The Isolation of Stress Granules From Plant Material.

Abstract

Stress granules (SGs) are ubiquitous nonmembrane-bound assemblies of protein and mRNA formed under stress conditions associated with stalled translation. SGs are evolutionarily conserved across eukaryotes. The canonical function of SGs is to selectively protect mRNAs and proteins from unfolding and prevent degradation induced by diverse environmental stresses. Moreover, sequestration into SGs provides an elegant way to regulate protein activities. Disassembly of SGs upon stress recovery is accompanied by the reactivation of protein translation and protein activities. The regulatory importance of SGs has been corroborated by recent studies describing the multiomics analysis of the composition of SGs from yeast, animal, and plant cells. Herein, we describe an isolation protocol of SGs that allows for the identification of proteins, mRNA, and metabolites sequestered into SG cores. Furthermore, the described protocols can be used to isolate other SG-like foci. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of SG-enriched fraction from plant material Basic Protocol 2: Affinity purification to isolate SGs Basic Protocol 3: Simultaneous extraction of proteins and metabolites from affinity-purified beads Basic Protocol 4: Protein digestion on affinity-purified beads Basic Protocol 5: Data analysis.