Condensation of Ded1p Promotes a Translational Switch from Housekeeping to Stress Protein Production.

Cells sense elevated temperatures and mount an adaptive heat shock response that involves changes in gene expression, but the underlying mechanisms, particularly on the level of translation, remain unknown. Here we report that, in budding yeast, the essential translation initiation factor Ded1p undergoes heat-induced phase separation into gel-like condensates. Using ribosome profiling and an in vitro translation assay, we reveal that condensate formation inactivates Ded1p and represses translation of housekeeping mRNAs while promoting translation of stress mRNAs. Testing a variant of Ded1p with altered phase behavior as well as Ded1p homologs from diverse species, we demonstrate that Ded1p condensation is adaptive and fine-tuned to the maximum growth temperature of the respective organism. We conclude that Ded1p condensation is an integral part of an extended heat shock response that selectively represses translation of housekeeping mRNAs to promote survival under conditions of severe heat stress.

eIF4F is a thermo-sensing regulatory node in the translational heat shock response.

Heat-shocked cells prioritize the translation of heat shock (HS) mRNAs, but the underlying mechanism is unclear. We report that HS in budding yeast induces the disassembly of the eIF4F complex, where eIF4G and eIF4E assemble into translationally arrested mRNA ribonucleoprotein particles (mRNPs) and HS granules (HSGs), whereas eIF4A promotes HS translation. Using in vitro reconstitution biochemistry, we show that a conformational rearrangement of the thermo-sensing eIF4A-binding domain of eIF4G dissociates eIF4A and promotes the assembly with mRNA into HS-mRNPs, which recruit additional translation factors, including Pab1p and eIF4E, to form multi-component condensates. Using extracts and cellular experiments, we demonstrate that HS-mRNPs and condensates repress the translation of associated mRNA and deplete translation factors that are required for housekeeping translation, whereas HS mRNAs can be efficiently translated by eIF4A. We conclude that the eIF4F complex is a thermo-sensing node that regulates translation during HS.

Surviving the heat: the role of macromolecular assemblies in promoting cellular shutdown.

During heat shock (HS), cells orchestrate a gene expression program that promotes the synthesis of HS proteins (HSPs) while simultaneously repressing the synthesis of other proteins, including growth-promoting housekeeping proteins. Recent studies show that mRNAs encoding housekeeping proteins, along with associated processing factors, form macromolecular assemblies during HS. These assemblies inhibit transcription, nuclear export, and translation of housekeeping mRNAs, and coincide with structural rearrangements in proteins. These findings reveal a mechanism linking temperature sensitivity through structural rearrangements and macromolecular assembly to the 'shut down' of housekeeping protein synthesis. This review delves into recent findings in yeast, with a focus on macromolecular assembly, offering perspectives into mechanisms that regulate gene expression during HS and how these processes may be conserved.

Quantitative proteomics identifies the universally conserved ATPase Ola1p as a positive regulator of heat shock response in Saccharomyces cerevisiae.

The universally conserved P-loop ATPase Ola1 is implicated in various cellular stress response pathways, as well as in cancer and tumor progression. However, Ola1p functions are divergent between species, and the involved mechanisms are only poorly understood. Here, we studied the role of Ola1p in the heat shock response of the yeast Saccharomyces cerevisiae using a combination of quantitative and pulse labeling-based proteomics approaches, in vitro studies, and cell-based assays. Our data show that when heat stress is applied to cells lacking Ola1p, the expression of stress-protective proteins is enhanced. During heat stress Ola1p associates with detergent-resistant protein aggregates and rapidly forms assemblies that localize to stress granules. The assembly of Ola1p was also observed in vitro using purified protein and conditions, which resembled those in living cells. We show that loss of Ola1p results in increased protein ubiquitination of detergent-insoluble aggregates recovered from heat-shocked cells. When cells lacking Ola1p were subsequently relieved from heat stress, reinitiation of translation was delayed, whereas, at the same time, de novo synthesis of central factors required for protein refolding and the clearance of aggregates was enhanced when compared with wild-type cells. The combined data suggest that upon acute heat stress, Ola1p is involved in the stabilization of misfolded proteins, which become sequestered in cytoplasmic stress granules. This function of Ola1p enables cells to resume translation in a timely manner as soon as heat stress is relieved.

Protocol to reconstitute translationally arrested heat shock mRNPs and condensates in vitro.

Heat shock (HS) coincides with the assembly of translationally arrested heat shock messenger ribonucleoprotein particles (HS-mRNPs) and condensates. Here, we present a protocol to reconstitute HS-mRNPs and HS condensates with eIF4G, eIF4E, Pab1p, and mRNA in vitro. In addition, we describe the necessary steps to measure the effect of HS-mRNPs and HS condensates on translation in yeast extracts. The protocol can be modified to study mRNPs and condensates assembled with other proteins and to study translation in extracts prepared from different cells. For complete details on the use and execution of this protocol, please refer to Desroches Altamirano et al.1.