Establishment of in vivo proximity labeling with biotin using TurboID in the filamentous fungus Sordaria macrospora

Abstract

Proximity-dependent biotin identification (BioID) has emerged as a powerful methodology to identify proteins co-localizing with a given bait protein in vivo. The approach has been established in animal cells, plants and yeast but not yet in filamentous fungi. BioID relies on promiscuous biotin ligases fused to bait proteins to covalently label neighboring proteins with biotin. Biotinylated proteins are specifically enriched through biotin affinity capture from denatured cell lysates and subsequently identified and quantified with liquid chromatography-mass spectrometry (LC–MS). In contrast to many other affinity capture approaches for studying protein–protein interactions, BioID does not rely on physical protein–protein binding within native cell lysates. This feature allows the identification of protein proximities of weak or transient and dynamic nature. Here, we demonstrate the application of BioID for the fungal model organism Sordariamacrospora (Sm) using the example of the STRIPAK complex interactor 1 (SCI1) of the well-characterized striatin-interacting phosphatase and kinase (SmSTRIPAK) complex as proof of concept. For the establishment of BioID in S.macrospora, a codon-optimized TurboID biotin ligase was fused to SCI1. Biotin capture of the known SmSTRIPAK components PRO11, SmMOB3, PRO22 and SmPP2Ac1 demonstrates the successful BioID application in S.macrospora. BioID proximity labeling approaches will provide a powerful proteomics tool for fungal biologists.

The interactome of the UapA transporter reveals putative new players in anterograde membrane cargo trafficking

“…This unique feature allows for the identification of protein proximities that are weak, transient, and dynamic, such as the interactions occurring during vesicular secretion. PDB approaches have been established in animal cells, plants, and recently fungi (for examples see Mair et al, 2019;Larochelle et al, 2019;Arora et al, 2020;Kershberg et al, 2022, Yang et al, 2022Hollstein et al, 2022;Fenech et al, 2023), but have not been used in A. nidulans. Also, to our knowledge, no study has used PDB methods for identifying the very rapid and transient interactions of a moving transmembrane cargo.…”