APEX2‐mediated proximity labeling resolves protein networks in Saccharomyces cerevisiae cells. (12th August 2019)

• Record Type: Journal Article
• Authors: Singer‐Krüger, Birgit; Fröhlich, Theresa; Franz‐Wachtel, Mirita; Nalpas, Nicolas; Macek, Boris; Jansen, Ralf‐Peter
• Is Part Of: FEBS journal. Volume 287:Number 2(2020)
• Page Start: 325
• Page End: 344
• Publication Date: 2019-08-12
• Subjects: chromatin -- melanocyte‐proliferating factor MYG1 -- mitochondrial matrix -- proximity labeling -- SILAC; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.15007
• Languages: English
• ISSNs: 1742-464X

Abstract

Abstract : Enzyme‐catalyzed proximity labeling (PL) with the engineered ascorbate peroxidase APEX2 is a novel approach to map organelle compartmentalization and protein networks in living cells. Current procedures developed for mammalian cells do not allow delivery of the cosubstrate, biotin‐phenol, into living yeast cells. Here, we present a new method based on semipermeabilized yeast cells. Combined with stable isotope labeling by amino acids in cell culture (SILAC), we demonstrate proteomic mapping of a membrane‐enclosed and a semiopen 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. Its human ortholog, melanocyte proliferating gene 1, is linked to developmental processes and dermatological diseases. A first characterization of the Yer156C neighborhood reveals an array of proteins linked to proteostasis and RNA binding. Thus, our approach establishes APEX2 PL as another powerful tool that complements the methods palette for the model system yeast. Abstract : Poximity labeling with engineered ascorbate peroxidase APEX2 in yeast has suffered from difficulties in the delivery of the cosubstrate, biotin‐phenol. This has been overcome with an efficient labeling protocol based on cellular semipermeabilization by gradual freezing. Upon targeting of APEX2 to the mitochondrial matrix or the nucleus, spatially restricted biotinylation is achieved resulting in proteomic maps of excellent quality.