Phosphorylation of the Yeast Nitrate Transporter Ynt1 Is Essential for Delivery to the Plasma Membrane during Nitrogen Limitation*
- Nitrogen Metabolism Group, Departamento de Bioquímica y Biología Molecular, Instituto de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de La Laguna, Astrofísico Fco. Sánchez s/n, E-38206 La Laguna, Tenerife, Canarias 38206, Spain
- 4 To whom correspondence should be addressed. Tel.: 34-922-318-406; Fax: 34-922-318-311; E-mail: jsiverio{at}ull.es.
Abstract
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.
Footnotes
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↵5 The abbreviations used are: Npr1, nitrogen permease reactivator 1 kinase; GFP, green fluorescent protein; TOR, target of rapamycin.
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↵6 J. A. K. W. Kiel, personal communication.
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This article is dedicated to Carlos Gancedo (IIB, Consejo Superior de Investigaciones Científicas, Madrid) for his long-standing support.
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↵* This work was supported in part by the Ministerio de Educación y Ciencia (MEC, Spain) (Grant BFU2007-60172/BMC to J. M. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 Both authors contributed equally to this work.
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↵2 Recipients of predoctoral fellowships from the MEC.
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↵3 Laboratory of Yeast Genetics and Cell Biology, The Rockefeller University, 1230 York Ave., New York, NY 10065.
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- Received March 19, 2008.
- Revision received August 14, 2008.
- The American Society for Biochemistry and Molecular Biology, Inc.
