Fig. 1.

Dim2 depletion blocks 90S biogenesis and causes a defect in Nob1 binding. a Yeast strain GAL::scDIM2 with integrated scEnp1-FTpA or scRio2-FTpA was grown in galactose (GAL) medium or shifted for 8 h to glucose (GLU)-containing medium before the indicated bait proteins were tandem affinity-purified in two consecutive steps involving the ProtA- in the first and the Flag-tag in the second step. TCA-precipitated final Flag eluates were analyzed by SDS-PAGE (4–12%) and Coomassie staining (upper panel) or western blotting (lower panel) using the indicated antibodies. The indicated major protein bands were identified by mass spectrometry. Note that Dim1 co-migrates with HA-Dim2. b Recombinant MBP (maltose-binding protein)-ctDim2 co-expressed with HIS₆-ctNob1 in E. coli BL21 cells was affinity-purified utilizing SP-Sepharose and subsequently Ni-NTA resin. The final eluate was fractionated by size-exclusion chromatography (SEC). Relevant SEC fractions (lanes 15–21) containing the heterodimer were analyzed by SDS-PAGE (4–12%) and Coomassie staining. c Schematic drawing of the different ctNob1 domains. The middle (MID) domain of Nob1 contains a highly conserved tryptophan at position W267, which was mutated and tested by binding to Dim2. d In vitro binding assay with immobilized GST-ctDim2 as bait and indicated HIS₆-ctNob1 prey constructs (input, lanes 1–5). BL21 extract was used as mock control (input, lane 6). Samples (lanes 7–12) are SDS eluates analyzed by SDS-PAGE and Coomassie staining. e Single-point mutation in the ctNob1-MID sequence causes loss of interaction with ctDim2. Recombinant HIS₆-ctDim2 co-expressed with either ctNob1 or ctNob1 W267G in E. coli BL21 cells were tandem-purified on SP-Sepharose and Ni-NTA beads. Input Ni-NTA, lanes 1 and 3. Final eluates (lanes 2 and 4), analyzed by SDS-PAGE (4–12%) and Coomassie staining. The experiments were performed at least twice with consistent results. Uncropped images are shown in Supplementary Fig. 9. S molecular weight protein standard, L load