Identified protein interactors of four predicted Microtubule Organizing Center (MTOC) components in Neurospora crassa by LC-MS/MS
The dataset provides numerous putative interactors of four proteins at the spindle pole body and/or septa in the fungus Neurospora crassa, obtained by Co-Immunoprecipitation with GFP-Magnetic agarose followed by nano LC-MS/MS.
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Four strains of Neurospora crassa were constructed for expressing fluorescent-labeled proteins: γ-tubulin-sGFP (main MTOC component), MZT-1-sGFP (putative MTOC component), dRFP-APS-2 (putative MTOC and septal component), and SPA-10-sGFP (known pore septal protein). The expression and cellular location of fusion proteins were observed by Confocal Fluorescent Microscopy. One wt strain and another expressing the GFP in the cytosol were included as controls of basal fluorescence and positive GFP expression, respectively. For sample preparation, the six strains were grown in MMV (1x Vogel's salts, 2% sucrose, 1.5% agar, and, for SPA-10-sGFP strain, also 500 µg/µL histidine and 300 µg/µL hygromycin) for three days, 150 rpm at 30°C in the dark. The mycelia were collected and ground in liquid nitrogen until a fine powder was obtained. 1 mg of biomass was suspended in 1 mL of ice-cold extraction buffer (100 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.5 mM EDTA, 2.5 mM, 1 mM PMSF PMSF, and protease inhibitor cocktail) by pipetting several times. The crude extracts were clarified by centrifugation at 4°C 12,000 rpm for 10 min and passing by the supernatant through a 0.22 µm syringe filter. Total protein amounts were determined by the Bradford method, and the protein fluorescence and integrity were checked on native PAGE (8% or 10%). The Co-Immunoprecipitation was carried out in 2 mL centrifuge tubes with 10-15 µL GFP-Trap®_M magnetic agarose beads (Cromotek) and 4-6 mg of native extracts in 1 mL volume. The mixture was incubated overnight at 4°C andin gentle mixing. Bounded proteins were separated in a magnetic tube rack and washed five times with 500 µl ice-cold extraction buffer. For elution, the magnetic beads were suspended in a new 1.5 centrifuge tube with 50 µl 2x SDS-Sample buffer (65.8 mM Tris-HCl, pH 6.8, 2.1% SDS, 26.3% glycerol, 10% 2-mercaptoethanol, 0.01% bromophenol blue) and boiled for 5 min at 95°C. The supernatants were analyzed in SDS-PAGE gels, Coomassie blue-stained. The gel loans of interest were aseptically cut and sent to the protein identification service by LC-MS/MS. The Neurospora crassa protein database was used for protein annotation.