Eisosome organization in the filamentous ascomycete Aspergillus nidulans.

Eisosomes are subcortical organelles implicated in endocytosis and have hitherto been described only in Saccharomyces cerevisiae. They comprise two homologue proteins, Pil1 and Lsp1, which colocalize with the transmembrane protein Sur7. These proteins are universally conserved in the ascomycetes. We identify in Aspergillus nidulans (and in all members of the subphylum Pezizomycotina) two homologues of Pil1/Lsp1, PilA and PilB, originating from a duplication independent from that extant in the subphylum Saccharomycotina. In the aspergilli there are several Sur7-like proteins in each species, including one strict Sur7 orthologue (SurG in A. nidulans). In A. nidulans conidiospores, but not in hyphae, the three proteins colocalize at the cell cortex and form tightly packed punctate structures that appear different from the clearly distinct eisosome patches observed in S. cerevisiae. These structures are assembled late during the maturation of conidia. In mycelia, punctate structures are present, but they are composed only of PilA, while PilB is diffused in the cytoplasm and SurG is located in vacuoles and endosomes. Deletion of each of the genes does not lead to any obvious growth phenotype, except for moderate resistance to itraconazole. We could not find any obvious association between mycelial (PilA) eisosome-like structures and endocytosis. PilA and SurG are necessary for conidial eisosome organization in ways that differ from those for their S. cerevisiae homologues. These data illustrate that conservation of eisosomal proteins within the ascomycetes is accompanied by a striking functional divergence.

A cryptic role of a glycolytic-gluconeogenic enzyme (aldolase) in amino acid transporter turnover in Aspergillus nidulans.

In Aspergillus nidulans the fbaA1013 mutation results in reduced or total loss of growth on glycolytic and gluconeogenic carbon sources, respectively. It also negatively affects growth on several amino acids (including L-proline, L-glutamate or L-aspartate) that the fungus can use as nitrogen source on glycolytic carbon sources. Complementation of the fbaA1013 mutation using an A. nidulans genomic library resulted in cloning of the fbaA gene, which encodes a putative fructose 1,6-biphosphate aldolase (FBA), an enzyme involved in both glycolysis and gluconeogenesis. The fbaA1013 mutation is a chromosome rearrangement in the 5' regulatory region of the fbaA gene resulting in reduced or total loss of transcription in response to glycolytic and gluconeogenic carbon sources respectively. The fbaA gene is essential for growth. A functional FbaA protein is necessary for plasma membrane localization of the AgtA acidic amino acid (L-glutamate/L-aspartate) transporter, as the fbaA1013 mutation results in targeting to and presumably subsequent degradation of AgtA in the vacuole. Our results support a novel role of the FbaA protein that is, involvement in the regulation of amino acids transporters.

EglD, a putative endoglucanase, with an expansin like domain is localized in the conidial cell wall of Aspergillus nidulans.

Although the process of conidial germination in filamentous fungi has been extensively studied, many aspects remain to be elucidated since the asexual spore or conidium is vital in their life cycle. Breakage and reformation of cell wall polymer bonds along with the maintenance of cell wall plasticity during conidia germination depend upon a range of hydrolytic enzymes whose activity is analogous to that of expansins, a highly conserved group of plant cell wall proteins with characteristic wall loosening activity. In the current study, we identified and characterized the eglD gene in Aspergillus nidulans, an expansin-like gene the product of which shows strong similarities with bacterial and fungal endo-beta1,4-glucanases. However, we failed to show such activity in vitro. The eglD gene is constitutively expressed in all developmental stages and compartments of A. nidulans asexual life cycle. However, the EglD protein is exclusively present in conidial cell walls. The role of the EglD protein in morphogenesis, growth and germination rate of conidia was investigated. Our results show that EglD is a conidial cell wall localized expansin-like protein, which could be involved in cell wall remodeling during germination.

Regulation of expression and kinetic modeling of substrate interactions of a uracil transporter in Aspergillus nidulans.

Early genetic evidence suggested that A. nidulans possesses at least one uracil transporter. A gene, named furD, was recently identified by reverse genetics and in silico approaches and we confirm here that it encodes a high-affinity, high-capacity, uracil transporter. In this work, we study the regulation of expression of FurD and develop a kinetic model describing transporter-substrate interactions. The furD gene is not expressed in resting conidiospores, is transcriptionally activated and reaches a peak during the isotropic growth phase of conidiospore germination, and stays at a basic low level in mycelium. Transcriptional expression is correlated to uracil transport activity. Expression in a strain blocked in uracil biosynthesis (pyrG-) is moderately increased and extended to later stages of germination. The presence of excess uracil in the medium leads to down-regulation of furD expression and FurD activity. A detailed kinetic analysis using a number of pyrimidine and purine analogues showed that FurD is able to recognize with high-affinity uracil (Km 0.45 microM), thymine (Ki 3.3 microM) and several 5-substituted analogues of uracil, and with moderate affinity uric acid and xanthine (Ki 94-99 microM). Kinetic evidence supports a model in which the positions N1-H, =O2, N3-H, =O4, as well as planarity play a central role for the substrate binding. This model, which rationalizes the unique specificity of FurD for uracil, is compared to and found to be very similar to analogous models for protozoan uracil transporters.