A proteomic analysis of 14-3-3 binding proteins from developing barley grains.
14-3-3 proteins are important eukaryotic regulatory proteins. Barley (Hordeum vulgare L.) 14-3-3A was over-expressed, immobilised and used to affinity purify 14-3-3 binding proteins from developing barley grains. Binding was shown to be phosphorylation-dependent. These proteins were fractionated by PAGE and identified by MALDI-TOF MS. In total, 54 14-3-3 binding proteins were identified, 49 of these interactions are novel to plants. These proteins fell into a number of functional categories. The largest category was for carbohydrate metabolism, including plastidic enzymes for starch synthesis and modification. 14-3-3 was shown to be present in isolated plastids. Four of five enzymes involved in sucrose biosynthesis from triose phosphates were identified, suggesting co-ordinated regulation of this pathway. Invertase and sucrose synthase, which break down sucrose to hexoses, were found. Sucrose synthase activity was shown to be inhibited by exogenous 14-3-3 in a dosage-dependent manner. The second-largest functional group was for proteins involved in stress and defence responses; for example, RGH2A, closely related to the MLA powdery mildew resistance protein, was found. This work illustrates the broad range of processes in which 14-3-3 may be involved, and augments previous data demonstrating key roles in carbohydrate metabolism and plant defence.
School of Life Sciences, Heriot-Watt University, Edinburgh, UK.
Ross D
RD
Alexander
Peter C
PC
Morris
D048948:14-3-3 Proteins / Q000032:analysis*; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D001467:Hordeum / Q000737:chemistry* / Q000254:growth & development*; D013058:Mass Spectrometry; D010449:Peptide Mapping; D010940:Plant Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D048948:14-3-3 Proteins; D010940:Plant Proteins; D020543:Proteome