APEX2-mediated proximity labeling resolves protein networks in Saccharomyces cerevisiae cells.
Ascorbate Peroxidases
/ chemistry
Cell Nucleus
/ metabolism
Isotope Labeling
/ methods
Mass Spectrometry
/ methods
Mitochondrial Proteins
/ metabolism
Protein Interaction Mapping
/ methods
Protein Interaction Maps
Proteomics
/ methods
Recombinant Proteins
/ chemistry
Saccharomyces cerevisiae
Saccharomyces cerevisiae Proteins
/ chemistry
SILAC
chromatin
melanocyte-proliferating factor MYG1
mitochondrial matrix
proximity labeling
Journal
The FEBS journal
ISSN: 1742-4658
Titre abrégé: FEBS J
Pays: England
ID NLM: 101229646
Informations de publication
Date de publication:
01 2020
01 2020
Historique:
received:
11
04
2019
revised:
18
06
2019
accepted:
17
07
2019
pubmed:
20
7
2019
medline:
21
10
2020
entrez:
20
7
2019
Statut:
ppublish
Résumé
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.
Substances chimiques
Mitochondrial Proteins
0
Recombinant Proteins
0
Saccharomyces cerevisiae Proteins
0
Ascorbate Peroxidases
EC 1.11.1.11
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
325-344Informations de copyright
© 2019 Federation of European Biochemical Societies.
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