Proximity-dependent biotin labelling in yeast using the engineered ascorbate peroxidase APEX2.
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.
Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, U.S.A.
Jiwon
J
Hwang
Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, U.S.A. peter.espenshade@jhmi.edu.
Peter J
PJ
Espenshade
D060387:Ascorbate Peroxidases / Q000378:metabolism*; D001710:Biotin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000201:enzymology / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology
D011994:Recombinant Proteins; D001710:Biotin; D060387:Ascorbate Peroxidases
APEX2; ascorbate peroxidase; protein–protein interaction; proximity-dependent biotinylation; sorbitol; yeast