Protein neighborhoods in living hyphae

Many protein interactions that control development are weak, transient or dynamic, and are easily lost when cells are broken up for classical affinity purification. Proximity-dependent biotin identification (BioID) avoids this problem. A promiscuous biotin ligase is fused to a bait protein and labels neighboring proteins with biotin inside the living cell. The biotinylated proteins are then enriched from denatured cell lysates and identified and quantified by liquid chromatography–mass spectrometry (LC–MS).
We established BioID for S. macrospora, using a codon-optimized TurboID biotin ligase. As proof of concept, the STRIPAK component SCI1 was used as bait and captured the known STRIPAK components PRO11, SmMOB3, PRO22 and SmPP2Ac1. A method description for in vivo proximity labeling with miniTurboID has been published. Applied to the STRIPAK complex, proximity labeling led to the identification of DOC1 and DOC2 as regulators of MAK2 signaling.