AgtA, the Dicarboxylic Amino Acid Transporter of Aspergillus nidulans , Is Concertedly Down-Regulated by Exquisite Sensitivity to Nitrogen Metabolite Repression and Ammonium-Elicited Endocytosis

Abstract

We identified agtA, a gene that encodes the specific dicarboxylic amino acid transporter of Aspergillus nidulans. The deletion of the gene resulted in loss of utilization of aspartate as a nitrogen source and of aspartate uptake, while not completely abolishing glutamate utilization. Kinetic constants showed that AgtA is a high-affinity dicarboxylic amino acid transporter and are in agreement with those determined for a cognate transporter activity identified previously. The gene is extremely sensitive to nitrogen metabolite repression, depends on AreA for its expression, and is seemingly independent from specific induction. We showed that the localization of AgtA in the plasma membrane necessitates the ShrA protein and that an active process elicited by ammonium results in internalization and targeting of AgtA to the vacuole, followed by degradation. Thus, nitrogen metabolite repression and ammonium-promoted vacuolar degradation act in concert to downregulate dicarboxylic amino acid transport activity.Amino acids can be utilized by saprophytic fungi as nitrogen and/or carbon sources. Their uptake is mediated by transmembrane proteins belonging to the specific fungal YAT (TC 2.A.3.10, yeast amino acid transporter) family, a member of the APC (amino acid polyamine choline) superfamily (25), which shows a wide range of substrate specificities and includes representatives in all realms of life (53). In Saccharomyces cerevisiae (51), 18 YAT transporters have been functionally characterized, while only a few have been studied in other organisms (8,25,35,57,67,68,69,72,76; Saccharomyces genome database at http://www.yeastgenome.org/). The YAT family transporters share a common predicted topology, comprising 12 transmembrane domains, and show, even among proteins with completely different substrate specificities, a high degree of sequence identity (2, 25). The transporters of the dicarboxylic amino acids glutamate and aspartate have been characterized in S. cerevisiae (DIP5; 47) and Penicillium chrysogenum (PcDIP5; 68) and are members of this family. In A. nidulans, specific dicarboxylic amino acid transport activity and certain aspects of its regulation were previously reported (24, 26, 42, 51, 52), but the corresponding gene(s) was not characterized.In fungi, the genes that encode transporters and enzyme activities involved in nitrogen source utilization are subject to tight transcriptional and/or posttranscriptional controls. Preferred nitrogen sources (such as ammonium and glutamine) repress the transcription of genes involved in the utilization of metabolically less favorable nitrogen sources such as nitrate, purines, or amino acids. In A. nidulans, nitrogen metabolite repression acts by inactivating the AreA GATA transcriptional activator (30) by a number of concurring mechanisms (36, 66). In S. cerevisiae, a transcriptional repression mechanism similar but not identical to the one described above for A. nidulans is operative (see reference 33 for a review).In addition to being subject to transcriptional regulati...

Eisosome Organization in the Filamentous AscomyceteAspergillus nidulans

“…However, the data presented in this article imply that if they play a role in endocytosis in A. nidulans, this role is minor or limited to specific cargos, which remain unidentified. Neither the endocytosis of FM4-64 nor the ammonium-elicited endocytosis of the AgtA (dicarboxylic amino acid transporter [5]; data not shown) or PrnB (the major proline transporter [41]; data not shown) are visibly affected in mycelia by a deletion of pilA, encoding the only eisosomal protein present in discrete foci in hyphae. It should be noted that although the AgtA and PrnB proteins of A. nidulans and the Hxt2 protein of S. cerevisiae are transporters with a homogeneous distribution in the plasma membrane, upon endocytosis Hxt2 accumulates in discrete plasma membrane-associated foci (52), while the two A. nidulans proteins do not (5).…”

“…nidulans is arguably, among the Pezizomycotina, the organism where membrane proteins and endocytosis have been better studied. In this organism, a number of transporters driven by their physiological promoters have been visualized in the cell membrane (UapC [48], PrnB [45], UapA and AzgA [35], AgtA [5], MstE [14], FcyB [51], UreA [M. Sanguinetti and A. Ramón, personal communication], and FurD [G. Diallinas and F. Borbolis, personal communication]). Load-and-chase experiments have shown that when the dye FM4-64 is endocytosed, it first appears in cortical punctate structures (36).…”

“…Load-and-chase experiments have shown that when the dye FM4-64 is endocytosed, it first appears in cortical punctate structures (36). Active research concerning membrane proteins (see reference 12 for a review), determination and maintenance of polarity during development (including the possible involvement of sphingolipids [23], which may be involved in signaling eisosomal protein phosphorylation in S. cerevisiae), and ongoing recent work on endocytosis (1,5,6,16,36,40) make A. nidulans an obvious model within the Pezizomycotina with which to study eisosomal presence, structure, and function. A description of the presence and fate of these organelles during asexual development is presented below.…”

“…PilA (but not PilB or SurG) is present in punctate structures in mycelia. The presence of ammonium results in endocytosis in the germlings and mycelia of a number of transporters involved in the utilization of nitrogen sources (5,35,49). We have investigated whether the ammonium-elicited endocytosis of the dicarboxylic amino acid transporter AgtA (5) was affected in a strain with the pilA deletion.…”

The Aspergillus nidulans Proline Permease as a Model for Understanding the Factors Determining Substrate Binding and Specificity of Fungal Amino Acid Transporters

“…Numerous YAT transporters can be identified in all sequenced genomes of fungi. Characterized members include 15 of the 16 characterized amino acid transporters of Saccharomyces cerevisiae (5,6), two proteins of Aspergillus nidulans, PrnB and AgtA (7,8), as well as transporters from Candida glabrata, Neurospora crassa, and Penicillium chrysogenum (9 -11). These proteins display different specificities and are subject to tight transcriptional and post-translational regulation (12)(13)(14).…”