Fig. 4

DMS analysis of USP8 Rhod domain binding peptides and predicted peptides from known interactors containing the (partial) motifs generated by DMS. (A, B) Heatmap representation of PSSMs generated by the peptide-phage display-based DMS analysis of the two indicated peptides. (C) SPOT array results of selected peptides from known interactors tested for binding to the Rhod domain. The highest scoring USP8 Rhod binding peptides based on peptide array analysis of 42 predicted ligands are shown. The amino acid residues of the predicted motifs are indicated in bold. The KIAA1614_{568 − 583} peptide was used a positive control, and the signal intensities were normalized to the higher intensity (positive control) and indicated as percentage. The SPOT array and additional information can be found in Supplementary Fig. S6 and Supplementary Table S8. (D) Peptide SPOT array alanine scanning of USP21_{156 − 171} peptide binding to the MIT domain. (E) Peptide SPOT array alanine scanning of TP63_{608 − 622} peptide binding to the MIT domain. (D, E) Signal intensities were normalized to wild type (Wt) and displayed as average percent signal change. (F) Schematic model of how SLiM-based interactions of the Rhod domain may contribute to substrate targeting of USP8, while the MIT domain interacts with motifs found in ESCRT-III proteins (e.g. CHMPs) and other proteins thereby contributing to the targeting of the protein to different cellular localizations (e.g. endosomes) and interaction partners