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 ⌬npr1, 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.

The Complete Genome Sequence of the Plant Growth-Promoting Bacterium Pseudomonas sp. UW4

“…The gene ppkB (PputUW4_03641), which is located immediately downstream of nasA , encodes a serine/threonine protein kinase. It has been demonstrated that a protein kinase carried out phosphorylation of the nitrate transporter and played an important role in nitrate deprivation response in A. thaliana and Hansenula polymorpha [101]–[102].…”

Internal Amino Acids Promote Gap1 Permease Ubiquitylation via TORC1/Npr1/14-3-3-Dependent Control of the Bul Arrestin-Like Adaptors

“…The phosphorylation status of different amino acid permeases is indeed reported to be reduced in npr1⌬ cells (46). In the yeast Hansenula polymorpha, furthermore, the Npr1 kinase ortholog is reported to phosphorylate the Ynt1 nitrate and nitrite permease under poor N supply conditions (50). This phosphorylation occurs at serine residues of a central intracellular loop and protects the permease against Ub-dependent downregulation (50,54).…”

“…In the yeast Hansenula polymorpha, furthermore, the Npr1 kinase ortholog is reported to phosphorylate the Ynt1 nitrate and nitrite permease under poor N supply conditions (50). This phosphorylation occurs at serine residues of a central intracellular loop and protects the permease against Ub-dependent downregulation (50,54). Yeast Npr1 is also essential to the transport activity of the Mep transporters rather than to their stability at the cell surface, suggesting that it might also control permeases via other mechanisms (5).…”

The balance of protein expression and degradation: an ESCRTs point of view

“…Phosphorylation of Rsp5, Arts, and nutrient transporters by Npr1 could account for the increase in transporter ubiquitination and subsequent endocytosis [13,14,22,24,27,28] but the effect of these phosphorylation events is controversial. A recent study found rapamycin-induced phosphorylation of Art proteins suppresses recruitment of Rsp5 and concomitant ubiquitination and degradation of cell-surface transporters [14].…”