Identification of Cytosol-Facing Organelle Membrane-Proximal Proteins via Proximity-Dependent Biotinylation

Organelle membrane (OM)-resident or -proximal proteins that face the cytosol play a critical role in the import and exchange of ions, metabolites, and proteins between subcellular compartments, thereby regulating organelle biogenesis and function. However, the identification and functional characterization of these proteins pose challenges due to their dynamic spatial and temporal nature. We introduce a proximity labeling system using biotin ligase-mediated protein biotinylation in live Arabidopsis thaliana cells. This chapter outlines a detailed step-by-step protocol for identifying cytosol-facing OM-resident or -proximal proteins of mitochondria, chloroplasts, and peroxisomes in plant cells. This protocol can be adapted to identify cytosol-facing OM-resident and/or proximal proteins of any subcellular compartments of interest, as well as to map their spatiotemporal profiles under various stress conditions. The protocol described includes the following key steps: (1) sample preparation; (2) optimization of biotin labeling conditions; (3) enrichment of biotinylated proteins for liquid chromatography-tandem mass spectrometry (LC-MS/MS); (4) validation of identified candidate proteins.