Ihm

control 2 5 10 BSA

micrograms 14-3-3

B. Cell wall bound acid invertase g 40

I 40

rog 5

control 2 5 10 BSA

micrograms 14-3-3

FIGURE 35.2. Effect of barley 14-3-3A on acid invertase activity in barley endosperm extracts. Developing endosperm extracts A (soluble, 60 |g protein) and B (cell wall bound, 35 |g protein) were preincubated for 30 mins with ATP (1 mM) and increasing concentrations of purified recombinant barley 14-3-3A protein or 10 |g BSA, and then invertase activity (release of glucose from sucrose) was assayed. Control: no 14-3-3 added. Data represent the mean of three replications. Error bars indicating the SD (Ross and Morris, unpublished data).

control 2 5 10 BSA

micrograms 14-3-3

FIGURE 35.2. Effect of barley 14-3-3A on acid invertase activity in barley endosperm extracts. Developing endosperm extracts A (soluble, 60 |g protein) and B (cell wall bound, 35 |g protein) were preincubated for 30 mins with ATP (1 mM) and increasing concentrations of purified recombinant barley 14-3-3A protein or 10 |g BSA, and then invertase activity (release of glucose from sucrose) was assayed. Control: no 14-3-3 added. Data represent the mean of three replications. Error bars indicating the SD (Ross and Morris, unpublished data).

employed; mammalian 14-3-3 workers have made extensive use of Y2H screens. Thus when affinity purification was used to isolate 200 14-3-3 binding proteins from mammalian cells, the largest group of proteins found (24%) were enzymes of primary and secondary metabolism; only about 15% of the proteins were associated with cell signaling, and 15% with TFs and RNA-binding proteins [47].

This bias of protein functional class being dependent on the analytical methodology was well-illustrated in a recent analysis of barley proteins. The Y2H system was directly compared with affinity chromatography of 14-3-3 binding partners from barley seedling [48]. One hundred thirty-two putative targets were isolated by the Y2H method and 30 by affinity chromatography. About 10% of the partners isolated by the Y2H method were also found by affinity chromatography. An interesting difference in the functional classification of interacting partners obtained from both approaches was noted; affinity purification yielded predominantly (44%) proteins concerned with metabolism, whereas the Y2H method found a large proportion of cell-signaling-related proteins (35%). One reason for this difference is that affinity purification will yield results that reflect the relative abundance of those proteins in the cell; proteins involved in signaling are much less abundant than those involved in cellular metabolism. In the Y2H method, the cDNA clones are all expressed from the same promoter, so that protein levels in the yeast cells will be approximately equalized; however, there will still be a bias in favor of those clones that are more highly represented in the cDNA library.

Interacting proteins can be analyzed in a more indirect manner by manipulating 14-3-3 expression in transgenic plants and monitoring changes in enzyme activity and metabolites in the transgenics. This approach has been successfully carried out in potato, where expression of specific 14-3-3 isoforms was down-regulated by antisense [49]. The most significant results found here were enhancements in NR and SPS enzyme activity, thus confirming previous in vitro data on these interactions.

Was this article helpful?

0 0
How To Win Your War Against Allergies

How To Win Your War Against Allergies

Not Able To Lead A Happy Life Because Of Excessive Allergies? Want To Badly Get Rid Of Your Allergy Problems, But Are Super Confused And Not Sure Where To Even Start? Don't Worry, Help Is Just Around The Corner Revealed The All-In-One Power Packed Manual Containing Ample Strategies And Little-Known Tips To Get Rid Of Any Allergy Problems That Are Ruining Your Life Learn How You Can Eliminate Allergies Completely Reclaim Your Life Once Again

Get My Free Ebook


Post a comment