Figure 3.

NEDD4L/WWP2-mediated monoubiquitination of ALIX. (A) Scheme of ALIX (also see Figure S11). (B) The reduction in ¹H_N/¹⁵N cross-peak heights of 100 μM ¹⁵N-labeled ubiquitin with 100 μM nonlabeled ALIX_348–702 (blue) and ALIX_1–868*^His (pink). (C) Pie-charts illustrating the average frequency of monoubiquitination in individual ALIX domains using NEDD4L (upper) and WWP2 (lower); n = 2. (D) Bar-chart of site-specific differences in monoubiquitination frequencies of ALIX residues brought out by NEDD4L (pink) and WWP2 (magenta); arrows mark significant differences. Only ubiquitinated residues are plotted. (E) Microscopy images of condensates of ALIX_1–868* and its WWP2-mediated monoubiquitinated counterpart (5% w/v PEG-4000), represented by circled no. 1 and 2; the same numbering scheme is used in the remaining panels. (F) Poor FRAP recoveries (<25%) for the freshly prepared condensates of ALIX_1–868*^His (blue) and its WWP2-mediated monoubiquitinated moieties (red), n = 3, the same coloring scheme in the remaining panels. (G) Fibrillization of monoubiquitinated ALIX_1–868* and the lack thereof for its nonubiquitinated species by ThT assays, n = 2. (H) Negative-stain EM analyses of fibrils formed by monoubiquitinated ALIX_1–868*^His. (I) SDS-PAGE analysis of pre- and postaggregated samples from G. Fibrillization of monoubiquitinated ALIX resulted in a decreased band intensity in lane-5 as compared to the preaggregated sample in lane-4.