Validation of TurboID constructs
For the establishment of BioID in S. macrospora, multiple plasmids were generated and transformed into wild-type or the Δsci1 SmSTRIPAK mutant (Supplementary Note 1, Supplementary Table S1 – Table S3). These transformed strains express either the SCI1‑TurboID fusion protein or an unfused TurboID ligase, the latter one serving as negative control (Figure 1). The TurboID ligase was codon-optimized according to the codon usage table of S. macrospora  (Supplementary Figure S1). In addition, a 3xHA-tag was C-terminally fused to TurboID for convenient detection in immunoblots. Furthermore, we constructed plasmids with and without a 10 amino acid GGGGSGGGGS linker at the N-terminus of TurboID. The expression of free TurboID is either controlled by the ccg1 constitutive promoter (pc) of the clock‑controlled gene 1 from Neurospora crassa or the xylose inducible Smxyl promoter (px) of the beta‑xylanase gene (SMAC_08023) from S. macrospora . Either the native sci1 promoter (p5’) or the constitutive ccg1 promoter controls expression of the SCI1‑TurboID fusion protein in the Δsci1 strain. Based on the amino acid sequence, the expected molecular masses were calculated for free TurboID (~ 40.0 kDa), the SCI1-TurboID fusion protein (~ 73.1 kDa), and the linker (~ 1.3 kDa). To verify expression and compare the protein levels among the free TurboID and SCI1-TurboID strains, all available strains were grown in liquid Sordaria Westergaard’s (SWG) medium for 4 days at 27 °C. After protein extraction, the crude protein extract was separated by SDS‑polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted using a monoclonal anti-HA antibody for signal detection (Supplementary Figure S2).
The free TurboID ligase and the SCI1-TurboID fusion proteins were detected at their expected molecular masses in the Western blot experiment (Supplementary Figure S2). In addition, the expression of TurboID was verified with an anti-BirA antibody (Supplementary Figure S3). For the SCI1-BioID experiments we decided to use the native sci1 promoter for authentic expression regulation to remain at the most physiological conditions for the SCI1-BioID experiments. The functionality of the SCI1-TurboID fusion protein was confirmed by complementation of a S. macrospora Δsci1 mutant. Deletion of sci1 causes sterility due to the inability to develop fruiting bodies . Hence, the transformation of Δsci1 with SCI1-TurboID constructs should rescue this deficiency and restore fertility. For the complementation analysis Δsci1, Δsci1::p5’-sci1-TurboIDect and Δsci1::p5’‑sci1‑L‑TurboIDect were grown on solid medium. Both deletion strains expressing the TurboID-tagged sci1 gene were complemented and developed mature fruiting bodies, which indicates that the SCI1-TurboID and SCI1-L-TurboID fusion proteins can fulfill the endogenous function of SCI1. Sexual structures such as female ascogonia and protoperithecia developed in the complemented deletion mutant in the same time as in the wild-type (Supplementary Figure S4). Furthermore, the linker did not affect the complementation (Supplementary Figure S5).
Finally, the ligase activity of TurboID was analyzed. Selected strains were grown in liquid Biomalt maize (BMM) complex medium and SWG (synthetic medium without biotin) for 3 and 5 days at 27 °C, respectively. Shortly before harvest, exogenous biotin was added to the liquid cultures to increase the potential of protein labeling proximal to the bait. This biotin boost was provided for 10 or 30 minutes with biotin solved in either BMM or SWG medium to a final concentration of 410 nM. A non boosted sample served as control and represented the level of biotinylation occurring throughout the growth without biotin boost. Strains grown in BMM were harvested after 3 days, whereas cultivation in SWG (without biotin) had to be prolonged to 5 days due to extremely low yields of mycelium. A streptavidin-HRP conjugate was used for the detection of biotinylated proteins (Supplementary Figure S6).
S. macrospora strains grown in complex BMM medium exhibit biotinylated proteins even without the supplementation of exogenous biotin during boosting. Supplementation of the untransformed wild-type strain with biotin led to a slight increase in natural protein biotinylation. Overexpression of the free TurboID by the ccg1 promoter resulted in strong background biotinylation after 10 min, which increased even further after 30 min of boosting. As this substantial increase in biotinylation was not detectable in the wild-type strain, this increased biotinylation is caused by the activity of TurboID. Expression of TurboID under control of the xylose inducible Smxyl promoter yields biotinylation levels similar to those in wild-type cells. When TurboID was fused to SCI1 and expression controlled by the sci1 promoter, only a slight increase in biotinylation was observed, similar to the biotinylation in wild-type cells. Hence, it is unclear whether the biotinylation in Δsci1::p5’‑sci1‑L-TurboIDect is the result of endogenous ligases or the SCI1‑TurboID fusion protein (Supplementary Figure S6).
When grown in synthetic SWG medium without biotin, biotinylated proteins were mostly absent (Supplementary Figure S6). Strains grown in SWG medium and boosted with biotin show similar biotinylation patterns as in BMM medium. Overexpression of free TurboID led to strong biotinylation upon boosting. In contrast, expression of free TurboID from the Smxyl promoter and expression of SCI1‑TurboID under control of the native sci1 promoter result in wild-type-like biotinylation levels after exogenous supplementation of biotin. In both media, biotinylation levels were higher when the boosting duration was prolonged from 10 to 30 minutes. Therefore, boosting in the following experiments was performed for 30 minutes.
We observed that growth of S. macrospora in SWG medium without biotin is not feasible for further experiments, as mycelium yields were extremely low and sexual development was inhibited. These results indicated that S. macrospora might be biotin auxotroph. Basic Local Alignment Search Tool (BLAST) searches with fungal genes of the biotin synthesis pathway revealed no homologs of de novo biotin biosynthesis genes in the genome of S. macrospora. Only a homolog of high-affinity plasma membrane H+-biotin (vitamin H) symporter SmVHT1 is encoded by S. macrospora. This result confirmed that S. macrospora relies on exogenous biotin supplementation for growth and development (Supplementary Figure S7).
For BioID experiments in S. macrospora, the following criteria were set for the desired medium: 1) full complementation of the life cycle, as indicated by discharged ascospores after 7‑9 days; 2) low concentration of biotin to avoid potential side effects of unspecific biotinylation by the TurboID ligase during vegetative growth. To find a suitable biotin concentration, S. macrospora was grown on SWG with 410, 200, 100, or 50 nM biotin (Supplementary Figure S8). Based on these results, strains for BioID experiments were grown in liquid SWG medium supplemented with 50 nM biotin for 4 days at 27 °C. Shortly before harvest of the mycelium, the cultures were boosted with SWG supplemented with 10 µM biotin for 30 min at RT.
TurboID proximity proteomics with the fusion protein SCI1-L-TurboID
The workflow of a BioID experiment with SCI1 as bait fused to TurboID is depicted in Figure 2. To reliably identify SCI1 proximal proteins and putative physical interactors of the SmSTRIPAK protein SCI1, the BioID experiment was performed with four biological replicates of Δsci1::p5'-sci1-L-TurboIDect (single spore isolates (ssi) 1.3, 1.6, 1.9, 1.10) and wt::pc-L-TurboIDect (ssi 1.7, 1.8, 1.9, 1.11). As the expression of free TurboID by the ccg1 overexpression promoter results in high biotinylation levels, the crude protein extract of the control strain was diluted 1:6 with extract of the wild-type strain (non boosted) to match the biotinylation level of the Δsci1::p5'-sci1-L-TurboIDect strain. Biotinylated proteins were enriched using Strep‑Tactin® Sepharose® beads and eluate proteins were afterwards separated with SDS‑PAGE. Proteins were digested with trypsin in-gel prior to liquid chromatography‑mass spectrometry (LC-MS) analysis.
The reproducibility of the four biological replicates from both strains was high, as indicated by mean Pearson correlations of 0.9556 among Δsci1::p5'-sci1-L-TurboIDect replicates, and 0.9676 among the control replicates. A total of 1185 proteins were identified. The Label Free Quantification (LFQ) intensities and MS/MS counts of proteins significantly enriched against the negative control (enrichment factor ≥ 4) are listed in Table 2. The SmSTRIPAK components SCI1, PRO11, SmMOB3, PRO22, and SmPP2Ac1 were identified among the top hits. One peptide of SmPP2Ac1 was found to be biotinylated at residue K308. The volcano plot in Figure 3 shows SmSTRIPAK components identified relative to the other enriched proteins dependent on statistical significance and log2(difference) values as described in Supplementary Table S4. Uncharacterized candidate proteins were identified using the UniProtKB BLAST. The BLAST results are listed in Supplementary Table S5.
In addition to the known SmSTRIPAK components, 4 other proteins were significantly enriched: a putative biotin apo-protein ligase (SMAC_00725), a signal recognition particle 54 kDa protein (SMAC_05070), a putative NAD‑dependent protein deacetylase (SMAC_01219), and a putative pre-mRNA-splicing factor SPF27. No phosphorylation sites (STY) were identified among these top hits.