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. The authors would like to thank K.
Kusaka for their support in chemical synthesis and compound characterisation; H. Matsuba for plasmid preparation; K. Gonda for mouse experiments; S.
Sugimoto for lipid oxidation analysis; and H. Utsunomiya for early-stage evaluation of nanobody-photosensitiser conjugates. We also thank Radu Aricescu and Michisuke Yuzaki for their contributions to the preparation of nanobodies.
We thank Laura Murray, PhD, from Edanz for editing a draft of this manuscript. This work was supported by the Japan Society for the Promotion of Science Grant-in-Aid for Specially Promoted Research 23H05405 to I.H.; the JST FOREST program (JPMJFR230G) to T.T.; the Japan Society for the Promotion of Science Grant-in-Aid for JSPS Fellows 21J23228 to M.T.; the Ministry of Education, Culture, Sports, Science and Technology (MEXT) scholarship to F.Y.T.V. Department of Chemical Science and Engineering, Graduate School of Engineering, Kyoto University, Kyoto, Japan Fátima Yuri Tanimura Valor, Mikiko Takato, Ayane Araki, Seiji Sakamoto & Tomonori Tamura Institute of Advanced Energy, Kyoto University, Kyoto, Japan Correspondence to Tomonori Tamura or Itaru Hamachi.
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