Abstract
Eukaryotic initiation factor 2A is a single polypeptide that acts to negatively regulate IRES-mediated translation during normal cellular conditions. We have found that eIF2A (encoded by YGR054w) abundance is reduced at both the mRNA and protein level during 6% ethanol stress (or 37°C heat shock) under conditions that mimic the diauxic shift in the yeast Saccharomyces cerevisiae. Furthermore, eIF2A protein is posttranslationally modified during ethanol stress. Unlike ethanol and heat shock stress, H2O2 and sorbitol treatment induce the loss of eIF2A mRNA, but not protein and without protein modification. To investigate the mechanism of eIF2A function we employed immunoprecipitation-mass spectrometry and identified an interaction between eIF2A and eEF1A. The interaction between eIF2A and eEF1A increases during ethanol stress, which correlates with an increase in IRES-mediated translation from the URE2 IRES element. These data suggest that eIF2A acts as a switch to regulate IRES-mediated translation, and eEF1A may be an important mediator of translational activation during ethanol stress.
Highlights
Eukaryotic initiation factor 2A, a single polypeptide, was purified based on its ability to direct binding of initiator methionyl-tRNA to the 40S ribosome in an AUG-dependent manner and its ability to catalyze poly(U)directed polyphenylalanine synthesis at low [Mg+2] [1]
Since the role of eukaryotic initiation factor 2A (eIF2A) remains elusive in translational control, and it is known to repress IRES-mediated translation, we sought to delineate whether protein expression of eIF2A is reduced under cellular stress conditions that might result in enhanced internal initiation of translation
All four treatments resulted in rapid mRNA turnover, as eIF2A mRNA became undetectable by 2 min after application of the stress (Figure 1B)
Summary
Eukaryotic initiation factor 2A (eIF2A), a single polypeptide, was purified based on its ability to direct binding of initiator methionyl-tRNA (met-tRNAi) to the 40S ribosome in an AUG-dependent manner and its ability to catalyze poly(U)directed polyphenylalanine synthesis at low [Mg+2] [1]. Comparative analysis indicated that eIF2A was less efficient at met-tRNAi delivery to the 40S ribosomal subunit on artificial templates, and was inactive using globin mRNA as a template for polypetide synthesis [3]. This initial work established the idea of competition between two distinct pathways for delivery of methionyl-tRNA to the 40S ribosomal subunit during translation initiation. Identification of a yeast homolog to eIF2A (corresponding to yeast gene YGR054w) reignited efforts to characterize eIF2A because of the potential for genetic dissection of the pathway for eIF2A-mediated regulation of translation [4]
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