Figure 4. M1-linked tetra-ubiquitin induces phase transition of NEMO in vitro.
(A) Domain structure of recombinant WT NEMO-GFP or Q330X NEMO-GFP. MBP (maltose-binding protein), TEV (Tobacco Etch Virus protease). (B) WT NEMO-GFP and Q330X NEMO-GFP do not phase-separate. Fusion proteins were incubated in the presence of TEV protease (1 h at RT) to cleave off the N-terminal MBP and the C-terminal His6 tag and then WT NEMO-GFP and Q330X NEMO-GFP were analyzed by fluorescent microscopy using a laser scanning microscope. (C) Concentration-dependent phase separation of WT NEMO and M1-linked tetra-ubiquitin (4×M1-ub). WT NEMO-GFP was incubated in presence of recombinant M1-linked tetra-ubiquitin (4×M1-ub) at the concentrations indicated and analyzed by fluorescence microscopy. Red empty circles: no phase separation; green solid circles: phase separation; sizes of the circles illustrate the sizes of liquid droplets. (D, E) WT NEMO but not Q330X NEMO undergoes LLPS in the presence of M1-linked tetra-ubiquitin. GFP-tagged (D, E) or untagged (E) WT NEMO and Q330X NEMO (5 µM in 10 mM Tris, pH 7.4, and 150 mM sodium chloride [NaCl]) were incubated in the presence or absence of 10 µM 4×M1-ub and analyzed by fluorescence microscopy (D) or bright-field microscopy (E). Shown are representative images; scale bar, 10 µm. (F) Preformed NEMO assemblies persist upon OTULIN-mediated hydrolysis of M1-linked tetra-ubiquitin. MBP-WT NEMO-GFP was incubated with the components indicated (lanes 3–9). In the reaction corresponding to lane 4, recombinant OTULIN was added together with TEV protease, whereas in the reaction corresponding to lane 5 OTULIN was added 1 h after TEV protease. Lane 1: M1-linked tetra-ubiquitin only. Lane 2: M1-linked tetra-ubiquitin incubated with OTULIN for 1 h. The samples were analyzed by SDS–PAGE and Coomassie blue staining (upper panel) and laser scanning microscopy (lower panel).
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