##Sample processing
D. discoideum AX4 wildtype cells (DictyStockCenter ID: DBS0237637) were first grown axenically in HL5-C (Formedium) to exponential phase. 3x108 cells were harvested at 400 x g, 5 min, and washed twice in 50 ml KK2. For the 0h time point (not developed), half the cells were harvested as before and stored at -80°C to await further processing for transcriptomics library preparation; the other half was harvested and stored at -80°C awaiting proteomics sample preparation. For the other time points, cells were plated on 92mm NN-Agar plates (1.2 g/l  KH2PO4, 0.48 g/l Na2HPO4⋅2H2O, 15 g/l agar) and harvested at the designated time points using Nunc Cell Scrapers (Thermo Fisher), into KK2 buffer; half the plate for transcriptomics and half the plate for proteomics. The cells were harvested as before and the cell pellets were frozen at -80°C until further processed for transcriptomics or proteomics sample preparation. Cell pellets were lysed in 150 µL of 1% β-octyl glucopyranoside and 6M urea containing lysis buffer using a sonication probe for 60 seconds (3 mm probe, pulse 1 s, amplitude 30%) according to a standard operating procedure. After homogenization, the samples were incubated for 90 min at 4°C during mild agitation. The lysates were clarified by centrifugation for 10 min (16 000 × g at 4°C). The supernatant containing extracted proteins was collected and further processed. The total protein concentration in the samples was measured using the DC Protein Assay (BioRad) with bovine serum albumin as standard. Aliquots corresponding to 35 µg of proteins were taken out for digestion. The proteins were reduced, alkylated, and on-filter digested by trypsin using 3kDa centrifugal spin filter (Millipore, Ireland). The collected peptide filtrate was vacuum centrifuged to dryness using a Speedvac system. The samples were dissolved in 100 µL 0.1% formic acid and further diluted 4 times. For LC-MS/MS analysis, the peptides were separated in reversed-phase on a C18-column with 150 min gradient and electrosprayed on-line to a Q Exactive Plus Orbitrap LC-MS/MS system (Thermo Scientific). Tandem mass spectrometry was performed applying Higher-energy collisional dissociation.
##Data processing
Label free quantification (LFQ) of the raw data was performed using FragPipe v20.0 (https://fragpipe.nesvilab.org/), which is powered by MSFragger (Kong et al., 2017). Analysis was performed with oxidation and lysine ubiquitination specified as variable modifications. Up to 3 missed cleavages were allowed. PSM validation performed with Percolator (Käll et al., 2007), and protein inference with ProteinProphet (da Veiga Leprevost et al., 2020). Data is filtered at 1% FDR at the PSM, ion, peptide, and protein levels. Site localization with PTM-Prophet. For quantification, a minimum of 1 ion was required for MaxLFQ determination with IonQuant, using match between runs (Yu et al., 2021).