|dc.description.abstract||Protein O-mannosyltransferase (Pmt) catalyzes the transfer of the first mannosyl residue from dolichyl phosphate activated mannose (Dol-P-Man) to specific serine/threonine residues of target proteins. It is becoming increasingly evident that protein O-mannosylation is essential for viability, cell wall integrity, signaling, morphogenesis, virulence and polar growth of fungi. Pmts are classified as members of three subfamilies named for the Saccharomyces cerevisiae proteins Pmt1, Pmt2 and Pmt4. Pmts are not active as monomers, rather they must be part of heteromeric or homomeric complexes to function. In S. cerevisiae Pmt 1 and Pmt2 form a heteromeric complex and Pmt 4 forms a homomeric complex. Most mannosylated proteins studied in yeasts are targeted to the membrane or cell wall or are secreted. Many of those are important for cell wall integrity and morphogenesis. In filamentous fungi, only a few Pmt-modified proteins have been identified. Although protein O-mannosylation and Pmt-modified proteins have been extensively studied in unicellular yeasts, little is known about roles of Pmts in polar growth and cell wall biogenesis of multicellular filamentous fungi. Discovery of novel cell wall proteins will benefit our knowledge of growth and development of multicellular fungi.
This dissertation describes the use of A. nidulans as a model organism to better understand molecular and biological roles of Pmts in filamentous fungi. A. nidulans contains 3 Pmts representing each subfamily, PmtA (Pmt 2 subfamily), PmtB (Pmt 1 subfamily) and PmtC (Pmt 4 subfamily). All three A. nidulans pmts were individually deleted. Single pmt mutants are viable and exhibit distinctive phenotypes. The pmtA pmtB mutant is the only viable double mutant. All pmt mutants are hypersensitive to cell wall perturbing agents and develop abnormal conidiophores suggesting that Pmt is involved in cell wall integrity and morphogenesis. A. nidulans Pmts form heteromeric complexes among the three subfamilies. In addition, PmtC forms a homomeric complex. AxlA, WscA and MsbA, A. nidulans orthologs of yeast proteins modified by Pmts, were used to compare Pmt substrate specificity. Each individual Pmt appears to carry substrate specificity independently from the other Pmts in the complex. Unlike Axl2 from S. cerevisiae, AxlA is not a substrate of Pmts in A. nidulans. In S. cerevisiae Wsc1 is modified by Pmt2, as a member of the Pmt1/2 complex. In contrast, A. nidulans WscA is modified by PmtA (Pmt2 subfamily) and PmtC (Pmt 4 subfamily) whether or not PmtB (Pmt 1 subfamily) is present. Unlike S. cerevisiae Msb2, which is modified by Pmt4, A. nidulans MsbA is modified by PmtA (subfamily 2) and Pmt B (subfamily 1), not PmtC (subfamily 4).
Our work shows that all three Pmts from A. nidulans can form heteromeric complexes with each other and that substrate specificity appears to be determined by individual Pmts within complexes.||