A proteomic analysis of 14‐3‐3 binding proteins from developing barley grains

Abstract

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.

A review of starch‐branching enzymes and their role in amylopectin biosynthesis

“…Recent studies with maize SBEIIb, the major isoform of SBEII in the starch-storing endosperm, have shown that the enzyme can be phosphorylated at three Ser sites by two plastidial Ca 21 -dependent protein kinase (CDPK) activities (14,70). Two of the phosphorylation sites (Ser 286 and Ser 297 ) are highly conserved among the SBEII class (see Fig.…”

“…2.4.1.1, or P-enzyme) was thought to be solely responsible for a-1,4-glucan chain elongation in glycogen and amylopectin, the branching activity was termed Q-enzyme. The relative Abbreviations: ae, amylose extender; BE, branching enzyme (1,4-a-glucan: 1,4-a-glucan 6-glucosyl transferase); C-terminus, carboxyl terminus; CBM48, family 48 carbohydrate-binding module; CDPK, Ca 21 -dependent protein kinase; DBE, debranching enzyme; DP, degree of polymerization; GBE, glycogen-branching enzyme; GH13, glycoside hydrolase family 13; MOS, maltooligosaccharides; NH 2 -terminus, amino terminus; SBE, starchbranching enzyme; Ser, serine; SP, starch phosphorylase; SS, starch synthase. This work is dedicated to the memory of Koushik Seetharaman.…”

Identification of a host 14-3-3 protein that interacts with Xanthomonas effector AvrRxv

“…In barley, 14-3-3 proteins are upregulated by powdery mildew [74]. Affinity purification of barley 14-3-3 binding partners indicated that the second largest class of interactors was involved in defense, including three different disease NBS-LRR resistance proteins [75]. This suggests that there is a direct role of 14-3-3 in resistance gene-mediated resistance signaling.…”

Oligomerization, Membrane Association, and in Vivo Phosphorylation of Sugarcane UDP-glucose Pyrophosphorylase

“…The amino acid sequence of ScUGPase-1 is strongly conserved among other plant UGPases (Fig. 3) and contains a serine at position 419 that is predicted to be a phosphorylation site and a 14-3-3 binding site (12). To evaluate ScUGPase-1 in relation to its phosphorylation status in vivo, we once again extracted the soluble and membrane fractions from sugarcane internode and leaf and immunoblotted the crude extract using the anti-pS419 antibody.…”

“…In addition, protein phosphorylation and the binding of 14-3-3 proteins could be other posttranslational modifications that are involved in regulating UGPase activity, subcellular localization, and protein turnover. Barley UGPase binds to 14-3-3 proteins in vivo (12), and yeast UGPase phosphorylation does not affect its activity but leads to a decreased glycogen content and an increased cell wall glucan content (13).…”