Supplementary Figure 3: Cue5 forms oligomers involving its CUE domain.
From: Receptor oligomerization guides pathway choice between proteasomal and autophagic degradation
a, Different regions of Cue5 were purified and subjected to cross-linking as in Fig. 4b. Representative image of two independent replicates is shown. b, Alignment of CUE domain sequences from Cue5, Cue2 and Vps9. The conserved amino acids important for dimerization are boxed. Residues L135-L136 and F109-P110 (in red) are shown in modelled structure of Cue5 CUE domain using UCSF chimera based on Vps9 CUE domain (PDB ID code 1P3Q). Purple: ubiquitin, light blue: CUE (monomer), light green: CUE (monomer). c, Amino acid replacements within the CUE domain of Cue5 impair self-interaction. GST-pull-down assays were performed using His-tagged Cue5 and GST-fusions of WT Cue5 or CUE domain mutant variants of Cue5 (Cue5-F109A, P110A; Cue5-L135A, L136A). A Cue5 variant harbouring amino acid replacements C-terminal of the bona fide CUE domain (Cue5-L174A, L175A) was used as control. Representative image of two independent replicates is shown. d, Amino acid replacements described above impair ubiquitin binding. Experiments were carried out similar as in c with purified proteins and yeast cell lysates. Representative image of two independent replicates is shown. Unprocessed original scans of blots are shown in Supplementary Fig. 5.
