José M. Siverio

Functional properties and differential mode of regulation of the nitrate transporter from a plant symbiotic ascomycete

Nitrogen assimilation by plant symbiotic fungi plays a central role in the mutualistic interaction established by these organisms, as well as in nitrogen flux in a variety of soils. In the present study, we report on the functional properties, structural organization and distinctive mode of regulation of TbNrt2 (Tuber borchii NRT2 family transporter), the nitrate transporter of the mycorrhizal ascomycete T. borchii. As revealed by experiments conducted in a nitrate-uptake-defective mutant of the yeast Hansenula polymorpha, TbNrt2 is a high-affinity transporter (K(m)=4.7 microM nitrate) that is bispecific for nitrate and nitrite. It is expressed in free-living mycelia and in mycorrhizae, where it preferentially accumulates in the plasma membrane of root-contacting hyphae. The TbNrt2 mRNA, which is transcribed from a single-copy gene clustered with the nitrate reductase gene in the T. borchii genome, was specifically up-regulated following transfer of mycelia to nitrate- (or nitrite)-containing medium. However, at variance with the strict nitrate-dependent induction commonly observed in other organisms, TbNrt2 was also up-regulated (at both the mRNA and the protein level) following transfer to a nitrogen-free medium. This unusual mode of regulation differs from that of the adjacent nitrate reductase gene, which was expressed at basal levels under nitrogen deprivation conditions and required nitrate for induction. The functional and expression properties, described in the present study, delineate TbNrt2 as a versatile transporter that may be especially suited to cope with the fluctuating (and often low) mineral nitrogen concentrations found in most natural, especially forest, soils.

The role of nitrate reductase in the regulation of the nitrate assimilation pathway in the yeast Hansenula polymorpha

The role of nitrate reductase (NR) in the regulation of the nitrate assimilation pathway was evaluated in the yeast Hansenula polymorpha. Posttranscriptional regulation of NR in response to reduced nitrogen sources and the effect of a heterologous NR on the transcriptional regulation of nitrate-assimilatory gene expression was examined. The strain bearing YNR1 (nitrate reductase gene) under the control of the methanol-induced MOX (methanol oxidase) promoter showed that NR is active in the presence of reduced nitrogen sources. In cells incubated with glutamine plus nitrate, rapamycin abolished nitrogen catabolite repression, NR activity being very similar to that in cells induced by nitrate alone. This reveals the involvement of the Tor-signalling pathway in the transcriptional regulation of H. polymorpha nitrate assimilation genes. To assess the role of NR in nitrate-assimilatory gene expression, different strains lacking YNR1, or both YNR1 and YNT1 (high-affinity nitrate transporter) genes, or expressing the tobacco NR under the YNR1 promoter, were used. Tobacco NR abolished the constitutive nitrate-assimilatory gene induction shown by an NR gene disruptant strain. Moreover, in strains lacking the high-affinity nitrate transporter and NR this deregulation disappeared. These facts discard the role of NR protein in the transcriptional induction of the nitrate-assimilatory genes and point out the involvement of the high-affinity nitrate transporter as a part of the nitrate-signalling pathway.

The role of Ynt1 in nitrate and nitrite transport in the yeast Hansenula polymorpha

Ynt1 is the only high‐affinity nitrate uptake system in Hansenula polymorpha. Nitrate uptake was directly correlated with the Ynt1 levels and shown to be independent of nitrate reductase (NR) activity levels. Ynt1 failed to transport chlorate and, as a result, strains lacking YNT1 were sensitive to chlorate, as is the wild‐type. Nitrite uptake in a wild‐type strain was partially inhibited by nitrate to levels shown by a YNT1‐disrupted strain in which, in turn, nitrite transport was not inhibited by nitrate. It is concluded that nitrite uptake takes place by two different transport systems: Ynt1 and a nitrite‐specific transporter(s). The nitrite‐specific transport system was induced by nitrate; consistently, no induction was observed in strains lacking the transcription factor YNA1, which is involved in nitrate and nitrite induction of the nitrate assimilatory structural genes. Ynt1 presents its optimal rate for nitrite uptake at pH 6, while pH 4 was optimal for the specific nitrite uptake system(s). At pH 5.5, the contribution of Ynt1 to high‐affinity nitrate and nitrite uptake was around 95% and 60%, respectively. The apparent Km of Ynt1 for nitrate and nitrite is in the µM range, as is the specific nitrite uptake system for nitrite. The analysis of the effect of the reduced nitrogen sources on nitrate assimilation revealed that glutamine inactivates nitrate and nitrite transport, dependent on Ynt1, but not the nitrite‐specific system. Copyright

One-step, PCR-mediated, gene disruption in the yeast Hansenula polymorpha.

Previous evidence based on the experience of our laboratory showed that one‐step gene disruption in the yeast Hansenula polymorpha is not straightforward. A systematic study of several factors which could affect gene disruption frequency was carried out. We found that the more critical factor affecting one‐step gene disruption in H. polymorpha is the length of the target gene region flanking the marker gene. Target gene regions of about 1 kb flanking the marker gene were necessary to obtain a disruption frequency of about 50%. However, the gene marker, either homologous or heterologous, the locus and the strain examined did not significantly affect the frequency of disruption; the highest disruption frequency obtained for the YNR1 gene was in the strain HMI39, using the Saccharomyces cerevisiae URA3 gene as a marker. Since long regions flanking the gene marker do not allow the easy PCR‐mediated strategies, developed for S. cerevisiae, to obtain constructs to disrupt a given gene in H. polymorpha, an alternative PCR strategy was developed. Copyright

Down-regulation of Eukaryotic Nitrate Transporter by Nitrogen-dependent Ubiquitinylation

In the yeast Hansenula polymorpha, the YNT1 gene encodes the high affinity nitrate transporter, which is repressed by reduced nitrogen sources such as ammonium or glutamine. Ynt1 protein is degraded in response to glutamine in the growth medium. Ynt1 disappears independently of YNT1 glutamine repression as shown in strains where YNT1 repression is abolished. Ynt1-green fluorescent protein chimera and a mutant defective in vacuolar proteinase A (Δpep4) showed that Ynt1 is degraded in the vacuole in response to glutamine. The central hydrophilic domain of Ynt1 contains PEST-like sequences whose deletion blocked Ynt1 down-regulation. Site-directed mutagenesis showed that Lys-253 and Lys-270, located in this sequence, were involved in internalization and subsequent vacuolar degradation of Ynt1. Ynt1-ubiquitin conjugates were induced by glutamine and not nitrate. We conclude that glutamine triggers Ynt1 down-regulation via ubiquitinylation of lysines in the central hydrophilic domain, and proteolysis in the vacuole.

A second Zn(II)2Cys6 transcriptional factor encoded by the YNA2 gene is indispensable for the transcriptional activation of the genes involved in nitrate assimilation in the yeast Hansenula polymorpha

Nitrate assimilation genes encoding a nitrate transporter (YNT1), nitrite reductase (YNI1), a Zn(II)2Cys6 transcriptional factor involved in nitrate induction (YNA1) and the nitrate reductase (YNR1) are clustered in the yeast Hansenula polymorpha. A second gene, termed YNA2 (yeast nitrate assimilation), was located seven nucleotides away from the 3′ region of YNR1 gene. The cluster is flanked by an ORF encoding a protein with similarity to glutathione‐S‐transferase on the YNT1 side and an ORF with similarity to Saccharomyces cerevisiae Rad3p on the YNA2 side. The disruption of YNA2 confers the resulting null mutant strain with inability to grow in nitrate. The YNA2 gene encodes a putative protein of 618 residues bearing in the N‐terminus the consensus sequence Cys–X2–Cys–X6–Cys–X5–16–Cys–X2–Cys–X6–8–Cys characteristic of the Zn(II)2Cys6 transcriptional factors. YNA2 is therefore a member of the H. polymorpha nitrate assimilation gene cluster which is transcribed in the opposite direction to the rest of the members. Yna2p shares about 27% similarity with the H. polymorpha Yna1p Zn(II)2Cys6 transcriptional factor involved in nitrate induction. Unlike the wild‐type, the yna2::URA3 strain showed no expression of the nitrate assimilation genes when incubated in nitrate for 2 h. With regard to YNA2 expression, similar YNA2 transcript levels were observed in ammonium and in ammonium plus nitrate, but about a four‐fold higher expression was observed in nitrate. However, this induction by nitrate of the YNA2 gene was not observed in the Δyna1::URA3 strain. On the contrary, the pattern of YNA1 expression was the same in the wild‐type as in the yna2::URA3 strain, indicating that YNA2 does not affect YNA1 expression. The nucleotide sequence Accession No. for YNA2 is AJ223294. Copyright

Phosphorylation of the Yeast Nitrate Transporter Ynt1 Is Essential for Delivery to the Plasma Membrane during Nitrogen Limitation

Ynt1 is the sole high affinity nitrate transporter of the yeast Hansenula polymorpha. It is highly regulated by the nitrogen source, by being down-regulated in response to glutamine by repression of the YNT1 gene and Ynt1 ubiquitinylation, endocytosis, and vacuolar degradation. On the contrary, we show that nitrogen limitation stabilizes Ynt1 levels at the plasma membrane, requiring phosphorylation of the transporter. We determined that Ser-246 in the central intracellular loop plays a key role in the phosphorylation of Ynt1 and that the nitrogen permease reactivator 1 kinase (Npr1) is necessary for Ynt1 phosphorylation. Abolition of phosphorylation led Ynt1 to the vacuole by a pep12-dependent end4-independent pathway, which is also dependent on ubiquitinylation, whereas Ynt1 protein lacking ubiquitinylation sites does not follow this pathway. We found that, under nitrogen limitation, Ynt1 phosphorylation is essential for rapid induction of nitrate assimilation genes. Our results suggest that, under nitrogen limitation, phosphorylation prevents Ynt1 delivery from the secretion route to the vacuole, which, aided by reduced ubiquitinylation, accumulates Ynt1 at the plasma membrane. This mechanism could be part of the response that allows nitrate-assimilatory organisms to cope with nitrogen depletion.

Reversible inactivation and binding to mitochondria of nitrate reductase by heat shock in the yeast Hansenula anomala

Heat shock from 25°C to 40°C of Hansenula anomalacells resulted in a rapid and reversible inactivation of the NADPH‐nitrate reductase (NR) activity. The inactive enzyme retained partial activity with the non‐physiological co‐substrates, reduced methyl viologen and reduced flavin mononucleotide. The inactive NR pelleted after centrifugation at 12,000 × g for 30 min and was associated with mitochondria. In untreated cells around 10% of the total NR is inactive and associated with mitochondria, while the active enzyme is soluble. In vitro, inactive NR could be partially dissociated from the mitochondria by incubating them at pH 11.5 or in the presence of 15 mM CHAPS.

Effect of nitrogen source on the levels of nitrate reductase in the yeast Hansenula anomala.

Levels of nitrate reductase (NR) protein in Hansenula anomala and Hansenula wingei were determined using specific antiserum raised against the enzyme from H. anomala. Extracts from nitrate-grown cells contained NR protein, while in those from cells grown on ammonium, glutamine or peptone, no cross-reacting material could be observed. Enzyme activity correlated with the levels of cross-reacting material. When nitrate was used as nitrogen source, NR was always present, even in cultures with ammonium, glutamine or peptone, although in these cases both the levels of activity and protein were lower. NR activity was consistently two to four times higher in cells grown in glucose than in cells grown in ethanol. Nitrate was required for NR induction, and deprivation of nitrate from nitrate-grown cells resulted in a rapid loss of NR activity.

Molecular Components of Nitrate and Nitrite Efflux in Yeast

ABSTRACT Some eukaryotes, such as plant and fungi, are capable of utilizing nitrate as the sole nitrogen source. Once transported into the cell, nitrate is reduced to ammonium by the consecutive action of nitrate and nitrite reductase. How nitrate assimilation is balanced with nitrate and nitrite efflux is unknown, as are the proteins involved. The nitrate assimilatory yeast Hansenula polymorpha was used as a model to dissect these efflux systems. We identified the sulfite transporters Ssu1 and Ssu2 as effective nitrate exporters, Ssu2 being quantitatively more important, and we characterize the Nar1 protein as a nitrate/nitrite exporter. The use of strains lacking either SSU2 or NAR1 along with the nitrate reductase gene YNR1 showed that nitrate reductase activity is not required for net nitrate uptake. Growth test experiments indicated that Ssu2 and Nar1 exporters allow yeast to cope with nitrite toxicity. We also have shown that the well-known Saccharomyces cerevisiae sulfite efflux permease Ssu1 is also able to excrete nitrite and nitrate. These results characterize for the first time essential components of the nitrate/nitrite efflux system and their impact on net nitrate uptake and its regulation.