Mobicol column

To assess in vitro binding, the GST-tagged bait protein (15 µg GST-CtRsa4-UBL, 15 µg GST-CtYtm1-UBL or 30 µg GST-CtKap104) was incubated with 15 µg wild-type CtMIDAS or respective MIDAS loop mutants (as indicated in Fig. 5c, d and Fig. 7a; Supplementary Fig. 6a, b) and/or 140 µg CtRea1-NAAA⁺ ring (amino acid residues 1–2390) and incubated in binding buffer (50 mM Tris, pH 7.5, containing 80 mM NaCl, 5 mM MgCl₂, 5% Glycerol, 2% DMSO, 2 mM Na₂SO₄, 0.01% NP-40, and 1 mM DTT) at 4 °C for 45 min. Nucleotides (ATP or AMPPNP, Sigma-Aldrich) or Rbin-1 (Axon Medchem) where added to a final concentration of 2 and 0.1 mM, respectively. Next, GST-tagged bait proteins and bound material were pulled-down by incubation with 70 µl GSH–agarose (Prontino, Macherey-Nagel) in Mobicol columns (MoBiTec), at 4 °C for 45 min. The flow-through was discarded and beads were washed five times with 500 µl binding buffer. Subsequently, elution was performed with the addition of 50 µl elution buffer (binding buffer containing 30 mM reduced GSH). Eluates were analyzed by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis on 4–12% polyacrylamide gels (NuPAGE, Invitrogen) and Coomassie staining.

GST pull-down assays were performed in 20 mM HEPES pH 7.5, 150 mM NaCl (or higher concentration, see description in the figure legends), 5% glycerol, and 0.1% NP-40. The bait protein was incubated with a 1.5 molar excess of prey protein in 500 µl buffer at 4 °C for 45 min. Glutathione agarose beads (Cytiva) were added and the samples were incubated for an additional 45 min at 4 °C. The wash was performed on 1 ml mobicol columns (MoBiTec) using buffer with the indicated salt concentration. Beads were washed twice with 500 µl buffer. Samples were eluted with 1× Laemmli SDS buffer and analyzed by SDS-PAGE followed by Coomassie staining.
For in vitro-binding experiments with the ctRed1 mutants, the proteins were co-expressed in E. coli Rosetta 2™ (DE3) strain (Novagen). Wild type and mutant variants of T4L-ctRed1_pep-His₆ were co-expressed with an untagged version of ctMtr4_SA. Lysis was performed in a buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl and 20 mM imidazole, followed by Ni-NTA purification. Samples from total, soluble, and elution fractions were analyzed by SDS-PAGE, followed by Coomassie staining.

An aliquot (peptide: TiO₂ resin = 1: 6) of TiO₂ (Titansphere TiO, GL Sciences, 5020-75000) was washed twice with 50% methanol (Fisher Chemical, A456-212) and twice with glycolic acid solution (1 M glycolic acid (Sigma Aldrich, 124737-25G), 70% ACN (VWR, 83639.320), 3% TFA (Thermo Fisher Scientific, 28903)). Peptides were dissolved in glycolic acid solution, mixed with the TiO₂ resin, incubated rotating at room temperature for 30 min, transferred onto Mobicol columns (MoBiTec, M1003, M2110), and shortly centrifuged in a table centrifuge to remove unphosphorylated peptides. The resin was washed twice with glycolic acid solution, twice with 200 μl 70%, ACN 3%, TFA, and twice with 1% ACN, 0.1% TFA. Phosphorylated peptides were eluted twice using 150 μl 300 mM ammonium hydroxide (VWR, 1.05432.1000); eluates were united and immediately acidified with conc. TFA to a pH of 2.5. Samples were desalted using a standard C18 StageTip protocol (32 (link)). The enrichment protocol resulted in a relative phosphorylated peptide abundance of 95.3%.

Pre-40S particles were purified using the AF Tsr1 as bait protein. Cells expressing C-terminally TAP-tagged Tsr1 (Tsr1-TAP) were grown in 2 L YPD medium at 30 °C and harvested at an OD₆₀₀ of ~1.8. TSR1-TAP strains expressing mutant variants of Ltv1 from LEU2 plasmids were grown in 4 L SDC medium lacking leucine and harvested at an OD₆₀₀ of ~0.9. Cell pellets were resuspended in lysis buffer containing 50 mM TRIS pH 7.5, 100 mM NaCl, 1.5 mM MgCl₂, 0.075% NP-40, 1 mM DTT, and 1× FY protease inhibitor cocktail (Serva). Cells were broken in the presence of glass beads by shaking in a bead mill (B. Braun homogenizer 853022) cooled with CO₂. Lysates were cleared by centrifugation at 4 °C for 10 and 30 min at 5000 and 14,000 rpm, respectively. Lysates were incubated with IgG-beads (GE Healthcare) on a rotating wheel at 4 °C for 75 min. Beads were washed with 5 mL lysis buffer w/o protease inhibitors, subsequently transferred into Mobicol columns (Mobitec) and washed with 10 mL lysis buffer. After resuspension in lysis buffer, pre-40S particles were eluted by incubation with TEV protease at room temperature (RT) on a rotating wheel for 90 min. Eluates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 4–12% polyacrylamide gels (Invitrogen) and Coomassie staining or western blotting with the indicated antibodies.