
Haselbeck, A. & Tanner, W. (1982). Dolichyl phosphate-mediated mannosyl transfer through liposomal membranes. Proc Natl Acad Sci U S A 79, 15201524.[Abstract]
Hausler, A., Ballou, L., Ballou, C. E. & Robbins, P. W. (1992). Yeast glycoprotein biosynthesis: MNT1 encodes an alpha-1,2-mannosyltransferase involved in O-glycosylation. Proc Natl Acad Sci U S A 89, 68466850.[Abstract]
Hayashida, S., Nakahara, K., Kuroda, K., Miyata, T. & Iwanaga, S. (1989). Structure of the raw-starch-affinity site on the Aspergillus awamori var. kawachi glucoamylase I molecule. Agric Biol Chem 53, 135141.
Horiuchi, H., Fujiwara, M., Yamashita, S., Ohta, A. & Takagi, M. (1999). Proliferation of intrahyphal hyphae caused by disruption of csmA, which encodes a class V chitin synthase with a myosin motor-like domain in Aspergillus nidulans. J Bacteriol 181, 37213729.
Kellner, E. M. & Adams, T. H. (2002). Mutations in sfdA and sfdB suppress multiple developmental mutations in Aspergillus nidulans. Genetics 160, 159168.
Lee, H. H., Park, J. S., Chae, S. K., Maeng, P. J. & Park, H. M. (2002). Aspergillus nidulans sod(VI)C1 mutation causes defects in cell wall biogenesis and protein secretion. FEMS Microbiol Lett 208, 253257.[CrossRef][Medline]
Lussier, M., Sdicu, A. M., Bussereau, F., Jacquet, M. & Bussey, H. (1997). The Ktr1p, Ktr3p, and Kre2p/Mnt1p mannosyltransferases participate in the elaboration of yeast O- and N-linked carbohydrate chains. J Biol Chem 13, 1552715531.[CrossRef]
Lussier, M., Sdicu, A. M. & Bussey, H. (1999). The KTR and MNN1 mannosyltransferase families of Saccharomyces cerevisiae. Biochim Biophys Acta 1426, 323334.[Medline]
Martin-Blanco, E. & Garcia-Bellido, A. (1996). Mutations in the rotated abdomen locus affect muscle development and reveal an intrinsic asymmetry in Drosophila. Proc Natl Acad Sci U S A 93, 60486052.
Momany, M., Westfall, P. J. & Abramowsky, G. (1999). Aspergillus nidulans swo mutants show defects in polarity establishment, polarity maintenance and hyphal morphogenesis. Genetics 151, 557567.
Motoyama, T., Fujiwara, M., Kojima, N., Horiuchi, H., Ohta, A. & Takagi, M. (1997). The Aspergillus nidulans genes chsA and chsD encode chitin synthases which have redundant functions in conidia formation. Mol Gen Genet 253, 520528.[CrossRef][Medline]
Mrsa, V. & Tanner, W. (1999). Role of NaOH-extractable cell wall proteins Ccw5p, Ccw6p, Ccw7p and Ccw8p (members of the Pir protein family) in stability of the Saccharomyces cerevisiae cell wall. Yeast 15, 813820.[CrossRef][Medline]
Pazur, J. H., Tominaga, Y., Forsberg, L. S. & Simpson, D. L. (1980). Glycoenzymes: an unusual type of glycoprotein structure for a glucoamylase. Carbohydr Res 84, 103114.[CrossRef][Medline]
Philip, B. & Levin, D. E. (2001). Wsc1 and Mid2 are cell surface sensors for cell wall integrity signaling that act through Rom2, a guanine nucleotide exchange factor for Rho1. Mol Cell Biol 21, 271280.
Reissig, J. L., Strominger, J. L. & Leloir, L. F. (1955). A modified colorimetric method for the estimation of N-acetylamino sugars. J Biol Chem 217, 959966.
Romero, P. A., Lussier, M., Veronneau, S., Sdicu, A. M., Herscovics, A. & Bussey, H. (1999). Mnt2p and Mnt3p of Saccharomyces cerevisiae are members of the Mnn1p family of alpha-1,3-mannosyltransferases responsible for adding the terminal mannose residues of O-linked oligosaccharides. Glycobiology 9, 10451051.
Sauer, J., Sigurskjold, B. W., Christensen, U., Frandsen, T. P., Mirgorodskaya, E., Harrison, M., Roepstorff, P. & Svensson, B. (2000). Glucoamylase: structure/function relationships, and protein engineering. Biochim Biophys Acta 1543, 275293.[Medline]
Sharma, C. B., Babczinski, P., Lehle, L. & Tanner, W. (1974). The role of dolicholmonophosphate in glycoprotein biosynthesis in Saccharomyces cerevisiae. Eur J Biochem 46, 3541.[Medline]
Shaw, B. D. & Momany, M. (2002). Aspergillus nidulans polarity mutant swoA is complemented by protein O-mannosyltransferase pmtA. Fungal Genet Biol 37, 263270.[CrossRef][Medline]
Strahl-Bolsinger, S., Immervoll, T., Deutzmann, R. & Tanner, W. (1993). PMT1, the gene for a key enzyme of protein O-glycosylation in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 90, 81648168.
Strahl-Bolsinger, S., Gentzsch, M. & Tanner, W. (1999). Protein O-mannosylation. Biochim Biophys Acta 1426, 297307.[Medline]
Timpel, C., Strahl-Bolsinger, S., Ziegelbauer, K. & Ernst, J. (1998). Multiple functions of Pmt1p-mediated protein O-mannosylation in the fungal pathogen Candida albicans. J Biol Chem 273, 2083720846.
Timpel, C., Zink, S., Strahl-Bolsinger, S., Schroppel, K. & Ernst, J. (2000). Morphogenesis, adhesive properties, and antifungal resistance depend on the Pmt6 protein mannosyltransferase in the fungal pathogen Candida albicans. J Bacteriol 182, 30633071.
van den Hondel, C. A., Punt, P. J. & van Gorcom, R. F. (1992). Production of extracellular proteins by the filamentous fungus Aspergillus. Antonie Van Leeuwenhoek 61, 153160.[Medline]
Wallis, G. L., Swift, R. J., Hemming, F. W., Trinci, A. P. & Peberdy, J. F. (1999). Glucoamylase overexpression and secretion in Aspergillus niger: analysis of glycosylation. Biochim Biophys Acta 1472, 576586.[Medline]
Willer, T., Amselgruber, W., Deutzmann, R. & Strahl, S. (2002). Characterization of POMT2, a novel member of the PMT protein O-mannosyltransferase family specifically localized to the acrosome of mammalian spermatids. Glycobiology 12, 771783.
Woo, P. C., Chong, K. T., Leung, A. S., Wong, S. S., Lau, S. K. & Yuen, K. Y. (2003). AFLMP1 encodes an antigenic cell wall protein in Aspergillus flavus. J Clin Microbiol 41, 845850.
Yelton, M. M., Hamer, J. E. & Timberlake, W. E. (1984). Transformation of Aspergillus nidulans by using a trpC plasmid. Proc Natl Acad Sci U S A 81, 14701474.[Abstract]
Yip, C. L., Welch, S. K., Klebl, F., Gilbert, T., Seidel, P., Grant, F. J., O'Hara, P. J. & MacKay, V. L. (1994). Cloning and analysis of the Saccharomyces cerevisiae MNN9 and MNN1 genes required for complex glycosylation of secreted proteins. Proc Natl Acad Sci U S A 91, 27232727.[Abstract]
Zakrzewska, A., Migdalski, A., Saloheimo, M., Penttila, M. E., Palamarczyk, G. & Kruszewska, J. S. (2003). cDNA encoding protein O-mannosyltransferase from the filamentous fungus Trichoderma reesei; functional equivalence to Saccharomyces cerevisiae PMT2. Curr Genet 43, 1116.[Medline]
Received 24 December 2003;
revised 5 February 2004;
accepted 1 March 2004.
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