Body of Abstract: Plant heat stress response involves rapid inhibition of protein synthesis rates leading to polysome disassembly. This is associated with the transient formation of cytoplasmic condensates, in which translation-stalled mRNAs accumulate together with translation initiation complexes and other stress-related proteins. So far it is unclear how translation and condensate formation is coordinated, whether condensates are only associated with translational repression or might also support translation of stress-related transcripts. Here, we focused on the conserved translation elongation factor complex eEF1B which is essential for recharging the EF1A complex during translation elongation. The plant eEF1B complex consists of three subunits, of which eEF1Bα and eEF1Bβ are the catalytic subunits harboring a conserved GTP/GDP exchange domain while the function of the third subunit, eEf1Bγ is less clear. By generating Arabidopsis mutants with strongly reduced eEf1Bγ levels, we demonstrated that eEf1Bγ is essential for efficient protein synthesis and thus growth and development at control conditions.
Since the eEF1B proteins were in proteomic studies identified as components of heat-induced stress granules we analyzed the in vivo localisation pattern of EF1B-GFP fusion proteins. We found that incubation of seedlings for 30 min at 38°C (HS) induced a global decrease of translation rates. The same treatment resulted in accumulation of each of the three eEF1B subunits to cytoplasmic foci. These condensates largely co-localised with the stress granule marker Poly(A)-binding protein 8 (PAB8). To understand the function of eEF1B containing condensates, we analyzed the interactome eEF1Bγ-GFP. Mass-Spectrometry analysis of eEF1Bγ-GFP-bound proteins at control conditions revealed a small set of proteins almost exclusively involved in translation elongation. At HS conditions eEF1Bγ associated with numerous additional proteins involved in mRNA binding, translation initiation and control of protein stability. Our data provide new insights into mechanisms coordinating protein translation with heat stress response and identify novel components of heat-induced cytoplasmic condensates.