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Functional characterization of the Arabidopsis thaliana nitrate transporter CHL1 in the yeast Hansenula polymorpha

Plant Molecular Biology volume 68, Article number: 215 (2008) Cite this article

Abstract

CHL1 (AtNRT1.1) is a dual-affinity nitrate transporter of Arabidopsis thaliana, in which phosphorylation at Thr 101 switches CHL1 from low to high nitrate affinity. CHL1 expressed in a Hansenula polymorpha high-affinity nitrate-transporter deficient mutant (Δynt1) restores nitrate uptake and growth. These events take place at nitrate concentrations as low as 500 μM, suggesting that CHL1 has a high-affinity for nitrate in yeast. Accordingly, CHL1 expressed in H. polymorpha presents a K m for nitrate of about 125 μM. The absence of nitrate, the CHL1 gene inducer, showed the high turnover rate of CHL1 expressed in yeast, which is counteracted by nitrate CHL1 induction. Furthermore, H. polymorpha strains expressing CHL1 become sensitive to 250 μM chlorate, as expected for CHL1 high-affinity behaviour. Given that CHL1 presented high affinity by nitrate, we study the role of CHL1 Thr101 in yeast. Strains producing CHL1Thr101Ala, unable to undergo phosphorylation, and CHL1Thr101Asp, where CHL1 phosphorylation is constitutively mimicked, were used. Yeast strains expressing CHL1Thr101Ala, CHL1Thr101Asp and CHL1 at the same rate showed that Δynt1CHL1Thr101Ala is strikingly unable to transport nitrate and contains a very low amount of CHL1 protein; however, Δynt1CHL1Thr101Asp restores nitrate uptake and growth, although no significant changes in nitrate affinity were observed. Our results show that CHL1-Thr101 is involved in regulating the levels of CHL1 expressed in yeast and suggest that the phosphorylation of this residue could be involved in targeting this nitrate transporter to the plasma membrane. The functional expression of CHL1 in H. polymorpha reveals that this yeast is a suitable tool for evaluating the real nitrate transport capacity of plant putative nitrate transporters belonging to different families and study their regulation and structure function relationship.

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Acknowledgements

The authors thank Yin-Fang Tsay for CHL1 cDNA, site-point mutation constructs, anti-CHL1 antiserum, and critical reading of the manuscript. This work was supported by Grants BFU2004-01012 and BFU2007-60172/BMC to José M. Siverio from the Ministerio de Educación y Ciencia (MEC) Spain. Yusé Martín and Francisco J. Navarro were recipients of doctoral fellowships from the MEC. We thank Guido Jones for English revision of the manuscript and A. Lancha for her suggestions. The group is member of the Network for Cooperative Research on Membrane Transport Proteins (REIT), co-funded by the Ministerio de Educación y Ciencia, Spain and the European Regional Development Fund (ERDF, Grant BFU2005-24983-E/BFI).

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  1. Francisco J. Navarro

    Present address: Laboratory of Yeast Genetics and Cell Biology, The Rockefeller University, 1230 York Av., New York, NY, 10065, USA

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  1. Grupo del Metabolismo del Nitrógeno, Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Enfermedades Tropicales y Salud Pública, Universidad de La Laguna, 38206 La Laguna, Tenerife, Canarias, Spain

    Yusé Martín, Francisco J. Navarro & José M. Siverio

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  1. Yusé Martín
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  2. Francisco J. Navarro
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  3. José M. Siverio
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Correspondence to José M. Siverio.

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Martín, Y., Navarro, F.J. & Siverio, J.M. Functional characterization of the Arabidopsis thaliana nitrate transporter CHL1 in the yeast Hansenula polymorpha . Plant Mol Biol 68, 215 (2008). https://doi.org/10.1007/s11103-008-9363-z

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  • DOI: https://doi.org/10.1007/s11103-008-9363-z

Keywords

  • Nitrate transport
  • CHL1 (AtNRT1.1)
  • Arabidopsis thaliana
  • Yeast
  • Hansenula polymorpha
  • Ynt1