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 degrees 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, H(2)O(2) 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.