Dictyostelium discoideum strains (Sup. Tab. 3 ) expressing either pDM317-GFP-Sac1 or pDM317-GFP63 were grown at 22 °C in HL5-C medium (ForMedium) supplemented with geneticin (G418, 5 μg/ml). Electroporation of D. discoideum was performed according to Paschke et al. 2018 with modification64 (link). The cell number was determined (Countess II F2, Thermo Fisher Invitrogen), and 3 ∙ 107 cells were used for each sample. Pulldowns were performed in biological independent triplicates (n = 3). Cells were pelleted and washed once in cold Soerensen-Sorbitol. Cell pellets were snap frozen in liquid nitrogen and lysed with glass beads in 500 µl GFP pulldown buffer (20 mM HEPES pH 7.4, 150 mM KOAc, 5% glycerol, 1% GDN, Roche Complete Protease Inhibitor Cocktail EDTA free, Roche) using a FastPrep (MP biomedicals). The supernatant was cleared at 21,000 g for 10 min and incubated for 10 min rotating at 4 °C together with 12.5 µl pre-equilibrated GFP-Trap beads (Chromotek). Beads were washed four times with GFP pulldown buffer at 2500 g for 2 min at 4 °C. Afterwards, they were washed two times with wash buffer (20 mM HEPES pH 7.4, 150 mM KOAc, 5% glycerol) at 2500 g for 2 min at 4 °C. Beads were further treated following the iST Sample Preparation Kit (PreOmics) protocol. Dried peptides were resuspended in 10 µl LC-Load, and 4 µl were used to perform reversed-phase chromatography as described above. Data were analyzed using MaxQuant (V2.2.0.0, https://www.maxquant.org/maxquant/ )65 (link),66 (link) with the corresponding FASTA database. Contaminants were identified based on the MaxQuant contaminants.fasta file. The resulting data were analyzed using Perseus (V2.0.7.0, www.maxquant.org/perseus )58 (link). Significance lines in the volcano plot of the Perseus software package corresponding to a given FDR were determined by a permutation-based method.59 (link) The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE60 (link) partner repository.
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