title: Npr1 Ser/Thr protein kinase links nitrogen source quality and carbon availability with the yeast nitrate transporter (Ynt1) levels.
abstract: Ynt1, the single high affinity nitrate and nitrite transporter of the yeast Hansenula polymorpha, is regulated by the quality of nitrogen sources. Preferred nitrogen sources cause Ynt1 dephosphorylation, ubiquitinylation, endocytosis, and vacuolar degradation. In contrast, under nitrogen limitation Ynt1 is phosphorylated and sorted to the plasma membrane. We show here the involvement of the Ser/Thr kinase HpNpr1 in Ynt1 phosphorylation and regulation of Ynt1 levels in response to nitrogen source quality and the availability of carbon. In Δnpr1, Ynt1 phosphorylation does not take place, although Ynt1 ubiquitin conjugates increase. As a result, in this strain Ynt1 is sorted to the vacuole, from both plasma membrane and the later biosynthetic pathway in nitrogen-free conditions and nitrate. In contrast, overexpression of NPR1 blocks down-regulation of Ynt1, increasing Ynt1 phosphorylation at Ser-244 and -246 and reducing ubiquitinylation. Furthermore, Npr1 is phosphorylated in response to the preferred nitrogen sources, and indeed it is dephosphorylated in nitrogen-free medium. Under conditions where Npr1 is phosphorylated, Ynt1 is not and vice versa. We show for the first time that carbon starvation leads to Npr1 phosphorylation, whereas Ynt1 is dephosphorylated and degraded in the vacuole. Rapamycin prevents this, indicating a possible role of the target of rapamycin signaling pathway in this process. We concluded that Npr1 plays a key role in adapting Ynt1 levels to the nitrogen quality and availability of a source of carbon.
authors.affiliation: Department of Biochemistry and Molecular Biology, Institute of Biomedical Technologies, Nitrogen Metabolism Group, Universidad de La Laguna, E-38206 La Laguna, Tenerife, Canarias, Spain.
authors.forename: Yusé
authors.initials: Y
authors.lastname: Martín
authors.forename: Yelvis V
authors.initials: YV
authors.lastname: González
authors.forename: Elisa
authors.initials: E
authors.lastname: Cabrera
authors.forename: Celia
authors.initials: C
authors.lastname: Rodríguez
authors.forename: José M
authors.initials: JM
authors.lastname: Siverio
mesh_terms: D015153:Blotting, Western; D002244:Carbon / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism*; D009584:Nitrogen / Q000378:metabolism*; D010766:Phosphorylation; D010843:Pichia / Q000201:enzymology / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D002352:Carrier Proteins; D005656:Fungal Proteins; D009566:Nitrates; D002244:Carbon; D011494:Protein Kinases; D009584:Nitrogen