Npr1 Ser/Thr Protein Kinase Links Nitrogen Source Quality and Carbon Availability with the Yeast Nitrate Transporter (Ynt1) Levels*

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.

tion pathway is highly conserved. Nitrate enters the cells via nitrate transporter(s), and it is then reduced to nitrite and then to ammonium by nitrate and nitrite reductase, respectively. Because reduction of nitrate to ammonium is a highly energydemanding process and nitrite is potentially toxic to the cells, several mechanisms have been found that regulate this pathway in response to nitrogen and carbon sources (1). In most organisms, genes encoding the enzymatic machinery for nitrate assimilation require nitrate for their induction and are subject to nitrogen catabolite repression (2,3). In fact, nitrate acts as an inducer once it enters the cells (4 -6), besides being the substrate. Thus, nitrate transporter regulation plays a key role in modulating nitrate assimilation gene expression. In this context, the Arabidopsis thaliana nitrate transporter CHL1 (NRT1.1) acts as a nitrate sensor independently of its nitrate transport activity (7). CHL1 undergoes phosphorylation at Thr-101, in response to low nitrate concentration. This phosphorylation is carried out by a calcineurin B-like-interacting protein kinase (CIPK23) that is involved in the primary nitrate response (8). Ynt1, the sole high affinity nitrate transporter in the yeast Hansenula polymorpha (9,10), is regulated according to the quality of nitrogen source. In the presence of preferred sources, such as glutamine or ammonium, Ynt1 is ubiquitinylated mainly at Lys-253 and Lys-270. This modification triggers Ynt1 endocytosis and vacuolar degradation (11). In contrast, when the cells are subjected to nitrogen deprivation, Ynt1 phosphorylation is essential for sorting it to the plasma membrane. The nonphosphorylated forms of Ynt1 are thus delivered to the vacuole from the biosynthetic pathway (12). Evidence so far obtained on Ynt1 does not allow us to conclude that it behaves as a nitrate sensor, in the same way as CHL1. However, when nitrate is scarce, its fine regulation by Npr1-mediated phosphorylation targets it to the plasma membrane, facilitating nitrate uptake and consequently nitrate assimilation gene induction (12). There is also strong evidence on the post-translational regulation of A. thaliana NRT2.1 and Aspergillus nidulans NrtA (13,14).
Plasma membrane permeases involved in nutrient transport have been extensively studied in Saccharomyces cerevisiae. They are internalized and degraded in the vacuole or stabilized at the plasma membrane in response to the quality and quantity of nutrients available in the medium. Ubiquitinylation via E3-ubiquitin ligase Rsp5p and phosphorylation are involved in cellular trafficking of such plasma membrane permeases (15)(16)(17)(18)(19). The Ser/Thr protein kinase termed nitrogen permease reactivator 1 (Npr1) plays an outstanding role in regulating nitrogen-related permeases such as Bap2 (19), Mep2 (20), Tat2, and Gap1. In this way, Npr1 is required for Gap1 plasma membrane sorting in the presence of nonpreferred nitrogen sources (15), while under the same conditions, Tat2 is degraded in the vacuole (21). Npr1 is also involved in the ubiquitinylation of Gap1 in an Rsp5-dependent manner (15). The knowledge of Npr1 beyond S. cerevisiae is very scarce. In addition to our work in H. polymorpha, in Candida albicans Npr1 has been reported to regulate the ammonium permease Mep2, unlike Mep1 (22).
Nutritional starvation is the most common stress that yeast cells face in nature. They use several mechanisms to counteract nutritional starvation and to restart the cell cycle when nutrients are newly available. Plasma membrane permeases undergo internalization and vacuolar degradation in response to carbon, nitrogen, or phosphate starvation. This has not been thoroughly studied. However, it is rather well established that tryptophan permease (Tat2) and uracil permease (Fur4) are degraded in response to carbon and nitrogen starvation (23,24). The high affinity phosphate transporter Pho84 is also degraded in external phosphate below 30 -40 M and under carbon starvation conditions (25)(26)(27). In contrast, Gap1 and HpYnt1 are remarkably stable under nitrogen starvation conditions (11,12,17).
Hall and co-workers (21) showed that Npr1 activity is controlled by the TOR 5 signaling pathway in response to nitrogen source quality. The inactivation of TOR by incubation in poor nitrogen sources provided a nonphosphorylated active form of Npr1. However, in good nitrogen sources where TOR is active, Npr1 is phosphorylated and inactive. Indeed, Npr1 has been shown to have 20 rapamycin-sensitive phosphorylation sites clustered in two regions within the N-terminal serine-rich domain (28). Growing evidence suggests that TORC1 is involved in vesicle trafficking and may also sense the cellular nutrient status in endogenous membrane structures. Accordingly, several components of TORC1 have been localized to the Golgi, endosome, or other vesicle compartments by different methods. In this sense, TORC1 affects late steps of endocytosis, and Pmr1, a Golgi-localized ATPase that transports Ca 2ϩ and Mn 2ϩ ions from the cytoplasm into the Golgi, down-regulates TORC1 but also Npr1, which is mostly cytosolic, although a portion is localized at the Golgi. Consistent with this, an increase in Gap1 was observed in npr1 mutants at the plasma membrane (29 -33). Moreover, in addition to nutrients, K ϩ has also been found to be involved in plasma membrane permease stability (34).
The post-translational regulation of nitrate transporters reveals its importance in modulating nitrate assimilation genes (8,12). Therefore, the study of nitrate transporter modifications in response to environmental and nutritional conditions is essential to achieve further understanding of nitrate assimilation control. Indeed, in H. polymorpha, an ORF encodes for a protein with a high similarity to ScNpr1, and its deletion abolishes Ynt1 phosphorylation (12). However, the role of HpNpr1 kinase in the post-translational regulation of Ynt1 was totally unknown until now. We found a new connection between carbon sources and Npr1; indeed, Npr1 is closely involved in the cellular fate of Ynt1, according to the nitrogen quality and carbon source availability. Npr1 undergoes a basal phosphorylation under nitrogen deprivation, which increases in good nitrogen sources and carbon starvation. Rapamycin also affects the phosphorylation of Npr1 and Ynt1 under carbon starvation, suggesting that the TOR kinase pathway is involved in Npr1 regulation in response to carbon availability.

EXPERIMENTAL PROCEDURES
Strains and Media-The H. polymorpha strains used in this study (supplemental Table SI) are all derivatives of NCYC495 leu2 Ϫ ura3 Ϫ . Cells were grown in YPD media (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose) or synthetic media containing 0.17% (w/v) yeast nitrogen base without amino acids and ammonium sulfate (YNB), 2% (w/v) glucose, and the appropriate nitrogen source for each experiment. In K ϩ experiments, cells were grown in synthetic K ϩ -free medium (YNB-F) (0.17% (w/v) yeast nitrogen base without amino acids, ammonium sulfate, and potassium (ForMedium TM United Kingdom, CYN7505) supplemented with 2% glucose, 5 mM ammonium sulfate, and 20 mM potassium chloride buffered at pH 5.5 with 50 mM MES-Tris. To induce nitrate assimilation genes at restricted K ϩ conditions, cells were then incubated in a homemade yeast nitrogen base following the Difco recipe but without amino acids, ammonium sulfate, or monobasic potassium phosphate. This was brought to pH 5.5 with 50 mM MES-Tris; 2% glucose and the indicated nitrogen source were then added.
To obtain yeast cultures bearing Ynt1, cells were grown in 0.17% (w/v) yeast nitrogen base without amino acids and ammonium sulfate (YNB), 2% (w/v) glucose, 5 mM ammonium chloride, centrifuged, and resuspended at 10 mg/ml in the same medium but without nitrogen for 1 h. After that, cells were incubated in 5 or 10 mM sodium nitrate for 1.5-2 h. In the experiments where the effect of carbon starvation on Ynt1 was studied, once the cells were incubated in nitrate they were transferred to fresh medium (YNB) without nitrate in the presence of 2% glucose (YG) or absence (Y). The presence or absence of nitrate did not affect these results. 6 Any modification of this procedure was indicated for each experiment. Whenever necessary, media were supplemented with 30 g/ml L-leucine, 20 g/ml uracil, or 100 g/ml Zeocin.
Npr1 Tagging-To tag Npr1 with an HA epitope, we constructed the pHANPR1LEU2 plasmid following the strategy described previously (21). First, we cloned a PCR fragment containing the NPR1 promoter and the initiation codon in pGEM-T easy (Promega, Madison, WI). Then the HA-NPR1 open reading frame without initiation codon but with the NPR1 terminator was cloned in the plasmid derived from this first step. This NPR1-tagged version was made by PCR with a primer containing the HA sequence at the 5Ј end. The LEU2 gene, used as auxotrophic marker, was inserted into the last plasmid.
Nitrate Uptake-High affinity nitrate uptake was determined by measuring depletion of extracellular nitrate as described previously (9). To determine the nitrate concentration enzy-matically, the purified H. polymorpha nitrate reductase enzyme was used (NECi, Lake Linden, MI).
Gene Disruption-NPR1, END4, and VPS27 were disrupted by replacing the chromosomal copy by one containing the partially deleted gene target sequence flanking the H. polymorpha auxotrophic markers URA3 or LEU2. Further information on constructing vectors and cassettes is available on request.
-Protein Phosphatase Treatment-50 g of proteins was incubated with 150 units of -protein phosphatase (New England Biolabs, Beverly, MA) for 25 min at 30°C as indicated (12).
Fluorescence Microscopy-To follow Ynt1 cellular localization, Ynt1 was fused to GFP as described previously (11). Images were obtained under an epifluorescence Nikon Eclipse 50i microscope equipped with a 1200F camera (Nikon, Kanagawa, Japan). Fig. S1.

Npr1 Kinase Is Essential to Maintain Ynt1 Levels under Nitrogen Limitation Conditions-A previous characterization of H. polymorpha ⌬npr1 is shown in the supplemental
Under nitrogen limitation, nitrate uptake in ⌬npr1 mutant falls abruptly, unlike the WT that remains stable longer (Fig.  1A). In the same way, Ynt1 levels in ⌬npr1 are strongly correlated with those observed for nitrate uptake (Fig. 1B). Degradation of Ynt1 in ⌬npr1 was almost fully prevented by deleting the PEP4 gene that encodes for the vacuolar proteinase A (Fig. 1B). As expected, Ynt1-GFP co-localizes with the vacuole in ⌬npr1 cells subjected to nitrogen deprivation, whereas in a WT strain, Ynt1 remains at the cell surface even after 80 min (Fig. 1C). These results show that under nitrogen deprivation Npr1 is involved in regulating Ynt1 levels by controlling its delivery to the vacuole for degradation.
In a ⌬npr1 Mutant, Ynt1 Is Sorted to the Vacuole from Endocytic and Biosynthetic Pathways-The role of Npr1 in controlling the cellular fate of Ynt1 required elucidation, in addition to the pathways involved. For this, we disrupted genes involved in targeting proteins, such as END4 and VPS27, to the vacuole. END4 is involved in actin organization and plasma membrane endocytosis (35). In ⌬end4, Ynt1 endocytosis was almost abolished ( Fig. 2A). VPS27 is an endosomal protein required for recycling Golgi proteins, forming luminal membranes and sorting ubiquitinated proteins destined for degradation to the vacuole. As a result, ⌬vps27 mutation prevents endocytosis, trans-Golgi network, and resident vacuolar proteins from being delivered to the vacuole (36). When strains combining disruptions of NPR1 with END4 or VPS27 were incubated in nitratecontaining medium and then transferred to one deprived of nitrogen, we observed that Ynt1 disappearance was greater in the double mutant ⌬npr1⌬end4 than in ⌬end4 but slower than in ⌬npr1. However, it remained significantly slower than in WT ( Fig. 1B) or ⌬end4 strains ( Fig. 2A). These observations were consistent with those obtained by fluorescence microscopy of Ynt1-GFP. Indeed, in ⌬npr1 after 40 min of nitrogen deprivation, all the fluorescence was localized in the vacuole, whereas in the ⌬npr1⌬end4 strain most was vacuolar but some was still at the cell surface (Fig. 2B). These results suggest that, in ⌬npr1, Ynt1 is sorted to the vacuole from both the plasma membrane and the biosynthetic pathway. To further confirm this, we tested Ynt1 degradation in a ⌬npr1⌬vps27 strain in response to nitrogen deprivation. In contrast with ⌬npr1⌬end4, ⌬vps27 almost suppresses NPR1 deletion, and Ynt1 levels are much more stable than in ⌬npr1⌬end4 ( Fig. 2A). Therefore, in response to nitrogen-free conditions, Npr1 is required for Ynt1 delivery to the plasma membrane and its further stabilization there.
We then asked whether the action of Npr1 on Ynt1 is restricted to nitrogen deprivation or whether it also affects the rate of Ynt1 appearance in response to nitrate. To test this, we estimated the rate of delivery of Ynt1 to the plasma membrane upon nitrate incubation. The cells were subjected to nitrogen starvation and then incubated in 0.5 mM nitrate. Afterward, nitrate uptake rate was measured, as an index of that delivery rate, showing a rapid increase (Fig. 2C) in WT. In contrast, nitrate uptake was slower to appear in ⌬npr1 and ⌬npr1⌬end4, being slightly higher in this latter mutant due to the stability of the Ynt1 present at the plasma membrane, as a result of ⌬end4 mutation. The behavior of the Ynt1-GFP was consistent with that observed for nitrate uptake appearance, so after incubation in nitrate, Ynt1-GFP was at the cell surface in WT. However, in ⌬npr1 and ⌬npr1⌬end4 mutants, Ynt1-GFP cellular distribution was heterogeneous, with a high cytosolic accumulation (Fig. 2D). Therefore, our results indicate that in nitrate and nitrogen-free conditions, Npr1 prevents sorting of newly synthesized Ynt1 to the vacuole, facilitating its delivery to the plasma membrane.
Phosphorylation of Ynt1 Is Mediated by Npr1-Nitrogen limitation induced phosphorylation of Ynt1 at Ser residues 244 and 246, located in the central hydrophilic domain. Under nitrogen limitation, a nonphosphorylatable form of Ynt1 (S244A and S246A) is missorted to the vacuole (12). The similarity in behavior of the nonphosphorylatable Ynt1 with ⌬npr1 prompted us to study the relationship between Ynt1 phosphorylation and Npr1 in detail. For this, we compared the levels of Ynt1 under nitrogen deprivation in ⌬npr1 and the Ynt1 2S3 A mutant. In both strains, Ynt1 disappearance (Fig. 3A) and the drop in nitrate uptake were very rapid. Ynt1-GFP was localized in the vacuole after a few minutes (data not shown). We then studied the phosphorylation state of Ynt1 in a ⌬npr1 mutant. For this, we disrupted the nitrate reductase gene (YNR1) (37) in a ⌬npr1 strain. The ⌬ynr1 mutant was unable to assimilate nitrate, which acts only as inducer and as a result ensures high levels of phosphorylated Ynt1. Immunoblots showed the presence of a slow mobility band of Ynt1 in ⌬ynr1, not detected in ⌬npr1⌬ynr1 (Fig. 3B). -Protein phosphatase treatment of total protein extracts confirmed that this band corresponds to a phosphorylated form of Ynt1 (Fig. 3B), allowing us to conclude that Npr1 is necessary for Ynt1 phosphorylation.
Further evidence on the role of Npr1 in Ynt1 phosphorylation was obtained in strains bearing several copies of NPR1 (nNPR1). In this strain, Ynt1 is phosphorylated even in gluta-

Npr1 Protein Kinase Regulates Ynt1 Levels
mine or high nitrate, unlike in WT where it is scarcely or not phosphorylated (Fig. 3C). To rule out this being a consequence of increased Ynt1 levels, the strain bearing the Ynt1 3K3 R version was used, which accumulated Ynt1, because it lacks the lysine residues required for efficient ubiquitinylation and further endocytosis. Moreover, it is worth noting that in a WT genetic background, Ynt1 3K3 R is phosphorylated like Ynt1 during nitrogen deprivation (data not shown). These results further support the involvement of Npr1 in Ynt1 phosphorylation.
The high levels of Ynt1 phosphorylation in the nNPR1 strain also allow us to determine the residues involved in its phosphorylation mediated by Npr1. We already showed that Ser-244 and Ser-246 are phosphorylated in response to nitrogen deprivation (12). Ynt1-phosphorylated forms were not detected in either Ynt1 2S3 A or in nNPR1Ynt1 2S3 A strains, but they were in nNPR1Ynt1 (Fig. 3D). We concluded that Ynt1 phosphorylation at Ser-244 and Ser-246 is mediated directly or indirectly by Npr1.
Npr1 Regulates Ynt1 Ubiquitinylation-We reasoned that if the effect of NPR1 disruption on Ynt1 was due entirely to the absence of Ynt1 phosphorylation, then replacing the serine phosphoacceptors by glutamic acid to mimic phosphorylation would reverse it. However, Ynt1 behaved the same in both ⌬npr1 and ⌬npr1 Ynt1 2S3 E strains under nitrogen limitation conditions, indicating that NPR1 deletion acts beyond Ynt1 phosphorylation (Fig. 4A). Despite this, in a WT genetic background, Ynt1 2S3 E reversed Ynt1 2S3 A in such a way that Ynt1 2S3 E levels were almost unaltered under nitrogen limitation, unlike Ynt1 2S3 A (12). In ⌬npr1, Ynt1 degradation was nevertheless counteracted when the Lys residues involved in Ynt1 ubiquitinylation (11) were mutated to Arg (Fig. 4A). This finding suggested a close relationship between Ynt1 ubiquitinylation and Npr1. To test this, the ubiquitinylated forms of Ynt1 in a ⌬npr1 mutant were determined by the use of ⌬npr1⌬ynr1 bearing the Ynt1 ⌬232-286 -Ct236 -286 (Ynt1-Ct) mutation, achieved by fusing the core sequence (236 -286) of the central cytosolic domain to the C terminus of Ynt1 ⌬232-286 (11). The mutation of the nitrate reductase gene (YNR1) ensures high levels of Ynt1, whereas Ynt1-Ct provides a robust Ynt1-ubiquitinylation assay (11). When the cells were incubated in nitrate, the ⌬npr1 mutant showed an increased amount of Ynt1 ubiquitin conjugates, as seen in WT cells after addition of glutamine. The nonphosphorylatable form of Ynt1 (Ynt1 2S3 A ) exhibited the same ubiquitinylation as the WT (Fig.  4B). These results indicate that Npr1 down-regulates the ubiquitinylation of Ynt1, independently of Ynt1 phosphorylation.
We then asked whether NPR1 in multicopy (nNPR1) further stabilizes Ynt1 by lowering the amount of ubiquitin conjugates. This was assayed on Ynt1 down-regulation in response to glutamine. In WT, the amount of Ynt1 falls abruptly, but in an nNPR1 it did not (Fig. 4C). Examination of Ynt1 ubiquitinylation in WT and the nNPR1 mutant revealed that the Ynt1 ubiquitin conjugates could not be detected in the nNPR1 strain, even after the addition of glutamine (Fig. 4D). These results support a close relationship between Ynt1 ubiquitinylation and Npr1. However, it is worth noting that the ubiquitinylation is independent of Ynt1 phosphorylation.
To gain further insights into the relationship between Npr1 and Rsp5, we constructed a loss of function rsp5-1 temperature-sensitive mutant strain (38). Most plasma membrane proteins in yeast undergo Rsp5-mediated ubiquitinylation and subsequent internalization (39). However, in rsp5-1, Ynt1 down-regulation was not affected (supplemental Fig. S2), possibly because the ubiquitinylation activity remaining in this temperature-sensitive mutant (rsp5-1) is enough to  ubiquitinylate Ynt1 and deliver it to the vacuole for degradation. In contrast, Gap1 (supplemental Fig. S1) and Hak1 7 increase their stability in response to preferred nitrogen sources or K ϩ in rsp5-1, confirming the phenotype of this mutant. Similarly, in the S. cerevisiae RSP5 conditional mutants rsp5-1 and spa1-1, the high affinity iron transporter Fet1-Fet3 and P i transporter Pho84 are unaffected in response to iron or P i abundance, respectively (40,41).
Ynt1 Levels Are Also Regulated by the Carbon Sources and Dependent on Npr1-It has been shown in our laboratory that, in addition to the nitrogen source, YNT1 gene expression is regulated by the carbon source. 8 Here, we investigated whether this regulation also takes place at the post-translational level. We have found that carbon starvation also down-regulates Ynt1 (Fig. 5A), which correlates with a fast dephosphorylation of Ynt1 (Fig. 5B), as occurs with glutamine (12). The presence of different carbon sources, such as 0.5% glucose, sucrose, ethanol, and glycerol, restores the levels of Ynt1 (Fig. 5C). Therefore, we concluded that carbon starvation down-regulates Ynt1 levels. We then asked if Npr1 is also involved in regulating Ynt1 levels in a medium lacking a carbon source. To test this, we compared the levels of Ynt1 in WT and in nNPR1 strains transferred to a carbon-free medium. In the nNPR1 strain, Ynt1 was clearly resistant to degradation (Fig. 5D), indicating that Npr1 is involved in adapting Ynt1 levels to carbon source availability. To further analyze the role of Ynt1 ubiquitinylation in response to carbon starvation, the strain bearing Ynt1-Ct was used. Ynt1 appears clearly ubiquitinylated in response to carbon starvation (Fig. 5E); however, ubiquitinylation disappears when NPR1 is overexpressed. Altogether our results clearly indicate that Npr1 also plays a key role in adapting Ynt1 levels to carbon source availability, acting on Ynt1 via its phosphorylation and ubiquitinylation.
We also explored whether calcineurin could play some role in signaling the effect of nitrogen and carbon sources on Ynt1 post-translational regulation. To address this, the strain ⌬cnb1 lacking the regulatory subunit of calcineurin was used (42). In the conditions studied, the Ynt1 phosphorylation or degradation pattern shows no significant differences between ⌬cnb1 and WT (supplemental Fig. S3).
Npr1 Phosphorylation State Is Regulated by Nitrogen Source Quality, Carbon Source, and K ϩ Availability-The effect of Npr1 on post-translational regulation of Ynt1 was clearly observed under nitrogen limitation and nitrate and carbon starvation. We also studied the Npr1 phosphorylation state to correlate it with its involvement in Ynt1 regulation, using a strain bearing a functional N-terminal HA-tagged Npr1. As shown in

. Ynt1 is down-regulated by carbon starvation, and this is counteracted by Npr1 overexpression.
A, Ynt1 is down-regulated after carbon withdrawal. Cells were incubated in YNB, 2% glucose, and 5 mM nitrate for 2 h and then transferred to the same medium without nitrogen, with 2% glucose (YG) and without (Y). B, in the absence of a carbon source, Ynt1 is rapidly dephosphorylated. Ynt1 SDS-PAGE mobility shift was determined in protein extracts from cells incubated for 2 h in 5 mM nitrate then transferred for 5 min to fresh medium with 2% glucose (YG) and without (Y). C, Ynt1 levels in cells incubated in different carbon sources. Cells were incubated in YNB plus 5 mM nitrate and 2% glucose for 2 h and then transferred to the same medium with 5 mM nitrate and the carbon sources indicated. D, overexpression of Npr1 stabilizes Ynt1 even in carbon-free medium. E, Ynt1 ubiquitinylation in a strain overexpressing NPR1. After 2 h of incubation in 5 mM nitrate, cells were nitrogen-deprived for 30 min and then transferred to a medium with no carbon source (Y, 0 min). Fig. 6A, the SDS-PAGE mobility of Npr1 depends on the nitrogen source. Thus, Npr1 from cells incubated in glutamine presented less mobility than that from cells incubated in nitrate or nitrogen-free media. -Protein phosphatase treatment of cell extracts confirmed the phosphorylation of Npr1 in response to glutamine. Moreover, this treatment of cell extract from nitro-gen-deprived cells revealed that Npr1 migrated faster than when not treated (Fig. 6B). This suggests that Npr1 may be phosphorylated in nonpreferred nitrogen sources, and addition of glutamine causes further phosphorylation. A close correlation was observed between glutamine-dependent Npr1 phosphorylation and Ynt1 dephosphorylation, such as in a ⌬npr1 mutant (Fig. 6A). Therefore, the capacity of Npr1 to directly or indirectly phosphorylate Ynt1 is regulated by the nitrogen sources, such that when Npr1 was phosphorylated no Ynt1 phosphorylation was observed.
The effect of rapamycin on the phosphorylation state of Npr1 and Ynt1 levels was tested by incubating H. polymorpha in glutamine plus 0.5-3 M rapamycin. Npr1 mobility was not affected while Ynt1 was being degraded, unlike those cells transferred to N-free medium (Fig. 6, C and D). Rapamycin mimicked nitrogen derepression for those genes subject to nitrogen catabolite repression (6). These results contrast with those obtained for ScNpr1, where rapamycin strongly modifies the nitrogen-dependent phosphorylation pattern of Npr1 (18).
As Npr1 is modulated by phosphorylation, we studied whether Npr1 was affected by carbon starvation. In cells subjected to carbon starvation, Npr1 was found to be phosphorylated (Fig. 7A), although less so than in glutamine (Fig. 6B), which strongly suggested that Npr1 is inactivated by phosphorylation. Thus, when Npr1 is phosphorylated, or disrupted, Ynt1 is not. However, Npr1 phosphorylation could also be involved in recognizing and binding its substrates. In contrast to what was observed in glutamine, 1 M rapamycin prevents Ynt1 degradation in response to carbon starvation (Fig. 7C). Consistent with this, rapamycin also prevented phosphorylation of Npr1 in carbon starvation (Fig. 7D), as well as Ynt1 dephosphorylation (Fig. 7E). These results suggest that Npr1 responds to carbon starvation in a TOR pathway-dependent manner.
We also explored whether K ϩ levels also affect Npr1 phosphorylation and Ynt1 levels. K ϩ has been found to be involved FIGURE 6. Nitrogen-dependent phosphorylation of Npr1. A, HA-Npr1 (upper panel) and Ynt1 (lower panel) mobility in SDS-PAGE using anti-HA and anti-Ynt1. Cells were incubated in 5 mM nitrate for 1.5 h and then transferred to a nitrogen-free medium or 5 mM glutamine for 1 h. B, HA-Npr1 SDS-PAGE mobility changes in response to different nitrogen sources are due to phosphorylation. A WT strain expressing HA-Npr1 was grown in ammonium and then transferred to glutamine or N-free medium for 1 h. Protein extracts were treated (ϩ) or nontreated (Ϫ) with -protein phosphatase. C, effect of rapamycin (rap) on the phosphorylation state of Npr1. Cells were collected after incubation in the indicated medium for 1 h, and HA-Npr1 was detected by immunoblot. D, effect of rapamycin treatment on glutamine-induced downregulation of Ynt1. WT was incubated in nitrate and then transferred to a medium without nitrogen or with 5 mM glutamine plus 0.5 or 3 M rapamycin, for 40 min. Pma1 was used as loading control. FIGURE 7. Carbon-dependent Npr1 and Ynt1 regulation is rapamycin-sensitive. A, HA-Npr1 SDS-PAGE mobility in carbon-starved cells. Cells were incubated in 5 mM nitrate for 1 h and then transferred to the same medium without nitrogen with 2% glucose (YG) or without (Y). B, HA-Npr1 SDS-PAGE mobility in extracts from carbon-starved cells treated with -protein phosphatase (ϩ) and not treated (Ϫ). C, rapamycin (Rap) prevents Ynt1 down-regulation in response to carbon starvation. Cells incubated in 5 mM nitrate for 2 h were transferred to the same medium without nitrogen and glucose (Y) in the presence and absence of 1 M rapamycin. D, rapamycin prevents Npr1 phosphorylation. HA-Npr1 SDS-PAGE mobility shift was determined in protein extracts from cells incubated in 5 mM nitrate for 1 h then transferred to fresh nitrogen-free medium in the presence of 2% glucose or its absence, with and without 1 M rapamycin. E, rapamycin prevents Ynt1 dephosphorylation in response to carbon starvation. Cells incubated for 2 h in 5 mM nitrate were transferred to nitrogen-free medium at the condition indicated for 5 min. Rapamycin was used at 1 M. AUGUST 5, 2011 • VOLUME 286 • NUMBER 31 in the stabilization of several permeases at the plasma membrane (34). A clear increase in the phosphorylation of Npr1 was observed in the absence of K ϩ but only in preferred nitrogen sources such ammonium or glutamine (Fig. 8, A and B). This makes it difficult to see the effect on Ynt1, because Ynt1 is only present in cells incubated in nitrate. However, because we have consistently observed that changes in Npr1 phosphorylation affect that of Ynt1 and its levels, we studied the effect of K ϩ absence on Ynt1. No significant changes were observed in Ynt1 by Western blotting, although nitrate uptake decreased (Fig.  8C), and this alteration in uptake could be due in part to electrochemical potential change in the absence of K ϩ . Indeed, addition of 20 mM KCl restored nitrate transport. We concluded that the absence of K ϩ alters the Npr1 phosphorylation state in preferred nitrogen sources; nevertheless, this condition does not seem to affect Ynt1 but does influence nitrate uptake. In Fig. 9, we summarize our current knowledge about the role of Npr1 in regulating Ynt1.

DISCUSSION
In this study, we uncover the role of Npr1 in regulating the Ynt1 levels in response to nitrogen source quality and carbon availability. This yeast protein kinase belongs to a subgroup involved in the regulation of several permeases (15,17,34,43). Hp⌬npr1 showed deficient growth in low ammonium or other nitrogen sources such as proline. However, contrary to expectations, Gap1 levels are not affected in the ⌬npr1 strain, indicating that this protein kinase is not involved in Gap1 activation in H. polymorpha (supplemental Fig. S1). Nevertheless, the poor growth of ⌬npr1 in ammonium suggests that ammonium permeases are also under control of H. polymorpha Npr1. The regulatory roles of HpNpr1 do not fully coincide with those observed in S. cerevisiae. Consistent with this, we found that  . Npr1 is involved in sorting and stabilizing Ynt1 at the plasma membrane. In preferred nitrogen sources or carbon starvation, Npr1 is polyphosphorylated, although Ynt1 is not phosphorylated, being therefore sorted via late endosome/multivesicular body (MVB) to the vacuole from the trans-Golgi network (TGN) (gray arrows). Under these conditions, Ynt1 located at the cell surface is ubiquitinylated (Ub), endocytosed, and then degraded in the vacuole. In contrast, in nitrate or nitrogen-free (N-free) medium, in the presence of a carbon source, Npr1 is dephosphorylated, although Ynt1 is phosphorylated and highly stable (black arrows). ⌬npr1 presents a high sensitivity to toxic cations such as Li ϩ or hygromycin B. 7 We observed that Npr1 is essential to maintain Ynt1 levels in nitrate and under nitrogen deprivation. Using the ⌬end4 and ⌬vps27 mutants involved in intracellular protein traffic, we showed that in the strain lacking Npr1, Ynt1 was routed to the vacuole from the later biosynthetic pathways and the plasma membrane. Therefore, Npr1 is involved in regulating Ynt1 levels by acting on both de novo synthesized Ynt1 present in the secretion pathway and that already in the plasma membrane. Furthermore, the following two experiments shed light on the poor growth of ⌬npr1 in low nitrate; (i) the amount of Ynt1 was found to be lower in ⌬npr1 after incubation in nitrate for 2 h, and (ii) the kinetics of Ynt1 reaching the plasma membrane was slower in ⌬npr1 than in WT (Fig. 2C).
The negative effect observed in ⌬npr1 on the levels of Ynt1 raises the question of the molecular mechanisms involved. The fact that Ynt1 was rapidly degraded in ⌬npr1 at the same rate as the unphosphorylatable Ynt1 2S3 A form strongly suggests that Npr1 is involved in phosphorylation of Ynt1. The use of strains overexpressing or lacking Npr1 clearly reveals that Npr1 is involved in the observed phosphorylation of Ynt1, which in turn takes place at Ser-244 and Ser-246. However, unlike CHL1 (7,8,44), Ynt1 phosphorylation does not seem to be involved in nitrate affinity changes or nitrate signaling. Overexpression of Npr1, by increasing the NPR1 copy number, causes an atypical phosphorylation of Ynt1, which takes place even in good nitrogen sources, unlike WT (Fig. 3C). This phosphorylation is not due to greater Ynt1 stability, because a highly stable nonubiquitinatable form of Ynt1, namely Ynt1 3K3 R (11), was not phosphorylated in good nitrogen sources, unlike nNPR1 strain. Our data contrast with those observed in S. cerevisiae Gap1, which is unphosphorylated and unstable in ⌬npr1; however, the increased stability of Gap1 because of RSP5 deletion causes the reappearance of Gap1 phosphorylation (15). This indicates that Npr1 phosphorylation is indirect. Using synthetic peptides, a (K/R)XXS(K/R) consensus sequence for the Npr1 phosphorylation site was found (45). This site is present in Ynt1 (Ser-246). However, in S. cerevisiae, Bul proteins, components of the RSP5 ubiquitin ligase complex, contain several perfect Npr1 consensus phosphorylation motifs (Bul1, 51 KGSSR 55 , 319 KVKSK 323 , and 849 KQHSK 853 , and Bul2, 446 KRTSK 450 ) (45). Moreover, in vitro and in vivo findings strongly suggest that the phosphorylation state and activity of the ␣-arrestin Aly2, involved in the intracellular traffic of Gap1, are directly regulated by Npr1 (46). Therefore, despite the presence of a putative Npr1 phosphorylation site in Ynt1, it seems that in general Npr1 does not phosphorylate the target permease it regulates.
Concerning the relationship between Npr1 and Ynt1 ubiquitinylation, the disruption effect of NPR1 was not reversed by Ynt1 2S3 E (Fig. 4A), in which phosphorylation is functionally and constitutively mimicked by mutating Ser residues to Asp (12). This suggests that Npr1 acts beyond Ynt1 phosphorylation and that Npr1 is probably not directly involved in Ynt1 phosphorylation. In turn, the nonubiquitinatable Ynt1 3K3 R overcomes the effect of NPR1 deletion on Ynt1 degradation, pointing to a link between Npr1 and Ynt1 ubiquitinylation (Fig.  4A). Consistent with this, deletion of NPR1 causes an increment in the Ynt1 ubiquitin conjugates, even in nitrogen starvation where Ynt1 is highly stable (Fig. 4B). Furthermore, overexpression of NPR1 protects Ynt1 from ubiquitinylation, even in good nitrogen sources. As our results show that the increase in Npr1 led to high Ynt1 phosphorylation and low ubiquitinylation, and completely the opposite when NPR1 was disrupted, at first glance it could be thought that phosphorylation blocks ubiquitinylation of Ynt1. However, we have not found any evidence for this. In fact, the opposite seems to be the case, because Ynt1 2S3 E was degraded in a ⌬npr1 mutant (Fig. 4A), unlike the WT strain, and the nonphosphorylatable form of Ynt1 2S3 A does not increase ubiquitinylation (Fig. 4B). Therefore, it can be concluded that the phosphorylation state of Ynt1 itself is not involved in regulating its ubiquitinylation, suggesting that Npr1 could be phosphorylating some auxiliary protein such as arrestin-related or ubiquitin-ligase adaptors or Bul1 or Bul2 (45)(46)(47), which could be hindering Ynt1 ubiquitinylation. The involvement of Npr1 in Ynt1 ubiquitinylation raises the question of the role of the ubiquitin ligase Rsp5 in this process. However, a thermo-sensitive rsp5-1 mutant in H. polymorpha affected Gap1 (supplemental Fig. S2) and the high affinity K ϩ transporter Hak1 degradation 6 but not Ynt1. To rule out the role of Rsp5 in Ynt1 ubiquitinylation, further experiments should be undertaken. DOA4 encodes for a deubiquitinylating enzyme required for ubiquitin homeostasis in yeast (48,49); its disruption does not affect Ynt1 down-regulation, unlike Gap1 (supplemental Fig. S2). However, as above, we cannot rule out the possibility of Ynt1 undergoing ubiquitinylation in ⌬doa4.
Carbon starvation also leads to a rapid decrease of Ynt1 levels. Most likely this obeys a general program to adapt cell enzymatic machinery to the adverse nutritional state. In S. cerevisiae, carbon or nitrogen starvation induces degradation of the tryptophan permease Tat2 and uracil permease Fur4 (23,24). Rapamycin also produces Tat2 degradation (23). We should highlight that although the precise reason why Ynt1 is degraded in response to carbon starvation is uncertain, apparently the mechanism involved is the same as when cells are transferred from nitrate to glutamine (11).
We also studied Npr1 phosphorylation in response to nitrogen sources and carbon and K ϩ starvation. In glutamine or other preferred nitrogen sources and in carbon starvation, Npr1 is highly phosphorylated, although in nitrogen limitation conditions or in nonpreferred nitrogen sources this phosphorylation almost disappears. In contrast to Npr1, Ynt1 was phosphorylated in nitrogen-free medium and nonphosphorylated in good nitrogen sources and carbon starvation (Figs. 6 and 7). In S. cerevisiae, Npr1 phosphorylation is dependent on the TOR signaling pathway; thus, rapamycin blocks this phosphorylation in good nitrogen sources (21,28). However, HpNpr1 phosphorylation in good nitrogen sources is not affected by rapamycin, despite it causing de-repression of the genes subjected to nitrogen catabolite repression (6). It also does not block Ynt1 degradation in response to glutamine (Fig. 6C). However, the effect of rapamycin on plasma membrane permeases is controversial. It has been shown that Gap1 is stable in rapamycin (23). However, when cells are grown in a sublethal concentration of rapamycin, Gap1 levels were greatly reduced, and this is explained by the increase in the internal concentration of amino acids (50). A striking difference in the response of Npr1/ Ynt1 to carbon starvation, unlike glutamine addition, is that rapamycin prevents Npr1 dephosphorylation, leading to a lower Ynt1 degradation rate. Therefore, it seems that in H. polymorpha the intracellular traffic of Ynt1 elicited by carbon starvation is regulated by the rapamycin-sensitive TOR complex. Nevertheless, it cannot be ruled out that an insensitive TOR complex could also be involved in the case of glutamine. Alternatively, under nutrient stress, like that produced by carbon starvation, rapamycin-sensitive TOR complex could be more essential for endocytosis and protein trafficking, thus affecting Ynt1 degradation (29). It is noteworthy that under these conditions, HpNpr1 phosphorylation is also impeded by rapamycin (Fig. 7D). In preferred nitrogen sources, Npr1 responded to K ϩ starvation with a higher degree of phosphorylation (Fig. 8), although no changes were observed in nonpreferred nitrogen sources. However, we observed no significant changes either in phosphorylation or stability of Ynt1. However, in K ϩ starvation, nitrate uptake was significantly reduced (Fig. 8). This could be due in part to the plasma electrochemical potential alteration, although a sound conclusion on the effect of K ϩ starvation on Ynt1 would need further research.
Another unexpected result was that during nitrogen deprivation conditions, Npr1 shows a basal phosphorylation that only disappears when protein extracts are treated with phosphatase (Fig. 6B). This could indicate that some of this Npr1 phosphorylation is nitrogen source-independent, which could explain some phenotypes of the ⌬npr1 mutant not being related to the nitrogen sources, such as Li ϩ and hygromycin sensitivity. 7 Npr1 phosphorylation events, up to 22 in S. cerevisiae (28), could be involved in regulating Npr1 activity and also in the recognition, binding, and phosphorylation of a broad set of substrates. Consistent with this, in the Saccharomyces Genome Data base, up to 67 records appear so far for NPR1 genetic or physical interactions.
The current knowledge about the regulation of the yeast high affinity nitrate/nitrite transporter Ynt1 is shown in Fig. 9. In summary, Npr1 plays a pivotal role in regulating Ynt1 levels according to nitrogen and carbon availability.