Abstract
In order for cells to remain viable, they need to have the capacity to survive during times of stress. Eukaryotic cells, in order to endure stress, form large and dynamic cytosolic aggregates called stress granules that act to sequester mRNA stalled during translation initiation. Previously, the human protein T Cell Intracellular Antigen 1 (TIA-1) was identified as the scaffold-forming protein for stress granules as well as basophilic inclusions found in individuals diagnosed with amyotrophic lateral sclerosis. We lack a clear understanding of how stress granules are assembled and disassembled. Using Saccharomyces cerevisae, an assay was developed that allows for monitoring of stress granule dynamics via confocal microscopy. Utilizing the TIA-1 orthologue in yeast, Poly-U binding protein 1 (Pub1), fused with green fluorescent protein (Pub1-GFP) stress granule assembly was observed at 19 ± 1.5 min following a stress stimulus (nutritional depletion) and disassembly 8 ± 2 min following reintroduction of nutrient-rich media. Using a Pub1!Q-GFP-His6Mx deletion strain lacking the Gln(Q) rich C-terminal of Pub1, stress granules were shown to assembly in the same time observed with Pub1-GFP. However, these structures did not disassemble. This is the first observation of a protein sequence critical to the disassembly of stress granules and is suggestive that a prion-like\r iv\r sequence is critical not to an aggregating phenomenon – but rather to trigger disassembly of a native, subcellular aggregate.