APEX2-mediated proximity labeling of mitochondrial matrix and chromatin in budding yeast

Enzyme-catalyzed proximity labeling (PL) with biotin is a novel approach to map organelle compartmentalization and protein interaction networks in living cells. Here, we present a new method based on semi-permeabilized yeast cells and the enzyme APEX2, an engineered ascorbate peroxidase. Combined with stable isotope labeling by amino acids in cell culture (SILAC), we demonstrate proteomic mapping of a membrane-enclosed and a semi-open compartment, the mitochondrial matrix and the nucleus. APEX2 PL revealed nuclear proteins that were previously not identified by conventional techniques. One of these, the Yer156C protein, is highly conserved but of unknown function. In follow-up experiments using quantitative PL with Yer156C-APEX2 we discovered an array of Yer156C interactors. Thus, our approach establishes APEX2 PL as an effective and versatile tool that complements the powerful methods palette for the model system yeast.