Transmembrane PhoxID: photoproximity labelling across the plasma membrane in vivo

Transmembrane proteins perform essential roles in cellular transport, signalling, and communication. The function and dynamics of these proteins are precisely regulated by interactions on both sides of the plasma membrane; thus, mapping the composition of these interactomes is a fundamental challenge in molecular biology. Proximity labelling methods are powerful tools for this purpose; however, existing approaches that rely on membrane-impermeable reactive species generally focus on either the extracellular or the intracellular region of transmembrane proteins. Here, we capitalise on the membrane permeability of singlet oxygen to carry out simultaneous proximity labelling of both the extra- and intracellular sides of transmembrane proteins using an extracellularly anchored photosensitiser. We apply this method, termed transmembrane PhoxID (tmPhoxID), to several receptors (GRID2, GABAAR, and GRM1) in the living mouse brain and successfully determine their specific membrane-proximal intracellular interactomes. Notably, network analysis of the identified proteins reveals that this method can characterise the native components of transsynaptic nanocolumns formed at parallel fibre–Purkinje cell synapses. Furthermore, our study reveals a previously uncharacterised GABAAR-CAMKV interaction in mice and human brains. Our results provide a proof of concept for transmembrane and transcellular proximity labelling, establishing a powerful platform for analysing the interactomes of transmembrane proteins. Transmembrane proteins interact with molecules on both sides of the cell membrane to regulate cellular functions. Here, the authors develop tmPhoxID, a proximity labelling method that simultaneously maps proteins near both sides of transmembrane proteins in living mouse brains.