A Proximity-Dependent Biotinylation Map of Cytosol-Facing Organelle Outer Membrane Proteome in Living Arabidopsis Cells

The cytosol-facing outer membrane (OM) of organelles communicates with other cellular compartments to exchange proteins, metabolites and signaling molecules. Cellular surveillance systems also target OM-resident proteins to control organellar homeostasis and ensure cell survival under stress. Using traditional approaches to discover OM proteins and identify their dynamically interacting partners remains challenging. In this study, we developed an OM proximity labeling (OMPL) system using biotin ligase-mediated proximity biotinylation to map the proximity proteome of the OMs of mitochondria, chloroplasts, and peroxisomes in living Arabidopsis ( Arabidopsis thaliana ) cells. We demonstrate the power of this system with the discovery of cytosolic factors and OM receptor candidates involved in local protein translation and translocation, membrane contact sites, and organelle quality control. This system also performed admirably for the rapid isolation of intact mitochondria and peroxisomes. Our data support the notion that TOM20-3 is a candidate for both a mitochondrial and a chloroplast receptor, and that PEX11D is a candidate for a peroxisome receptor for the coupling of protein translation and import. OMPL-generated OM proximity proteomes are valuable sources of candidates for functional validation and suggest directions for further investigation of important questions in cell biology.