Phosphorylation of the alpha-subunit of plant eukaryotic initiation factor 2 prevents its association with polysomes but does not considerably suppress protein synthesis

• Published in: Plant science (Limerick) 2022-04, Vol.317, p.111190-111190, Article 111190
• Main Authors: Zhigailov, Andrey V., Stanbekova, Gulshan E., Nizkorodova, Anna S., Galiakparov, Nurbol N., Gritsenko, Dilyara A., Polimbetova, Nailya S., Halford, Nigel G., Iskakov, Bulat K.
• Format: Article
• Language: English
• Subjects: 48S preinitiation complex; Arabidopsis (Arabidopsis thaliana) phosphomimetic and non-phosphorylatable AteIF2α; Arabidopsis - genetics; Arabidopsis - metabolism; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Eukaryotic Initiation Factor-2 - genetics; Eukaryotic Initiation Factor-2 - metabolism; GCN2; Phosphorylation; Polyribosomes - metabolism; Polyribosomes formation; Protein Biosynthesis; Transient expression in tobacco; Wheat germ cell-free system
• ISSN: 0168-9452; 1873-2259
• DOI: 10.1016/j.plantsci.2022.111190

•Phosphomimetic and unphosphorylatable versions of AteIF2α expressed in N. benthamiana.•Expression of phosphomimetic AteIF2α causes small reduction in polysomes.•Expression of unphosphorylatable AteIF2α causes small increase in polysomes.•Phosphomimetic AteIF2α does not associate with polysomes.•Phosphorylation of plant eIF2α does not result in strong suppression of translation. Phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) and subsequent inhibition of protein synthesis is a major survival response to different stresses in animal and yeast cells. However, the role of this regulatory mechanism in plants is not unambiguously established to date. Here we describe a slight reduction of polysome abundance in Nicotiana benthamiana after the transient expression of a cDNA, AteIF2α(S56D), encoding a phosphomimetic form of Arabidopsis thaliana eIF2α. In contrast, the expression of a cDNA, AteIF2α(S56A), that encodes a non-phosphorylatable form of AteIF2α caused slightly elevated polysome formation compared to the control. Recombinant AteIF2α(S56A) was detected in association with 40S ribosomal subunit-containing complexes and also in the polysomal fraction, while recombinant AteIF2α(S56D) was detected mainly in complex with 40S subunits. Intentional phosphorylation of TaeIF2α induced by L-histidinol in a wheat germ (Triticum aestivum) cell-free extract did not reduce the abundance of polysomes. Interestingly, the phosphorylated TaeIF2(αP) was not detected in the polysomal fraction, similar to AteIF2α(S56D) in the in vivo experiment. Using mRNAs with a ‘Strepto-tag’ in the 3′ untranslated region, the 48S pre-initiation complexes isolated from histidinol-treated wheat germ extracts were shown to contain phosphorylated TaeIF2(αP). Thus, the phosphorylation of plant eIF2 does not greatly affect its ability to participate in the initiation of mRNA translation, in contrast to animals and yeast, in which eIF2α phosphorylation results in profound suppression of protein synthesis.