Enzymatic-based proximity labeling approaches based on activated esters or phenoxy radicals have been widely used for mapping subcellular proteome and protein interactors in living cells. However, activated esters are poorly reactive which leads to a wide labeling radius and phenoxy radicals generated by peroxide treatment may disturb redox-sensitive pathways. Herein, we report a photoactivation-dependent proximity labeling (PDPL) method designed by genetically attaching photosensitizer protein miniSOG to a protein of interest. Triggered by blue light and tunned by irradiation time, singlet oxygen is generated, thereafter enabling spatiotemporally-resolved aniline probe labeling of histidine residues. We demonstrate its high-fidelity through mapping of organelle-specific proteomes. Side-by-side comparison of PDPL with TurboID reveals more specific and deeper proteomic coverage by PDPL. We further apply PDPL to the disease-related transcriptional coactivator BRD4 and E3 ligase Parkin, and discover previously unknown interactors. Through over-expression screening, two unreported substrates Ssu72 and SNW1 are identified for Parkin, whose degradation processes are mediated by the ubiquitination-proteosome pathway.

Protein interaction networks underpin the fundamental cellular processes. Enzymatic-based proximity labeling (PL) approaches based on activated esters or phenoxy radicals have been widely used for mapping subcellular proteome and protein interaction partners in living cells. However, activated esters are poorly reactive which can lead to a wide labeling radius and phenoxy radicals generated by peroxide treatment may disturb redox sensitive cellular process. Herein, we report a photoactivation-dependent proximity labeling (PDPL) method designed by genetically attaching photosensitizer protein miniSOG to the protein of interest (POI). Under the trigger of blue light, singlet oxygen is generated to enable labeling by a chemical probe with temporal resolution. Irradiation time could regulate the labeling radius that allows for spatial resolution. Open search for modification sites revealed selective modification of histidine residues. We demonstrated the high-fidelity and spatiotemporal resolved mapping of organelle-specific proteome. Side-by-side comparison of PDPL with TurboID revealed a more specific and deeper proteomic coverage by PDPL. Furthermore, PDPL was applied in disease-related proteins, the transcription coactivator BRD4 and E3 ligase Parkin. We validated known interactors and novel interactions for BRD4 and Parkin. Meanwhile, two novel substrates Ssu72 and SNW1 were identified for Parkin. The degradation process of these two proteins is mediated by ubiquitination-proteosome pathway. Together, these data establish PDPL as a general method capable of tracking subcellular and protein interaction networks in cells with high spatiotemporal resolution.