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

Abstract

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 K m 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.

Physiological and Biochemical Characterization of AnNitA, the Aspergillus nidulans High-Affinity Nitrite Transporter

“…A similar conclusion was arrived at from previous studies of nitrite uptake by Hansenula polymorpha (17) and A. nidulans (22). However, another possibility is that the conjugate acid of nitrite (nitrous acid), rather than the nitrite anion per se, might permeate the cell membrane to support growth.…”

NRT2.4 and NRT2.5 Are Two Half-Size Transporters from the Chlamydomonas NRT2 Family

“…The Hansenula strains correspond to WT strain (open circles), ∆ynt1 mutant (closed diamonds) and three CrNRT2.4::∆ynt1 transformants (L1-3) (closed circles, squares, and triangles, respectively). Nitrite uptake was performed at pH 6.5 from cells induced for 90 min in minimal medium without nitrogen [40]. Cell density was adjusted at 660 nm and corresponded to 10 mg/mL (fresh weight).…”

“…The ORF for NRT2.4 was cloned into the yeast pYNR vector and used to complement the Hansenula polymorpha ynt::URA3 Leu2 mutant. This mutant has no YNT1, the only NRT2 transporter described in this yeast, and is affected in nitrate and nitrite transport [40]. Three yeast transformants expressing the Chlamydomonas NRT2.4 (yntcrNRT2.4 L1-3) were assayed for nitrite transport activity under the conditions previously described [40].…”

“…This mutant has no YNT1, the only NRT2 transporter described in this yeast, and is affected in nitrate and nitrite transport [40]. Three yeast transformants expressing the Chlamydomonas NRT2.4 (yntcrNRT2.4 L1-3) were assayed for nitrite transport activity under the conditions previously described [40]. Yeast cells were induced for 90 min in YG medium without nitrogen, and then nitrite transport activity was measured at pH 6.5 and using 100 M nitrite.…”

“…Experiments were done at least by duplicate. The Hansenula polymorpha strains WT and ∆ynt1 mutant were previously described, as the conditions for growth and assay for nitrite transport activity [40].…”