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

Jiwon Hwang¹, Peter J Espenshade²

¹Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, U.S.A.

The Biochemical Journal

|June 9, 2016

Summary

We developed a new method for proximity-dependent biotin labeling in yeast using engineered ascorbate peroxidase (APEX2). This technique enables proteomic analysis in yeast, expanding APEX2 applications to diverse organisms.

Keywords:

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

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Area of Science:

Biochemistry
Molecular Biology
Proteomics

Background:

Engineered ascorbate peroxidase (APEX2) is a powerful tool for identifying protein-protein interactions in mammalian cells via proximity-dependent biotinylation.
The APEX2 system allows for rapid, temporally controlled labeling of proteins with biotin-phenol (BP) upon addition of H2O2.
Existing APEX2 methods have not been adapted for use in yeast, limiting its application in this important model organism.

Purpose of the Study:

To establish APEX2-mediated biotin labeling methods for live yeast cells.
To enable proteomic studies and the identification of protein complexes in yeast.

Main Methods:

Developed specific conditions for APEX2-mediated biotin labeling in Schizosaccharomyces pombe (high osmolarity) and Saccharomyces cerevisiae (cell wall disruption).
Utilized APEX2 fused to a protein of interest to covalently tag proximal proteins with BP in live yeast cells.
Employed affinity purification of biotinylated proteins followed by mass spectrometry (MS) for identification.

Main Results:

Successfully demonstrated APEX2-mediated proximity-dependent biotin labeling in both S. pombe and S. cerevisiae.
Identified specific cell treatments (high osmolarity or cell wall disruption) that enable live-cell labeling in yeast.
Validated the APEX2 system's ability to target and label proteins based on proximity in yeast.

Conclusions:

Established robust methods for APEX2-mediated biotin labeling in yeast, overcoming previous limitations.
These methods pave the way for large-scale proteomic analyses and the study of dynamic protein interactions in yeast.
The adapted APEX2 approach is potentially applicable to other cell-walled organisms like bacteria and plants.