The Arabidopsis 14-3-3 Family - Evolution, Expression, Localization, and Target Specificity

Sammanfattning: In plants, 14-3-3 proteins are key regulators of primary metabolism and membrane transport. It was previously thought that the 14-3-3 isoforms were not very specific with regard to target proteins, but more recent data suggest that the specificity may be high. Therefore, identification, localization, and characterization of all 14-3-3 isoforms in the model plant Arabidopsis are important. We identified all 14-3-3 genes in the fully sequenced Arabidopsis genome and partly characterized and localized the novel isoforms. We found three new 14-3-3 genes and determined the total number of genes to 15. Two of the novel genes were found to be transcribed and named grf11and 12, and the corresponding new 14-3-3 isoforms were named omicron and iota, respectively, and thereby 13 genes have now been shown to be expressed. Omicron is expressed in leaves, roots, and flowers, whereas the gene coding for iota is specifically expressed in flowers. We furthermore analysed the evolutionary history of the 14-3-3 family in plants, and particularly in Arabidopsis, which currently is the largest and most complete 14-3-3 family. We then used the interaction between 14-3-3 and the plasma membrane H+ pumping ATPase in Arabidopsis as a model system to elucidate potential isoform specificity in 14-3-3 binding. Considering the large number of 14-3-3 and H+-ATPase isoforms in Arabidopsis (13 and 11 expressed genes, respectively), specificity in binding may exist between 14-3-3 and H+-ATPase isoforms. We could show that the H+-ATPase is the main target for 14-3-3 binding at the plasma membrane, and that all twelve 14-3-3 isoforms tested bind to the H+-ATPase in vitro. Mass peptide fingerprinting identified two H+-ATPase isoforms, AHA1 and 2, as major isoforms in leaf plasma membranes. Using specific antibodies for nine of the 14-3-3 isoforms, we could show that most of the 14-3-3 isoforms are present in leaves, but that isolated plasma membranes lacked three of these isoforms, which suggests some specificity in the 14-3-3/ H+-ATPase interaction in vivo. We could also calculate that under ?unstressed? conditions less than one percent of total 14-3-3 in the cell is attached to the H+-ATPase. However, during a condition requiring full activation of H+ pumping several percent of total 14-3-3 may be engaged in activation of the H+-ATPase.