Mapping of cytosol-facing organelle outer membrane proximity proteome by proximity-dependent biotinylation 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. However, the OM proximity proteomes have never been mapped in plant cells since 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 identify the proximity proteins of the OMs of mitochondria, chloroplasts, and peroxisomes in living Arabidopsis (Arabidopsis thaliana) cells. Using this approach, we mapped the OM proximity proteome of these three organelles under normal conditions and examined the effects of the ultraviolet-B (UV-B) or high light (HL) stress on the abundances of OM proximity proteins. We demonstrate the power of this system with the discovery of cytosolic factors and OM receptor candidates potentially involved in local protein translation and translocation. The candidate proteins that are involved in mitochondrion-peroxisome, mitochondrion-chloroplast, or peroxisome-chloroplast contacts, and in the organellar quality control system are also proposed based on OMPL analysis. 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.