Protein O-Mannosyltransferases B and C Support Hyphal Development and Differentiation in Aspergillus nidulans

Aspergillus nidulans possesses three pmt genes encoding protein O-d-mannosyltransferases (Pmt). Previously, we reported that PmtA, a member of the PMT2 subfamily, is involved in the proper maintenance of fungal morphology and formation of conidia (T. Oka, T. Hamaguchi, Y. Sameshima, M. Goto, and K. Furukawa, Microbiology 150:1973-1982, 2004). In the present paper, we describe the characterization of the pmtA paralogues pmtB and pmtC. PmtB and PmtC were classified as members of the PMT1 and PMT4 subfamilies, respectively. A pmtB disruptant showed wild-type (wt) colony formation at 30°C but slightly repressed growth at 42°C. Conidiation of the pmtB disruptant was reduced to approximately 50% of that of the wt strain; in addition, hyperbranching of hyphae indicated that PmtB is involved in polarity maintenance. A pmtA and pmtB double disruptant was viable but very slow growing, with morphological characteristics that were cumulative with respect to either single disruptant. Of the three single pmt mutants, the pmtC disruptant showed the highest growth repression; the hyphae were swollen and frequently branched, and the ability to form conidia under normal growth conditions was lost. Recovery from the aberrant hyphal structures occurred in the presence of osmotic stabilizer, implying that PmtC is responsible for the maintenance of cell wall integrity. Osmotic stabilization at 42°C further enabled the pmtC disruptant to form conidiophores and conidia, but they were abnormal and much fewer than those of the wt strain. Apart from the different, abnormal phenotypes, the three pmt disruptants exhibited differences in their sensitivities to antifungal reagents, mannosylation activities, and glycoprotein profiles, indicating that PmtA, PmtB, and PmtC perform unique functions during cell growth.Protein glycosylation, which is a major posttranslational modification, plays essential roles in eukaryotic cells from fungi to mammals (19). N-linked oligosaccharides in glycoproteins that share relatively common structures are structurally classified into high-mannose, complex, and hybrid types (3). O-linked oligosaccharides in glycoproteins are diverse with respect to their sugar components and the mode of sugar linkages among the eukaryotic organisms (8, 19). O mannosylation, which is commonly found in the glycoproteins of fungi, has been extensively studied in the budding yeast Saccharomyces cerevisiae (4, 21, 35). The initial reaction of mannose transfer to serine and threonine residues in proteins is catalyzed by protein O-d-mannosyltransferase (Pmt) in the endoplasmic reticulum (ER), where dolichyl phosphate-mannose is required as an immediate sugar donor (4). In the Golgi complex, O mannosylation in S. cerevisiae is linearly elongated by up to five mannose residues by mannosyltransferases (Mnt) that utilize GDP-mannose as the mannosyl donor. At least six Pmt-encoding genes (PMT1 to -6), three α-1,2-Mnt-encoding genes (KRE2, KTR1, and KTR3), and three α-1,3-Mnt-encoding genes (MNN1, MNT2, and MNT3) are known to be involved in O mannosylation in S. cerevisiae (21, 31, 45).The Pmt family of proteins can be classified into the PMT1, PMT2, and PMT4 subfamilies based on phylogeny (6). Proteins of the PMT1 subfamily form a heteromeric complex with proteins belonging to the PMT2 subfamily, and PMT4 subfamily proteins form a homomeric complex (7). Simultaneous disruptions of three different types of PMT genes were lethal (4), suggesting that each class provided a unique function for O mannosylation. Yeasts other than S. cerevisiae, such as Schizosaccharomyces pombe (38, 41), Candida albicans (29), and Cryptococcus neoformans (28), possess three to five pmt genes, which have been characterized. Several studies provide evidence that protein O mannosylation modulates the functions and stability of secretory proteins and thereby affects the growth and morphology of these yeasts. O mannosylation by Pmt2 in S. cerevisiae (ScPmt2) provides protection from ER-associated degradation and also functions as a fail-safe mechanism for ER-associated degradation (11, 13, 23). Likewise, in C. albicans, CaPmt1- and CaPmt4-mediated O mannosylation specifically protects CaSec20 from proteolytic degradation in the ER (40). Cell wall integrity is maintained in S. cerevisiae by increased stabilization and correct localization of the sensor proteins ScWsc and ScMid2 due to O mannosylation by ScPmt2 and ScPmt4 (20). Similarly, the stability and localization to the plasma membrane of axial budding factor ScAxl2/Bud10 is enhanced by ScPmt4-mediated O mannosylation, increasing its activity (32). ScPmt4-mediated O glycosylation also functions as a sorting determinant for cell surface delivery of ScFus1 (30). CaPmt4-mediated O glycosylation is required for environment-specific morphogenetic signaling and for the full virulence of C. albicans (29).With respect to filamentous fungi like Aspergillus that develop hyphae in a highly ordered manner, which then differentiate to form conidiospores, little is known about the function and synthetic pathway of the O-mannose-type oligosaccharides. O-Glycans in glycoproteins of Aspergillus include sugars other than mannose, and their structures have been determined (8). The initial mannosylation catalyzed by Pmts is found in Aspergillus and occurs as in yeasts (8).We characterized the pmtA gene of Aspergillus nidulans (AnpmtA), belonging to the PMT2 subfamily, and found that the mutant exhibited a fragile cell wall phenotype and alteration in the carbohydrate composition, with a reduction in the amount of skeletal polysaccharides in the cell wall (26, 33). Recently, the Afpmt1 gene belonging to the PMT1 family of Aspergillus fumigatus, a human pathogen, was characterized. AfPmt1 is crucial for cell wall integrity and conidium morphology (46).In this study, we characterize the pmtB and pmtC genes of A. nidulans to understand their contribution to the cell morphology of this filamentous fungus. We also demonstrate that the PmtA, PmtB, and PmtC proteins have distinct specificities for protein substrates and function differently during cell growth of filamentous fungi.