The Phosphorylation State of the Wheat Translation Initiation Factors eIF4B, eIF4A, and eIF2 Is Differentially Regulated during Seed Development and Germination

Hanh Le,Karen S Browning,Daniel R Gallie

doi:10.1074/jbc.273.32.20084

Abstract

The translation initiation factors (eIF) 4B and eIF2 are phosphoproteins whose phosphorylation state differs between mature seed and leaves. We examined the isoforms of eIF4B and the alpha and beta subunits of eIF2 during the development and germination of wheat seed to determine whether the differences in their phosphorylation state are because of tissue-specific regulation or occur concomitant with changes in protein synthetic activity during development. eIF2alpha underwent phosphorylation through several intermediate isoforms that correlated with the increase and subsequent reduction in protein synthetic activity characteristic of seed development. eIF2beta and eIF4B, present as highly phosphorylated isoforms during early seed development, underwent dephosphorylation during late development. eIF4B was rapidly phosphorylated within 20 h of germination, whereas eIF2alpha did not undergo dephosphorylation until 48-60 h of growth. A third factor, eIF4A, was predominantly nonphosphorylated throughout most of seed development and germination. These observations suggest that the phosphorylation state of eIF2alpha, eIF2beta, and eIF4B is developmentally regulated in a way that correlates with the changes in protein synthetic activity but that some differences were also observed.

Highlights

The translation initiation factors 4B and eIF2 are phosphoproteins whose phosphorylation state differs between mature seed and leaves
We examined the isoforms of eIF4B and the ␣ and ␤ subunits of eIF2 during the development and germination of wheat seed to determine whether the differences in their phosphorylation state are because of tissue-specific regulation or occur concomitant with changes in protein synthetic activity during development. eIF2␣ underwent phosphorylation through several intermediate isoforms that correlated with the increase and subsequent reduction in protein synthetic activity characteristic of seed development. eIF2␤ and eIF4B, present as highly phosphorylated isoforms during early seed development, underwent dephosphorylation during late development. eIF4B was rapidly phosphorylated within 20 h of germination, whereas eIF2␣ did not undergo dephosphorylation until 48 – 60 h of growth
A third factor, eIF4A, was predominantly nonphosphorylated throughout most of seed development and germination. These observations suggest that the phosphorylation state of eIF2␣, eIF2␤, and eIF4B is developmentally regulated in a way that correlates with the changes in protein synthetic activity but that some differences were observed

Summary

EXPERIMENTAL PROCEDURES

Antibody Preparation—eIF4A and eIF2 [31] were purified from wheat germ (commercially prepared wheat embryos) as described. Polyclonal antibodies to eIF2 and recombinant eIF4B and eIF4A were produced in rabbits and affinity-purified as described [33, 36]. The nitrocellulose membranes were blocked in 5% milk, 0.01% thimerosal in TPBS (0.1% Tween 20, 13.7 mM NaCl, 0.27 mM KCl, 1 mM Na2HPO4, and 0.14 mM KH2PO4) followed by incubation with primary antibodies diluted 1:2000 for eIF4A and 1:1000 for eIF4B, eIF2␣, and eIF2␤ in TPBS with 1% milk for 1.5 h. The blots were washed twice with TPBS and incubated with goat anti-rabbit horseradish peroxidase-conjugated antibodies (Southern Biotechnology Associates, Inc.) diluted to 1:10,000 for 1 h. The pH range of the IEF gel following isoelectric focusing was determined from the measurements of 5 mm sections of a control gel soaked in 1 ml of 15 mM NaCl

RESULTS

Regulation of the Phosphorylation State of Wheat eIF

DISCUSSION