APEX Proximity Labeling of Stress Granule Proteins

Abstract

Ascorbate peroxidase (APEX)-catalyzed proximity labeling has been recently established as a robust approach to uncover localized protein environments and transient protein-protein interactions occurring across mammalian cells. This molecular tool enables improved identification of individual proteins localized to and involved in specific cellular and subcellular pathways and functions. Engineering of an APEX2 fusion protein into the endogenous loci of proteins of interest enables directed biotinylation of neighboring polypeptides and mRNAs. This results in identification of subcellular and context-dependent proteomes or transcriptomes via quantitative mass spectrometry or RNA sequencing, respectively. Here, we describe the utility of APEX-mediated proximity labeling to recover components of stress granules (SGs) by endogenous tagging of well-established SG-associated proteins.

Keywords: APEX2; Affinity purification; Biotinylation; Proximity labeling; Quantitative mass spectrometry; RNA binding protein; Stress granule; Tandem mass tag.