Tobacco Nia2 cDNA functionally complements a Hansenula polymorpha yeast mutant lacking nitrate reductase. A new expression system for the study of plant proteins involved in nitrate assimilation

Tobacco Nia2 cDNA functionally complements a Hansenula polymorpha yeast mutant lacking nitrate... An integrative expression vector based on promoter and terminator transcriptional sequences from the Hansenula polymorpha nitrate reductase gene (YNR1) has been developed to express nitrate assimilation plant genes in the nitrate assimilatory yeast H. polymorpha. Using this vector a plant nitrate reductase cDNA (tobacco Nia2) was expressed for the first time in a nitrate assimilatory yeast. The heterologous nitrate reductase produced retained its biochemical and physiological properties such as its NADH-dependent nitrate reductase activity, and allowed growth in nitrate containing media in a strain lacking endogenous nitrate reductase activity. In the transgenic strain, maximum tobacco nitrate reductase activity was about 70% of that presented in the wild-type. On the other hand, the disappearance of nitrate reductase activity correlated with that of the enzyme protein in response to the addition of ammonium to the medium and took place more rapidly in the transgenic strain than in the wild-type. Nitrate reductase activity of the recombinant strain assayed in the presence of Mg2+ was about 30% of that observed when assayed with EDTA. This result, together with a decreased growth rate in nitrate, suggests that tobacco nitrate reductase could be partially inactivated in H. polymorpha by phosphorylation and binding of 14-3-3-like proteins. These results show that H. polymorpha is a useful yeast heterologous expression system for studying plant proteins involved in nitrate assimilation. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Molecular Biology Springer Journals

Tobacco Nia2 cDNA functionally complements a Hansenula polymorpha yeast mutant lacking nitrate reductase. A new expression system for the study of plant proteins involved in nitrate assimilation

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Publisher
Kluwer Academic Publishers
Copyright
Copyright © 2002 by Kluwer Academic Publishers
Subject
Life Sciences; Biochemistry, general; Plant Sciences; Plant Pathology
ISSN
0167-4412
eISSN
1573-5028
D.O.I.
10.1023/A:1019814505677
Publisher site
See Article on Publisher Site

Abstract

An integrative expression vector based on promoter and terminator transcriptional sequences from the Hansenula polymorpha nitrate reductase gene (YNR1) has been developed to express nitrate assimilation plant genes in the nitrate assimilatory yeast H. polymorpha. Using this vector a plant nitrate reductase cDNA (tobacco Nia2) was expressed for the first time in a nitrate assimilatory yeast. The heterologous nitrate reductase produced retained its biochemical and physiological properties such as its NADH-dependent nitrate reductase activity, and allowed growth in nitrate containing media in a strain lacking endogenous nitrate reductase activity. In the transgenic strain, maximum tobacco nitrate reductase activity was about 70% of that presented in the wild-type. On the other hand, the disappearance of nitrate reductase activity correlated with that of the enzyme protein in response to the addition of ammonium to the medium and took place more rapidly in the transgenic strain than in the wild-type. Nitrate reductase activity of the recombinant strain assayed in the presence of Mg2+ was about 30% of that observed when assayed with EDTA. This result, together with a decreased growth rate in nitrate, suggests that tobacco nitrate reductase could be partially inactivated in H. polymorpha by phosphorylation and binding of 14-3-3-like proteins. These results show that H. polymorpha is a useful yeast heterologous expression system for studying plant proteins involved in nitrate assimilation.

Journal

Plant Molecular BiologySpringer Journals

Published: Oct 13, 2004

References

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