Experimental Results And Applications

Due to its relative abundance in all cell types, 14-3-3 has been fortuitously identified in a number of plant proteomic studies not directly aimed at 14-3-3 [for example, see reference 43]. However, the plant 14-3-3 proteome has been specifically investigated in Arabidopsis 14-3-3 suspension culture cells. Proteins encoded by 11 of the 13 Arabidopsis genes were positively identified. All the N-termini of analyzed isoforms were found to be acetylated (as is the case for mammalian 14-3-3), which may play a role in promoting protein stability. Other modifications such as phosphorylation or the C-terminal cleavage, as described for barley 14-3-3A, were not found, but could not be ruled out in this study [15]. Nine isoforms were also identified by MALDI-TOF-MS from Arabidopsis suspension culture cells after partial purification and 2-DGE [36]. Subcellular location of 14-3-3 has been investigated by a number of means including the use of GFP fusions in transgenic plants; this study showed that different isoforms showed distinct subcellular locations, which could be disrupted by means of chemical agents that intervene with 14-3-3/client binding. This confirms that individual isoforms have binding specificity and shows that subcellular location of 14-3-3 is driven by interactions with the binding partners.

The isoform specificity of tobacco 14-3-3 binding to SPS has been analyzed by screening for interacting partners in a Y2H assay; 12 SPS-interacting clones were identified, representing two 14-3-3 isoforms. Deletion analysis of these clones indicated that the C-terminus of 14-3-3 mediates binding specificity [44]. Y2H analysis was also used to investigate the interactions between barley 14-3-3 and ABA-responsive transcription factors of the ABI5 family, clearly illustrating the isoform specificity of these interactions [45].

Affinity purification of cauliflower proteins to immobilized yeast 14-3-3 and amino acid sequencing of peptide fragments resulted in the identification of nine different proteins, including enzymes of carbohydrate metabolism, nitrogen metabolism, and also a protein involved in floral development [40]. A similar approach, together with 14-3-3 overlays, was used to analyze 14-3-3 binding proteins from Arabidopsis cell suspension cells, which uncovered a nutrient sensing role for 14-3-3s. Sugar starvation of the suspension culture cells resulted in loss of 14-3-3 binding and the proteolytic cleavage of the client proteins; 14-3-3 binding is thought to protect these proteins from cleavage [46]. Also using an affinity chromatography approach, the binding of barley grain proteins to immobilized barley 14-3-3A was shown to be phosphorylation-dependent since hardly any binding was detected when extracts were dephosphorylated with AP [41]. After extensive washing, 54 of the binding proteins were identified, of which the largest group (31%) was involved in carbohydrate metabolism (in some respects not surprising because this is the major function of the barley grain). Four of the five enzymes involved in sucrose biosynthesis (TPI, FBPase, S(6) PS, and S(6) PS phosphatase) were identified, which suggests the coordinated regulation of sucrose biosynthesis by 14-3-3 proteins. For the enzyme sucrose syn-thase, it could be demonstrated that activity in barley grain extract was reduced by addition of exogenous 14-3-3. A similar example is shown in Figure 35.2, where the activity of barley grain soluble and cell-wall-bound acid invertase in crude extracts is reduced by addition of recombinant 14-3-3A.

As well as proteins concerned with metabolism, a large functional group of 14-3-3 clients was composed of proteins related to abiotic and biotic plant defense. Of particular interest are proteins of the NBS-LRR class, characteristic of plant disease resistance genes (R genes) encoding receptors to pathogen avirulence gene products; three proteins belonging to this category were found. It was previously found that 14-3-3 gene expression is up-regulated by powdery mildew infection [11], and the Mla powdery mildew resistance locus is also known to encode an NBS-LRR protein. This suggests an important role for 14-3-3 in the regulation of disease signal trans-duction for powdery mildew and perhaps for other pathogens. The barley NBS-LRR proteins described in the public databases were screened for potential 14-3-3 binding sites, and at least 30 were found that contained such motifs, sometimes in many copies (up to 20 motifs in one protein).

Affinity purification experiments on 14-3-3 client proteins from plant extracts have isolated predominantly proteins concerned with cellular metabolism. It was once thought that there is a marked difference in the type of client protein in animal and plant cells, with 14-3-3 clients from animal cell being primarily proteins concerned with cell signaling; however, this difference may be attributable to the techniques

A. Soluble acid invertase

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