The Npr1 kinase controls biosynthetic and endocytic sorting of the yeast Gap1 permease

Membrane trafficking of the general amino acid permease (Gap1) of Saccharomyces cerevisiae is under nitrogen regulation. In cells growing on proline or urea as the sole nitrogen source, newly synthesized Gap1 is delivered to the plasma membrane, where it accumulates. Upon addition of NH(4)(+), a preferential nitrogen source, Gap1 is endocytosed and targeted to the vacuole, where it is degraded. This down-regulation requires ubiquitination of the permease, and this ubiquitination is dependent on the essential Npi1/Rsp5 ubiquitin ligase. In this study, we investigated the role of the Npr1 kinase in the regulation of Gap1 trafficking. We show that Npr1 is required for stabilization of Gap1 at the plasma membrane: when an npr1(ts) mutant growing on proline is shifted to the restrictive temperature, Gap1 down-regulation is triggered, as it is when NH(4)(+) is added to wild-type cells. The fate of newly synthesized Gap1 en route to the plasma membrane is also under Npr1 control: in an npr1Delta mutant, neosynthesized Gap1 is sorted from the Golgi to the vacuole without passing via the plasma membrane. Similar direct sorting of neosynthesized Gap1 to the vacuole was observed in wild-type cells grown on NH(4)(+). Finally, Gap1 is phosphorylated in NPR1 cells, but this phosphorylation is not strictly dependent on Npr1. Our results show that Npr1 kinase plays a central role in the physiological control of Gap1 trafficking and that this control is exerted not only on Gap1 present at the plasma membrane but also on Gap1 late in the secretory pathway. Npr1 belongs to a subgroup of protein kinases, some of which are reported to exert a positive control on the activity of other permeases. We propose that these kinases also function as regulators of permease trafficking.     

Role of the Npr1 kinase in ammonium transport and signaling by the ammonium permease Mep2 in Candida albicans

The ammonium permease Mep2 induces a switch from unicellular yeast to filamentous growth in response to nitrogen limitation in Saccharomyces cerevisiae and Candida albicans. In S. cerevisiae, the function of Mep2 and other ammonium permeases depends on the protein kinase Npr1. Mutants lacking NPR1 cannot grow on low concentrations of ammonium and do not filament under limiting nitrogen conditions. A G349C mutation in Mep2 renders the protein independent of Npr1 and results in increased ammonium transport and hyperfilamentous growth, suggesting that the signaling activity of Mep2 directly correlates with its ammonium transport activity. In this study, we investigated the role of Npr1 in ammonium transport and Mep2-mediated filamentation in C. albicans. We found that the two ammonium permeases Mep1 and Mep2 of C. albicans differ in their dependency on Npr1. While Mep1 could function well in the absence of the Npr1 kinase, ammonium transport by Mep2 was virtually abolished in npr1Δ mutants. However, the dependence of Mep2 activity on Npr1 was relieved at higher temperatures (37°C), and Mep2 could efficiently induce filamentous growth under limiting nitrogen conditions in npr1Δ mutants. Like in S. cerevisiae, mutation of the conserved glycine at position 343 in Mep2 of C. albicans to cysteine resulted in Npr1-independent ammonium uptake. In striking contrast, however, the mutation abolished the ability of Mep2 to induce filamentous growth both in the wild type and in npr1Δ mutants. Therefore, a mutation that improves ammonium transport by Mep2 under nonpermissible conditions eliminates its signaling activity in C. albicans.     

The General Amino Acid Permease FfGap1 of Fusarium fujikuroi Is Sorted to the Vacuole in a Nitrogen-Dependent,but Npr1 Kinase-Independent Manner

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.