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.
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A schematic representation of the potential effect (in low intracellular Ca2+ conditions) of the Ca2+-binding, EF-hand-containing cytoskeletal protein EFhd2 on kinesin-mediated transport and actin dynamics in presynaptic compartments. For a detailed overview of the links between EFhd2 and neurodegeneration, see the review by Mielenz and Gunn-Moore in this issue (Volume 473, Issue 16, pages 2429–2437). - PDF Icon PDF LinkFront Matter
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Research Article|
August 11 2016
Proximity-dependent biotin labelling in yeast using the engineered ascorbate peroxidase APEX2
Jiwon Hwang;
Jiwon Hwang
*Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, U.S.A.
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Peter J. Espenshade
Peter J. Espenshade
1
*Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, U.S.A.
1To whom correspondence should be addressed (email peter.espenshade@jhmi.edu).
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Publisher: Portland Press Ltd
Received:
February 11 2016
Revision Received:
June 03 2016
Accepted:
June 07 2016
Accepted Manuscript online:
June 07 2016
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society
2016
Biochem J (2016) 473 (16): 2463–2469.
Article history
Received:
February 11 2016
Revision Received:
June 03 2016
Accepted:
June 07 2016
Accepted Manuscript online:
June 07 2016
Citation
Jiwon Hwang, Peter J. Espenshade; Proximity-dependent biotin labelling in yeast using the engineered ascorbate peroxidase APEX2. Biochem J 15 August 2016; 473 (16): 2463–2469. doi: https://doi.org/10.1042/BCJ20160106
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