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Biochem J. 2016 Aug 15;473(16):2463-9. doi: 10.1042/BCJ20160106. Epub 2016 Jun 7.

Proximity-dependent biotin labelling in yeast using the engineered ascorbate peroxidase APEX2.

Author information

1
Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, U.S.A.
2
Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, U.S.A. peter.espenshade@jhmi.edu.

Abstract

The engineered ascorbate peroxidase (APEX2) has been effectively employed in mammalian cells to identify protein-protein interactions. APEX2 fused to a protein of interest covalently tags nearby proteins with biotin-phenol (BP) when H2O2 is added to the cell culture medium. Subsequent affinity purification of biotinylated proteins allows for identification by MS. BP labelling occurs in 1 min, providing temporal control of labelling. The APEX2 tool enables proteomic mapping of subcellular compartments as well as identification of dynamic protein complexes, and has emerged as a new methodology for proteomic analysis. Despite these advantages, a related APEX2 approach has not been developed for yeast. Here we report methods to enable APEX2-mediated biotin labelling in yeast. Our work demonstrated that high osmolarity and disruption of cell wall integrity permits live-cell biotin labelling in Schizosaccharomyces pombe and Saccharomyces cerevisiae respectively. Under these conditions, APEX2 permitted targeted and proximity-dependent labelling of proteins. The methods described herein set the stage for large-scale proteomic studies in yeast. With modifications, the method is also expected to be effective in other organisms with cell walls, such as bacteria and plants.

KEYWORDS:

APEX2; ascorbate peroxidase; protein–protein interaction; proximity-dependent biotinylation; sorbitol; yeast

PMID:
27274088
PMCID:
PMC5290329
DOI:
10.1042/BCJ20160106
[Indexed for MEDLINE]
Free PMC Article

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