Tazebay U H, Sophianopoulou V, Cubero B, Scazzocchio C, Diallinas G
Institut de Génétique et Microbiologie, IGM, Université Paris-Sud, Centre d'Orsay, France.
FEMS Microbiol Lett. 1995 Oct 1;132(1-2):27-37. doi: 10.1111/j.1574-6968.1995.tb07806.x.
In the filamentous fungus Aspergillus nidulans, L-proline uptake is mediated by the product of the prnB gene which codes for a member of a family of amino acid transporters found both in pro- and eukaryotes. Regulation of prnB gene expression has previously been studied in great detail at the molecular level. However, no studies have addressed possible post-transcriptional controls or the kinetic characterisation of the PrnB transporter. Here we develop a rapid and efficient method for direct uptake measurements of proline in germinating conidiospores of A. nidulans. We make use of this method and Northern blot analyses in parallel to study the regulation of PrnB expression both at the level of prnB message accumulation and at a post-transcriptional level. These studies show that (i) pathway-specific and wide-domain regulatory systems, previously shown to control prnB gene expression in multicellular mycelia, also operate in unicellular conidia committed to germination; and (ii) PrnB activity is regulated in response to the nitrogen source present in the medium and the level of internally accumulated proline or other amino acids. We also characterise kinetically the PrnB transporter and a secondary proline transport system. Our results open new possibilities for studies using unicellular conidiospores of filamentous fungi and constitute a necessary first step for a subsequent structure-function analysis of the PrnB transporter.
在丝状真菌构巢曲霉中，L-脯氨酸的摄取由prnB基因的产物介导，该基因编码一种在原核生物和真核生物中均存在的氨基酸转运蛋白家族成员。此前已在分子水平上对prnB基因表达的调控进行了详细研究。然而，尚未有研究涉及可能的转录后调控或PrnB转运蛋白的动力学特性。在此，我们开发了一种快速有效的方法，用于直接测量构巢曲霉萌发分生孢子中脯氨酸的摄取。我们利用该方法并同时进行Northern印迹分析，以研究PrnB表达在prnB mRNA积累水平和转录后水平的调控。这些研究表明：（i）先前已证明在多细胞菌丝体中控制prnB基因表达的途径特异性和广域调控系统，在致力于萌发的单细胞分生孢子中也起作用；（ii）PrnB活性根据培养基中存在的氮源以及内部积累的脯氨酸或其他氨基酸的水平进行调节。我们还对PrnB转运蛋白和次生脯氨酸转运系统进行了动力学表征。我们的结果为利用丝状真菌的单细胞分生孢子进行研究开辟了新的可能性，并为随后对PrnB转运蛋白进行结构-功能分析奠定了必要的第一步。
