Pfannmüller Andreas, Wagner Dominik, Sieber Christian, Schönig Birgit, Boeckstaens Mélanie, Marini Anna Maria, Tudzynski Bettina
Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität Münster, Münster, Germany.
Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Neuherberg, Germany.
PLoS One. 2015 Apr 24;10(4):e0125487. doi: 10.1371/journal.pone.0125487. eCollection 2015.
The rice pathogenic fungus Fusarium fujikuroi is well known for the production of a broad spectrum of secondary metabolites (SMs) such as gibberellic acids (GAs), mycotoxins and pigments. The biosynthesis of most of these SMs strictly depends on nitrogen availability and of the activity of permeases of nitrogen sources, e.g. the ammonium and amino acid permeases. One of the three ammonium permeases, MepB, was recently shown to act not only as a transporter but also as a nitrogen sensor affecting the production of nitrogen-repressed SMs. Here we describe the identification of a general amino acid permease, FfGap1, among the 99 putative amino acid permeases (AAPs) in the genome of F. fujikuroi. FfGap1 is able to fully restore growth of the yeast gap1∆ mutant on several amino acids including citrulline and tryptophane. In S. cerevisiae, Gap1 activity is regulated by shuttling between the plasma membrane (nitrogen limiting conditions) and the vacuole (nitrogen sufficiency), which we also show for FfGap1. In yeast, the Npr1 serine/threonine kinase stabilizes the Gap1 position at the plasma membrane. Here, we identified and characterized three NPR1-homologous genes, encoding the putative protein kinases FfNpr1-1, FfNpr1-2 and FfNpr1-3 with significant similarity to yeast Npr1. Complementation of the yeast npr1Δ mutant with each of the three F. fujikuroi NPR1 homologues, resulted in partial restoration of ammonium, arginine and proline uptake by FfNPR1-1 while none of the three kinases affect growth on different nitrogen sources and nitrogen-dependent sorting of FfGap1 in F. fujikuroi. However, exchange of the putative ubiquitin-target lysine 9 (K9A) and 15 (K15A) residues of FfGap1 resulted in extended localization to the plasma membrane and increased protein stability independently of nitrogen availability. These data suggest a similar regulation of FfGap1 by nitrogen-dependent ubiquitination, but differences regarding the role of Fusarium Npr1 homologues compared to yeast.
水稻致病真菌藤仓镰孢菌以产生多种次生代谢产物（SMs）而闻名，如赤霉素（GAs）、霉菌毒素和色素。这些SMs中的大多数生物合成严格依赖于氮的可用性以及氮源通透酶的活性，例如铵和氨基酸通透酶。最近发现三种铵通透酶之一的MepB不仅作为转运蛋白起作用，还作为影响氮抑制SMs产生的氮传感器。在这里，我们描述了在藤仓镰孢菌基因组中的99个假定氨基酸通透酶（AAPs）中鉴定出一种通用氨基酸通透酶FfGap1。FfGap1能够完全恢复酵母gap1∆突变体在包括瓜氨酸和色氨酸在内的几种氨基酸上的生长。在酿酒酵母中，Gap1活性通过在质膜（氮限制条件）和液泡（氮充足）之间穿梭来调节，我们对FfGap1也进行了同样的展示。在酵母中，Npr1丝氨酸/苏氨酸激酶稳定Gap1在质膜上的位置。在这里，我们鉴定并表征了三个NPR1同源基因，它们编码与酵母Npr1具有显著相似性的假定蛋白激酶FfNpr1-1、FfNpr1-2和FfNpr1-3。用藤仓镰孢菌的三个NPR1同源物中的每一个对酵母npr1Δ突变体进行互补，导致FfNPR1-1部分恢复铵、精氨酸和脯氨酸的摄取，而这三种激酶均不影响藤仓镰孢菌在不同氮源上的生长以及FfGap1的氮依赖性分选。然而，FfGap1假定的泛素靶向赖氨酸9（K9A）和15（K15A）残基的交换导致其在质膜上的定位延长且蛋白质稳定性增加，而与氮的可用性无关。这些数据表明FfGap1通过氮依赖性泛素化受到类似的调节，但与酵母相比，镰孢菌Npr1同源物的作用存在差异。
