eIFiso4G Augments the Synthesis of Specific Plant Proteins Involved in Normal Chloroplast Function.

The plant-specific translation initiation complex eIFiso4F is encoded by three genes in Arabidopsis (Arabidopsis thaliana)-genes encoding the cap binding protein eIFiso4E (eifiso4e) and two isoforms of the large subunit scaffolding protein eIFiso4G (i4g1 and i4g2). To quantitate phenotypic changes, a phenomics platform was used to grow wild-type and mutant plants (i4g1, i4g2, i4e, i4g1 x i4g2, and i4g1 x i4g2 x i4e [i4f]) under various light conditions. Mutants lacking both eIFiso4G isoforms showed the most obvious phenotypic differences from the wild type. Two-dimensional differential gel electrophoresis and mass spectrometry were used to identify changes in protein levels in plants lacking eIFiso4G. Four of the proteins identified as measurably decreased and validated by immunoblot analysis were two light harvesting complex binding proteins 1 and 3, Rubisco activase, and carbonic anhydrase. The observed decreased levels for these proteins were not the direct result of decreased transcription or protein instability. Chlorophyll fluorescence induction experiments indicated altered quinone reduction kinetics for the double and triple mutant plants with significant differences observed for absorbance, trapping, and electron transport. Transmission electron microscopy analysis of the chloroplasts in mutant plants showed impaired grana stacking and increased accumulation of starch granules consistent with some chloroplast proteins being decreased. Rescue of the i4g1 x i4g2 plant growth phenotype and increased expression of the validated proteins to wild-type levels was obtained by overexpression of eIFiso4G1. These data suggest a direct and specialized role for eIFiso4G in the synthesis of a subset of plant proteins.

Discovery and characterization of conserved binding of eIF4E 1 (CBE1), a eukaryotic translation initiation factor 4E-binding plant protein.

In many eukaryotes, translation initiation is regulated by proteins that bind to the mRNA cap-binding protein eukaryotic translation initiation factor 4E (eIF4E). These proteins commonly prevent association of eIF4E with eIF4G or form repressive messenger ribonucleoproteins that exclude the translation machinery. Such gene-regulatory mechanisms in plants, and even the presence of eIF4E-interacting proteins other than eIF4G (and the plant-specific isoform eIFiso4G, which binds eIFiso4E), are unknown. Here, we report the discovery of a plant-specific protein, conserved binding of eIF4E 1 (CBE1). We found that CBE1 has an evolutionarily conserved eIF4E-binding motif in its N-terminal domain and binds eIF4E or eIFiso4E in vitro CBE1 thereby forms cap-binding complexes and is an eIF4E-dependent constituent of these complexes in vivo Of note, plant mutants lacking CBE1 exhibited dysregulation of cell cycle-related transcripts and accumulated higher levels of mRNAs encoding proteins involved in mitosis than did WT plants. Our findings indicate that CBE1 is a plant protein that can form mRNA cap-binding complexes having the potential for regulating gene expression. Because mammalian translation factors are known regulators of cell cycle progression, we propose that CBE1 may represent such first translation factor-associated plant-specific cell cycle regulator.