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Protein extraction and sample preparation were performed as described in Hollstein et al. (2025). In brief, biotinylated proteins were enriched from denatured cell lysates by affinity purification with Strep-Tactin® Sepharose® (iba Lifescience GmbH, Germany). Biotinylated proteins were released from the resin by adding a buffer containing 50 mM biotin for competitive elution. After chloroform methanol precipitation (Wessel & Flügge, 1984) of the eluated proteins, SDS-PAGE and in gel digestion with trypsin were applied to digest the proteins (Shevchenko et al., 1996). Peptides were desalted with the C18 stage tip purification protocol according to Rappsilber et al. (2003). Samples containing Strep-Tactin® Sepharose® enriched proteins were not fractionated, whereas the complex input controls were PAGE-separated into seven fractions in-gel trypsin digestion and the LC-MS analysis. Dried peptide samples were reconstituted in 20 µl LC-MS sample buffer (2 % acetonitrile, 0.1 % formic acid). 2 to 8 µl of each sample were subjected to reverse phase liquid chromatography for peptide separation using an RSLCnano Ultimate 3000 system (Thermo Fisher Scientific): Peptides were loaded on an Acclaim PepMap 100 pre-column (100 µm x 2 cm, C18, 5 µm, 100 Å; Thermo Fisher Scientific) with 0.07 % trifluoroacetic acid at a flow rate of 20 µL/min for 3 min. Analytical separation of peptides was done on an Acclaim PepMap RSLC column (75 µm x 50 cm, C18, 2 µm, 100 Å; Thermo Fisher Scientific) at a flow rate of 300 nL/min. The solvent composition was gradually changed within 94 min from 96 % solvent A (0.1 % formic acid) and 4 % solvent B (80 % acetonitrile, 0.1 % formic acid) to 10 % solvent B within 2 minutes, to 30 % solvent B within the next 58 min, to 45 % solvent B within the following 22 min, and to 90 % solvent B within the last 12 min of the gradient. All solvents and acids had Optima grade for LC-MS (Fisher Chemical). Eluting peptides were on-line ionized by nano-electrospray (nESI) using the Nanospray Flex Ion Source (Thermo Fisher Scientific) at 1.5 kV (liquid junction) and transferred into a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). Full scans in a mass range of 300 to 1650 m/z were recorded at a resolution of 30,000 followed by data-dependent top 10 HCD fragmentation at a resolution of 15,000 (dynamic exclusion enabled). LC-MS method programming and data acquisition was performed with the XCalibur 4.0 software (Thermo Fisher Scientific).  Hollstein, L. S., Groth, S., Schmitt, K., Valerius, O., & Pöggeler, S. (2025). In vivo proximity-labeling with miniTurboID to screen for protein-protein interactions in the filamentous ascomycete Sordaria macrospora. MethodsX, 14, 103351.  Wessel, D., & Flügge, U. I. (1984). A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Analytical Biochemistry, 138(1), 141–143.  Shevchenko, A., Wilm, M., Vorm, O., & Mann, M. (1996). Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Analytical Chemistry, 68(5), 850–858.  Rappsilber, J., Ishihama, Y., & Mann, M. (2003). Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Analytical Chemistry, 75(3), 663–670."],"repository":["Pride"],"quantification_method":[""],"modification":[""],"data_protocol":["The MaxQuant software (Cox & Mann, 2008; v1.6.10.43) was configured with standard parameters for protein identification and label free relative quantification using the MaxLFQ algorithm. The S. macrospora genome v04-derived protein database in FASTA format (Breuer et al., 2024) was used for the database search. Biotinylation of lysine and phosphorylation (at serine, threonine, or tyrosine) were considered as variable modifications. The number of maximum missed cleavage sites was set to 3 and trypsin/P was used for digestion. The proteinGroups.txt output file from MaxQuant was processed in Perseus v1.6.15.0 (Tyanova et al., 2016). Cox, J., & Mann, M. (2008). Maxquant enables high peptide identification rates, individualized p.P.B.-range mass accuracies and proteome-wide protein quantification. Nature Biotechnology, 26(12), 1367–1372.  Breuer, J., Busche, T., Kalinowski, J., & Nowrousian, M. (2024). Histone binding of ASF1 is required for fruiting body development but not for genome stability in the filamentous fungus Sordaria macrospora. MBio, 15(1), e0289623.  Tyanova, S., Temu, T., Sinitcyn, P., Carlson, A., Hein, M. Y., Geiger, T., Mann, M., & Cox, J. (2016). The Perseus computational platform for comprehensive analysis of (prote)omics data. Nature Methods, 13(9), 731–740."],"omics_type":["Proteomics"],"labhead":["Dr. Prof. Stefanie Pöggeler"],"instrument_platform":[""],"submission_type":["PARTIAL"],"labhead_affiliation":["Georg-August-Universität Göttingen, Institute of Microbiology and Genetics, Department of Genetics of Eukaryotic Microorganisms"],"species":["Sordaria Macrospora"],"publication":["10.1371/JOURNAL.PBIO.3003969"],"submitter_mail":["ovaleri@gwdg.de"],"submitter_affiliation":["Georg-August-Universität Göttingen"],"submitter_country":["Germany"],"additional_accession":[]},"is_claimable":false,"name":"Proximity labeling of the SmSTRIPAK complex in Sordaria macrospora using the SCI1 subunit as bait to identify its SmSTRIPAK-dependent microenvironment","description":"In this study, we used the in vivo proximity labeling biotin identification (BioID; Roux et al., 2012) method to capture proteins of the microenvironment of the striatin interacting kinase and phosphatase complex of the filamentous fungus Sordaria macrospora (SmSTRIPAK). For that, the TurboID biotin ligase (Branon et al., 2018) was fused to the STRIPAK complex interactor 1 (sci1; SMAC_05559), a subunit of the SmSTRIPAK complex (Hollstein et al., 2022). We applied two control strains for relative quantification to reduce false positive hits. The first control expressed a free unfused TurboID ligase under the control of the constitutive promoter of the clock controlled gene 1 (ccg1) from Neurospora crassa. For the second control, we crossed the strain expressing the sci1-TurboID fusion gene (native sci1 promoter) with the Δpro11 (Bloemendal et al., 2012) deletion strain. The pro11 gene encodes a striatin homolog, which serves as the main scaffold of the SmSTRIPAK complex. Therefore, the second control setup enables the comparison of the proxiome of SCI1-TurboID in the presence or absence of the assembled SmSTRIPAK complex (Δpro11). We generated additional input controls of the crude protein extracts with aliquots taken before biotin affinity purification.  Roux, K. J., Kim, D. in, Raida, M., & Burke, B. (2012). A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. The Journal of Cell Biology, 196(6), 801–810. Branon, T. C., Bosch, J. A., Sanchez, A. D., Udeshi, N. D., Svinkina, T., Carr, S. A., Feldman, J. L., Perrimon, N., & Ting, A. Y. (2018). Efficient proximity labeling in living cells and organisms with TurboID. Nature Biotechnology, 36(9), 880–887.  Hollstein, L. S., Schmitt, K., Valerius, O., Stahlhut, G., & Pöggeler, S. (2022). Establishment of in vivo proximity labeling with biotin using TurboID in the filamentous fungus Sordaria macrospora. Scientific Reports, 12(1), 17727.  Bloemendal, S., Bernhards, Y., Bartho, K., Dettmann, A., Voigt, O., Teichert, I., Seiler, S., Wolters, D. A., Pöggeler, S., & Kück, U. (2012). A homologue of the human STRIPAK complex controls sexual development in fungi. Molecular Microbiology, 84(2), 310–323.","dates":{"publication":"2026-08-19","submission":"2025-10-08"},"accession":"PXD069198","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:377960","NEWT:1131","NEWT:35795","NEWT:2042546","NEWT:259447","NEWT:295546","NCBITaxon:2719036","NEWT:1334565","NEWT:625351","NEWT:258594","NCBITaxon:1280","NEWT:112503","NEWT:1129","NEWT:309807","NEWT:59753","NEWT:89184","NEWT:309800","NEWT:281395","NEWT:57577","NEWT:247480","NEWT:1211601","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:592321","NEWT:1073882","NEWT:6819","NEWT:77173","NEWT:112509","NEWT:475174","NEWT:111205","NEWT:1599","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:12215","NEWT:1590","NEWT:10034","NEWT:33114","NEWT:1596","NEWT:661410","NEWT:638632","NEWT:224326","NEWT:294671","NEWT:376619","NEWT:1748418","NCBITaxon:79857","NEWT:1096976","NEWT:3765","NEWT:3767","NEWT:1589","NEWT:1907535","NEWT:135622","NEWT:4615","NEWT:67352","NEWT:672127","NEWT:35786","NEWT:1580","NEWT:399784","NEWT:96731","NEWT:656064","NEWT:1582","NEWT:3760","NEWT:1584","NEWT:2086595","NEWT:36630","NEWT:1585","NEWT:383379","NEWT:381198","NEWT:418106","NCBITaxon:12721","NEWT:10029","NEWT:913645","NEWT:2492962","NEWT:641809","NEWT:1392488","NEWT:38815","NEWT:317447","NEWT:4688","NEWT:3357","NEWT:111225","NEWT:7719","NEWT:29073","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:1562698","NEWT:79329","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:295105","NEWT:29064","NEWT:108061","NEWT:60711","NEWT:2020713","NEWT:224308","NEWT:273791","NEWT:3347","NEWT:49928","NEWT:3349","NEWT:160621","NEWT:2502409","NEWT:212790","NEWT:110368","NEWT:55350","NEWT:1916073","NEWT:1310161","NEWT:77133","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:1912919","NEWT:410658","NEWT:5516","NEWT:44688","NEWT:44689","NEWT:370038","NEWT:498211","NEWT:345076","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:33554","NEWT:246196","NEWT:246197","NEWT:145458","NEWT:1310173","NEWT:200795","NEWT:37919","NEWT:2579791","NEWT:171719","NEWT:44685","NEWT:161934","NEWT:156587","NEWT:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","NEWT:260499","NEWT:410661","NEWT:329854","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:2649329","NEWT:1143","NEWT:1287689","NEWT:1094343","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:150808","NEWT:1182590","NEWT:3712","NEWT:3711","NEWT:3714","NCBITaxon:37162","NEWT:270643","NEWT:3715","NEWT:1538","NEWT:2065263","NEWT:199225","NEWT:177437","NEWT:717966","NEWT:10891","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:118696","NEWT:34865","NEWT:52283","NEWT:28174","NEWT:284812","NEWT:8175","NEWT:43330","NEWT:980415","NEWT:1603293","NEWT:44664","NEWT:43346","NEWT:3702","NEWT:66440","NEWT:1245466","NEWT:3704","NEWT:244366","NEWT:1246791","NEWT:176597","NEWT:118694","NEWT:2850","NEWT:118691","NEWT:34871","NEWT:9006","NEWT:33548","NEWT:52299","NEWT:8165","NEWT:96794","NEWT:34878","NEWT:3708","NEWT:332648","NEWT:44670","NEWT:926571","NEWT:536231","NEWT:376219","NEWT:219813","NEWT:1510","NEWT:460519","NEWT:1515","NEWT:572307","NEWT:1432138","NEWT:2714896","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1348799","NEWT:1608994","NEWT:33995","NEWT:55784","NEWT:1303443","NEWT:9483","NEWT:485","NEWT:56636","NEWT:2709072","NEWT:2499525","NEWT:1122981","NEWT:2853422","NEWT:1122984","NEWT:487","NEWT:1538553","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:45972","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1185650","NEWT:216778","NEWT:294607","NEWT:1502","NEWT:128017","NEWT:2836","NEWT:376686","NEWT:237610","NEWT:95486","EFO:0001352","NEWT:1385720","NEWT:119562","NEWT:1883446","NEWT:31345","NEWT:1233435","NEWT:118223","NEWT:11757","NEWT:987059","NEWT:109760","NEWT:29031","NEWT:1945864","NEWT:317047","NEWT:1509","NEWT:491","NEWT:460523","NEWT:45954","NEWT:235443","NEWT:1579","NEWT:133894","NEWT:1757312","NEWT:85694","NEWT:109786","NEWT:1922344","NEWT:108458","NEWT:5936","NEWT:85692","NEWT:321967","NEWT:320637","NEWT:1078283","NEWT:3750","NEWT:983964","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:279010","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:1116234","NEWT:415978","NEWT:1255228","NEWT:410289","NEWT:191701","NEWT:373153","NEWT:1640278","NEWT:9054","NEWT:472","NEWT:940825","NEWT:29459","NEWT:529507","NEWT:1071661","NEWT:1380096","NEWT:28128","NEWT:470","NEWT:41122","NEWT:5911","NEWT:1328300","NEWT:748693","NCBITaxon:50557","NEWT:39251","NEWT:29491","NEWT:101841","NEWT:446","NEWT:153481","NEWT:2014887","NEWT:33952","NEWT:11723","NEWT:445","NEWT:153009","NEWT:261756","NEWT:166314","NEWT:166313","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:215402","NEWT:1547","NEWT:1290454","NEWT:3726","NEWT:906914","NEWT:81231006","NEWT:30455","NEWT:28141","NEWT:9031","NEWT:27292","NEWT:1050722","NEWT:3728","NEWT:108931","NEWT:1293497","NEWT:1055524","NEWT:548","NEWT:150475","NEWT:267872","NEWT:381666","NEWT:172269","NEWT:9534","NEWT:2126348","NEWT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