Subcellular localization and interaction network of the mRNA decay activator Pat1 upon UV stress

Abstract

To identify nucleo-cytoplasmic shuttle proteins that relocate to the nucleus upon UV stress, we selected 18 targets on the basis of their conservation amongst eukaryotes and their relatively poor functional description. Their relocation was assayed using quantitative nuclear relocation assay (QNR). We focused on Pat1, a component of the cytoplasmic foci called processing bodies (p-bodies), because it had the strongest response to the stress. We verified that Pat1 accumulates in the nucleus after GFP tagging and fluorescence microscopy. Using tandem affinity purification coupled to a mass spectrometry shotgun detection and quantitation approach, we explored the dynamics of Pat1 protein-protein interaction network after UV stress. We have shown that Pat1 co-purifies with Dhh1 specifically upon UV stress. We observed that the nuclear accumulation of Pat1 upon UV stress is abolished in a dhh1∆ strain. These data provide the first evidence that Dhh1 is required for Pat1 nuclear relocation after UV stress.

Keywords: Pat1; QNR assay; mRNA decay; nuclear localization; nucleo-cytoplasmic shuttling; stress response.