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APEX2‐mediated proximity labeling resolves protein networks in Saccharomyces cerevisiae cells
Author(s) -
SingerKrüger Birgit,
Fröhlich Theresa,
FranzWachtel Mirita,
Nalpas Nicolas,
Macek Boris,
Jansen RalfPeter
Publication year - 2020
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.15007
Subject(s) - stable isotope labeling by amino acids in cell culture , proteostasis , saccharomyces cerevisiae , biology , yeast , exoribonuclease , microbiology and biotechnology , biochemistry , interactome , compartment (ship) , gene , proteomics , rna , rnase p , oceanography , geology
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.