In-cell structural insights into fungal ER stress responses

Approximately a third of a cell’s proteome is folded in the endoplasmic reticulum (ER). The accumulation of misfolded proteins in the ER results in ER stress. The unfolded protein response (UPR) has evolved to attenuate ER stress by increasing the cell’s folding capacity and reducing the folding load in the ER. In metazoans, the latter is notably achieved through mRNA translation inhibition. However, S. cerevisiae lacks the corresponding pathways, and its reliance on translational downregulation in response to ER stress remains largely unexplored. In this study we combined cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to investigate the effect of ER stress on the translational machinery of S. cerevisiae. At the ultrastructural level, we describe changes in organelle morphology, confirming ER volume expansion and the activation of autophagy. Further structural analysis reveals that ER stress triggers an increase in the relative abundance of inactive 80S ribosomes (up to 25% of the total), both in the cytosol and at the ER. Finally, we visualise the binding of the elongation factors eEF2, eIF5A and eEF3 to inactive ribosomes. Our work provides evidence that ER stress in S. cerevisiae leads to modest translation inhibition, which might contribute to alleviating ER stress.