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 Sordaria macrospora (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.

Figure 1

Schematic illustration of constructs used in BioID experiments. For BioID experiments, multiple constructs for the SCI1-TurboID fusion protein and the unfused TurboID control have been generated. The TurboID sequence was codon-optimized according to the codon usage table of S. macrospora. Expression is controlled by different promoters: p5’, native sci1 promoter; pc, promoter of the clock-controlled gene 1 (ccg1) from N. crassa for overexpression; px, xylose inducible promoter (Smxyl) of the beta-xylanase gene (SMAC_08023) from S. macrospora; L, 11 amino acid linker (MGGGGSGGGGS) attached to N-terminus of TurboID; TtrpC, terminator of the anthranilate synthase gene from A. nidulans; a scalebar is indicated.

Figure 2

Western blot detection of TurboID constructs using an anti-HA antibody. Expression of the (A) free TurboID and (B) SCI1-TurboID fusion proteins in S. macrospora wt and Δsci1 was determined by Western blot hybridization using a monoclonal anti-HA antibody, which detects the C-terminal 3xHA tag of TurboID. Red arrows indicate (A) free TurboID (38.5 kDa) and (B) SCI-TurboID fusion proteins (71.6 kDa). Expression of the constructs is controlled by either the ccg1 overexpression promoter from N. crassa (pc), the native sci1 promoter (p5’), or the xylose inducible Smxyl promoter (px). Untransformed wt was used as negative control. S. macrospora strains were grown in liquid BMM for 3 days at 27 °C. 18 µL of crude protein extract were loaded, and Ponceau red staining was used as loading control. Blots and Ponceau red staining in (A,B) show the identical membrane, exposure was identical for all parts of the gel. Figure shows cropped blots. Full length blots and Ponceau red staining are depicted in Supplementary Fig. S2. ect, ectopically integrated; ssi, single-spore isolate.

Figure 3

Western blot analysis of TurboID activity in recombinant S. macrospora strains. The activity of free TurboID and SCI1-TurboID fusion proteins in S. macrospora was determined by Western blot-like hybridization using Streptavidin-HRP. Streptavidin binds biotinylated proteins, and HRP is used for signal detection via chemiluminescence. Expression of the constructs is controlled by either the ccg1 overexpression promoter from N. crassa (pc), the native sci1 promoter (p5’), or the xylose inducible Smxyl promoter (px). Untransformed wt shows endogenous biotinylation of S. macrospora. 2.25 µL crude protein extract were loaded onto the gel. Ponceau red staining was used as loading control. A) Strains were grown in liquid BMM medium for 3 days at 27 °C. Before harvest, BMM supplemented with biotin was added (final concentration = 410 nM) for 10 or 30 min. B) Strains were grown in liquid SWG medium (1 g/L arginine, without biotin) for 5 days at 27 °C. Shortly before harvest, SWG supplemented with biotin was added (final concentration = 410 nM) for 10 or 30 min. Blots and Ponceau red staining in (A,B) show the identical membrane, exposure was identical for all parts of the gel. Figure shows cropped blots. Full length blots and Ponceau red staining are depicted in Supplementary Fig. S6. ect, ectopically integrated.

Figure 4

Workflow of BioID experiment with SCI1 as bait. For the establishment of BioID and identification of putative protein interaction partners of the SmSTRIPAK complex, the TurboID biotin ligase was fused to the bait protein SCI1 (C-terminally). Exogenous addition of biotin (yellow stars) leads to promiscuous biotinylation of proteins in the proximity of the SCI1-TurboID fusion protein. Distant proteins are not biotinylated. After 30 min of labeling, the cells are lysed, denatured, and biotinylated proteins are purified through Strep-Tactin Sepharose beads. The proteins are separated by SDS-PAGE. Peptides for LC–MS analysis are obtained by tryptic in-gel digestion.

Figure 5

Volcano plot of proteins identified in the SCI1-BioID experiment. The volcano plot depicts the differences of log₂ transformed LFQ intensities between Δsci1::p5'-sci1-L-TurboID^ect and the control (wt::pc-L-TurboID^ect diluted 1:6 with wild-type crude protein extract) on the x-axis. The −log₁₀(p-value) is plotted on the y-axis. The gray dotted threshold lines indicate a p-value of 0.05 (horizontal) and a log₂(difference) of −2 or + 2 (vertical). Missing values were imputed in four repetitions. Proteins that were significantly enriched (log₂(difference) ≥ 2) in the SCI1-L-TurboID strain in all four imputations are marked with a red square.