Identification in vitro of a post-translational regulatory site in the hinge 1 region of arabidopsis nitrate reductase

Wenpei Su, Steven C. Huber, Nigel M. Crawford

Research output: Contribution to journalArticle

Abstract

Nitrate reductase (NR) is rapidly inactivated by phosphorylation of serine residues in response to loss of light or reduction in CO2 levels. To identify sites within NR protein that play a role in this post-translational regulation, a heterologous expression system and an in vitro inactivation assay for Arabidopsis NR were developed. Protein extracts containing NR kinases and inhibitor proteins were prepared from an NR-defective mutant that had lesions in both the NIA1 and NIA2 NR genes of Arabidopsis. Active NR protein was produced in a Pichia pastoris expression system. Incubation of these two preparations resulted in a Mg-ATP-dependent inactivation of NR that was reversed with EDTA. Mutant forms of NR were constructed, produced in P. pastoris, and tested in the in vitro inactivation assay. Six conserved serine residues in the hinge 1 region of NR, which separates the molybdenum cofactor and heme domains, were specifically targeted for mutagenesis because they are located in a potential regulatory region identified as a target for NR kinases in spinach. A change in Ser-534 to aspartate was found to block NR inactivation; changes in the other five serines had no effect. The aspartate that replaced Ser-534 did not appear to mimic a phosphorylated serine but simply prevented the NR from being inactivated. These results identify Ser-534, located in the hinge 1 of NR and conserved among higher plant NRs, as an essential site for post-translational regulation in vitro.

Original languageEnglish (US)
Pages (from-to)519-527
Number of pages9
JournalPlant Cell
Volume8
Issue number3
StatePublished - Mar 1 1996

Fingerprint

Nitrate Reductase
nitrate reductase
Arabidopsis
serine
Serine
inactivation
Pichia pastoris
aspartic acid
In Vitro Techniques
Aspartic Acid
phosphotransferases (kinases)
proteins
mutants
lesions (plant)
Proteins
Spinacia oleracea
Pichia
Nucleic Acid Regulatory Sequences
molybdenum
site-directed mutagenesis

ASJC Scopus subject areas

  • Plant Science
  • Cell Biology

Cite this

Identification in vitro of a post-translational regulatory site in the hinge 1 region of arabidopsis nitrate reductase. / Su, Wenpei; Huber, Steven C.; Crawford, Nigel M.

In: Plant Cell, Vol. 8, No. 3, 01.03.1996, p. 519-527.

Research output: Contribution to journalArticle

Su, Wenpei ; Huber, Steven C. ; Crawford, Nigel M. / Identification in vitro of a post-translational regulatory site in the hinge 1 region of arabidopsis nitrate reductase. In: Plant Cell. 1996 ; Vol. 8, No. 3. pp. 519-527.
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