Proximity Protein Labeling In Dictyostelium With Engineered Ascorbic Acid Peroxidase 2

Jamie A Takashima; Helena A Woroniecka; Pascale G Charest

doi:10.14440/jbm.2023.396

Proximity Protein Labeling In Dictyostelium With Engineered Ascorbic Acid Peroxidase 2

J Biol Methods. 2023 Mar 16:10:e99010002. doi: 10.14440/jbm.2023.396. eCollection 2023.

Authors

Jamie A Takashima^{1

2}, Helena A Woroniecka¹, Pascale G Charest^{1

3}

Affiliations

¹ Department of Chemistry and Biochemistry, University of Arizona, Tucson AZ, USA.
² Current address: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena CA, USA.
³ Department of Molecular and Cellular Biology, University of Arizona, Tucson AZ, USA.

Abstract

To fully understand any cellular process, we not only need to identify the proteins implicated, but also how the protein network is structurally and spatially organized and changes over time. However, the dynamic nature of many protein interactions involved in cellular signaling pathways continues to be the bottleneck in mapping and studying protein networks. Fortunately, a recently developed proximity labeling method using engineered ascorbic acid peroxidase 2 (APEX2) in mammalian cells allows the identification of weak and/or transient protein interactions with spatial and temporal resolution. Here, we describe a protocol for successfully using the APEX2-proximity labeling method in Dictyostelium, using the cAMP receptor cAR1 as example. Coupled to the identification of the labeled proteins by mass spectrometry, this method expands Dictyostelium's proteomics toolbox and should be widely useful for identifying interacting partners involved in a variety of biological processes in Dictyostelium.

Keywords: APEX; Biotinylation; Dictyostelium discoideum; Engineered ascorbic peroxidase; Protein-protein interaction; Proximity labeling.

Abstract

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