Methodology And Strategy

Different experimental objectives have been addressed by the methods described here: first, to examine the 14-3-3 isoform proteome in a given species; second, to screen for novel interacting partners; and third, to focus on the individual client protein, in order to investigate the mechanisms of 14-3-3 protein binding. Examples of these strategies are given in the next section. Critical to the approaches involving PPIs is the correct use of controls to show specificity of interaction. Thus, use of unrelated proteins, or proteins mutated in their binding sites, shows specificity of interaction rather than "sticky" proteins. Since the most common 14-3-3 binding sites are phos-phorylated, client binding should be phosphorylation-dependent; therefore treatment of client proteins with phosphatase should greatly reduce binding. The Arabidopsis 14-3-3 proteome has been analyzed by 2-DGE fractionation of cell extracts using a pH 3-6 IPG strip followed by immunoblotting of the 2D gels with a 14-3-3 specific antibody to identify the family of isoforms. This was followed by endopro-tease digestions, HPLC, and ESI-MS/MS of peptides for isoform identification [15]. Similarly, Arabidopsis suspension-cultured cells extracts were fractionated by column chromatography followed by 2-DGE and trypsin digestion and MALDI-TOF-MS analysis for identification of 14-3-3s [36]. A quantitative proteomics approach has employed SIL of Arabidopsis for an analysis of 14-3-3 binding to the NR and SPS 14-3-3-binding motifs [37]. Another analytical tactic has been to create transgenic plants designed to express specific 14-3-3 genes as GFP fusions in order to monitor the subcellular location of the proteins [38].

The Y2H system [39] has been used both to screen for novel interacting partners and to analyze the binding of a specific client protein. To search for novel partners, an activation domain-tagged cDNA library in yeast is needed, which allows many interactions to be quickly screened. However a careful analysis of positives from the Y2H is required because overexpression of proteins can yield artifactual associations.

Far Westerns or 14-3-3 overlays have been used to visualise 14-3-3 binding on blots. Here, plant extracts are separated by SDS-PAGE, transferred to nitrocellulose membranes, and hybridized with digoxigenin-labeled 14-3-3, which can be detected immunologically. The use of phosphopeptides corresponding to a strongly binding 14-3-3 site (e.g., ARAApSAPA) to outcompete the binding illustrates the specificity of the interaction. Co-immunoprecipitation with 14-3-3 antisera also has been used to monitor protein interactions with 14-3-3 [40].

The use of immobilized recombinant 14-3-3 for affinity purification from plant extracts was first described by reference 40. In these experiments, bound proteins from cauliflower extracts (this was used because bulk quantities of tissue are available from a close relative of Arabidopsis) were bound to immobilized yeast 14-3-3 and specifically eluted with a 14-3-3 binding site phosphopeptide. The eluted proteins were subjected to anion exchange chromatography and SDS-PAGE, followed by excision of stained protein bands and trypsin digestion. Peptides were then separated by RP chromatography and sequenced in order to identify the proteins. In a similar approach, the spectrum of barley grain client proteins binding to barley 14-3-3A protein was investigated by immobilizing the recombinant 14-3-3 to sepharose and using this matrix in affinity chromatography experiments. In order to simplify separation and analysis of bound proteins, the grain extracts were fractionated by ion exchange chromatography prior to affinity chromatography. After extensive washing of the affinity matrix, proteins were eluted with SDS-PAGE sample buffer, fractionated by SDS-PAGE, CBB stained, and identified by MALDI-TOF-MS [41]. The presence of a potential 14-3-3 binding site in a given protein sequence can also be used to predict binding of that protein. The limit dextrinase inhibitor protein contains a mode I motif and has been shown to bind 14-3-3 in a phosphorylation-dependent manner by immunoblotting after 14-3-3 affinity chromatography [42].

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