Regulation of expression and kinetic modeling of substrate interactions of a uracil transporter inAspergillus nidulans

Abstract

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.

UreA, the major urea/H+ symporter in Aspergillus nidulans

“…In agreement with transport assays, epifluorescence microscopy of germlings of a strain carrying a UreA-GFP fusion shows that the appearance of fluorescence in the membrane occurs also after 2 h of culture (not shown). Interestingly and at variance to what has been observed for other transporters (Amillis et al, , 2007Tazebay et al, 1997), uptake of urea is repressed by ammonium during the period of conidial isotropic growth. No saturable [ 14 C]-urea uptake was detected at any stage or N-source in the DureA strain.…”

“…[ 14 C]-urea uptake in minimal media (MM) was assayed in germinating conidiospores of A. nidulans concentrated at 10 7 conidiospores/100 lL, at 37°C, pH 6.8, as previously described (Amillis et al, 2007;Cecchetto et al, 2004;Papageorgiou et al, 2008). Initial velocities were measured at 1-2 min of incubation substrates.…”

Completing the purine utilisation pathway of Aspergillus nidulans

“…When one gene is involved in more than one step, the complete entry is shown once and only the gene symbol and database entry is shown opposite additional steps. Experimental identification of each gene is described in: nadA (Ribard et al, 2003); hxA (Glatigny and Scazzocchio, 1995); hxB (Amrani et al, 1999(Amrani et al, , 2000; xanA (Cultrone et al, 2005); uaZ ; azgA (Cecchetto et al, 2004); uapA (Gorfinkiel et al, 1993); uapC (Diallinas et al, 1995); fcyB (Vlanti and Diallinas, 2008); furD (Amillis et al, 2007;Hamari et al, 2009); furA (Hamari et al, 2009); ureA (Abreu et al, 2010); uaX and uaW are described in this article. alX (Hamari et al, 2009) aaX and the putative ureidoglycolase gene were identified by similarity with the cognate genes of S. cerevisiae, confirmed for alX and aaX by their known chromosomal locations.…”

pyrG is required for maintaining stable cellular uracil level and normal sporulation pattern under excess uracil stress in Aspergillus nidulans

“…In response to exogenous uracil, the transcriptional level of FUR4, which encodes a uracil permease for uracil transport in S. cerevisiae [12], is downregulated [13,14]. Similarly, exogenous uracil triggers the transcriptional down-regulation of A. nidulans furD, a gene encoding Fur4p-like uracil permease [15]. At the posttranslational level, the presence of exogenous uracil accelerates the degradation of Fur4p by enhancing its ubiquitylation [14].…”

“…At the posttranslational level, the presence of exogenous uracil accelerates the degradation of Fur4p by enhancing its ubiquitylation [14]. Tight control of the cellular uracil level is believed to be important to maintain a low ratio of uracil/thymine to prevent the incorporation of uracil into DNA [15]. Replacement of thymine with uracil in regulatory regions of genomic DNA can disrupt the specificity of protein-DNA interactions [16].…”