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Proteomic Mapping by APEX2-Catalyzed Proximity Labeling in Saccharomyces cerevisiae Semipermeabilized Cells
Birgit Singer-Krüger1, Ralf-Peter Jansen2
1Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany. birgit.singer-krueger@uni-tuebingen.de.
Methods in Molecular Biology (Clifton, N.J.)
|May 6, 2022
Summary
This study introduces a proximity labeling (PL) method using engineered ascorbate peroxidase (APEX2) for proteomic mapping in yeast. The approach utilizes semipermeabilized cells to efficiently label subcellular compartments like the nucleus.
Area of Science:
- Proteomics
- Cell Biology
- Biochemistry
Background:
- Enzyme-catalyzed proximity labeling (PL) is crucial for mapping protein networks and subcellular compartments in living cells.
- Engineered ascorbate peroxidase (APEX2) offers a powerful tool for PL applications.
- Challenges exist in delivering APEX2 substrates like biotin-phenol into cells for effective labeling.
Purpose of the Study:
- To develop a proximity labeling protocol for budding yeast using APEX2.
- To overcome the limitations of cellular permeability for APEX2 substrates.
- To enable high-resolution proteomic mapping of subcellular compartments, such as the yeast nucleus.
Main Methods:
- Utilized semipermeabilized yeast cells to enhance biotin-phenol delivery.
- Employed a ratiometric three-state stable isotope labeling by amino acids in cell culture (SILAC) approach.
- Focused on optimized proteomic sample preparation for yeast.
Main Results:
- Successfully adapted APEX2-based proximity labeling for budding yeast.
- Demonstrated the efficacy of semipermeabilized cells in overcoming substrate delivery issues.
- Achieved high-resolution mapping of the yeast nuclear proteome.
Conclusions:
- The developed protocol provides an efficient method for proximity labeling in yeast.
- Semipermeabilization is a key strategy for improving PL efficiency in yeast.
- This technique facilitates detailed proteomic analysis of subcellular compartments.
