Extended Data Fig. 6: Identification of ASDURF and POLR2E as constitutive PFDL subunits.
From: DIP-MS: ultra-deep interaction proteomics for the deconvolution of protein complexes
a. Coelution of ASDURF with the PFDL and the PFDL containing PAQosome complex within the PFDN2 and UXT DIP-MS experiments. The MS2 protein intensity was rescaled within the sections (PAQosome section: Fraction 5 – 25 and PFDL section 25 – 45) due to high signal differences between the PFDL and PAQosome complexes. b. Interactome derived from AP-MS of core-subunits of the R2TP complex (blue background), PFDL (gray background), and canonical prefoldin (red background). Interactions are filtered only for high-confidence (Log2FC ≥ 5 and Saint score ≥0.99) and showing only interactions between core-subunits. Baits are depicted as octagons, and red edges represent interactions with ASDURF (shown in red). ASDURF was only recovered with subunits of the PFDL and PFDL containing PAQosome complexes. c. Structural alignment of ASDURF AlphaFold model to canonical PFD, PFDL, and CCT/TRiC subunits. The TM-score was either normalized to ASDURF (blue circles) or the second structure (green circles). The mean of the two normalized TM-score is reported (red circles). A significant threshold of 0.5 TM-score was applied (dotted line). Filled circles indicate high structural similarity, empty circles indicate lower to no structural similarity. d. TM-score and RMSD for Prefoldin subunits (n = 9) and the negative control (subunits of CCT/TRiC, R2TP and adaptor proteins, n = 15). The TM-score for prefoldin subunits indicate strong structural similarities between all PFD subunits and ASDURF. The RMSD of the alignment (2 Å) indicates that the predicted ASDURF model structure shares strong similarities with PFD and PFDL subunits. The structural alignment was performed once for each subunit (n = 1). Data are presented as mean values +/- SEM. e. AlphaFold2 pLDDT score divided by 100 of URI1 (AF-O94763-F1) plot by residue. Very low pLDDT/100 score (<0.4) indicate IDRs. At the bottom prediction of IDRs and protein binding regions employing flDPnn indicate large regions IDR within URI1 which explains the poor predictions of these regions in the AlphaFold2 model. f. Structural model of the PFDL complex. Subunits are colored. Upper part shows a side view of the complex, the lower part shows a top view, showing the stacked β-sheets.
