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.

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.

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