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Single amino acid variation in barley 14‐3‐3 proteins leads to functional isoform specificity in the regulation of nitrate reductase
Author(s) -
Sinnige Mark P.,
Roobeek Ilja,
Bunney Tom D.,
Visser Antonie J.W.G.,
Mol Jos N.M.,
Boer Albertus H.
Publication year - 2005
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/j.1365-313x.2005.02599.x
Subject(s) - gene isoform , biochemistry , amino acid , isozyme , biology , 14 3 3 protein , function (biology) , mutagenesis , enzyme , phosphorylation , microbiology and biotechnology , gene , mutation
Summary The highly conserved family of 14‐3‐3 proteins function in the regulation of a wide variety of cellular processes. The presence of multiple 14‐3‐3 isoforms and the diversity of cellular processes regulated by 14‐3‐3 suggest functional isoform specificity of 14‐3‐3 isoforms in the regulation of target proteins. Indeed, several studies observed differences in affinity and functionality of 14‐3‐3 isoforms. However, the structural variation by which isoform specificity is accomplished remains unclear. Because other reports suggest that specificity is found in differential expression and availability of 14‐3‐3 isoforms, we used the nitrate reductase (NR) model system to analyse the availability and functionality of the three barley 14‐3‐3 isoforms. We found that 14‐3‐3C is unavailable in dark harvested barley leaf extract and 14‐3‐3A is functionally not capable to efficiently inhibit NR activity, leaving 14‐3‐3B as the only characterized isoform able to regulate NR in barley. Further, using site directed mutagenesis, we identified a single amino acid variation (Gly versus Ser) in loop 8 of the 14‐3‐3 proteins that plays an important role in the observed isoform specificity. Mutating the Gly residue of 14‐3‐3A to the alternative residue, as found in 14‐3‐3B and 14‐3‐3C, turned it into a potent inhibitor of NR activity. Using surface plasmon resonance, we show that the ability of 14‐3‐3A and the mutated version to inhibit NR activity correlates well with their binding affinity for the 14‐3‐3 binding motif in the NR protein, indicating involvement of this residue in ligand discrimination. These results suggest that both the availability of 14‐3‐3 isoforms as well as binding affinity determine isoform‐specific regulation of NR activity.