The variable C-terminus of 14-3-3 proteins mediates isoform-specific interaction with sucrose-phosphate synthase in the yeast two-hybrid system.
Sucrose-6-phosphate synthase (SPS) is a target for 14-3-3 protein binding in plants. Because several isoforms of the 14-3-3 protein are expressed in plants, I investigated which isoforms have the ability to bind SPS. Two 14-3-3 isoforms (T14-3d and a novel isoform designated T14-3 g) were found to interact with SPS from tobacco (Nicotiana tabacum L.) in a two-hybrid screen. To further address the question of isoform specificity of 14-3-3s, four additional isoforms were tested for their ability to interact with SPS in the yeast two-hybrid system. The results clearly revealed large differences in affinity between individual 14-3-3 isoforms toward SPS. Deletion analysis suggested that these differences were mediated by the variable C-terminus of 14-3-3s. Site-directed mutagenesis of candidate 14-3-3 binding sites on SPS demonstrated that interaction could be independent of a phosphorylated serine residue within conserved binding motifs in the yeast system. These findings suggest that the large number of 14-3-3 isoforms present in plants reflects functional specificity.
Börnke
Frederik
F
Institut für Pflanzengenetik und Kulturpflanzenforschung, Corrensstrasse 3, Gatersleben 06466, Germany. boernke@ipk-gatersleben.de
D048948:14-3-3 Proteins / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D018076:DNA, Complementary; D005964:Glucosyltransferases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology; D020798:Two-Hybrid System Techniques
D048948:14-3-3 Proteins; D018076:DNA, Complementary; D020033:Protein Isoforms; D005964:Glucosyltransferases; C022698:sucrose-phosphate synthase