| Publication note | We demonstrate that the evolutionarily conserved low-complexity protein, BuGZ, undergoes phase transition or coacervation to promote assembly of both spindles and their associated components. BuGZ forms temperature-dependent liquid droplets alone or on microtubules in physiological buffers. Coacervation in vitro or in spindle and spindle matrix depends on hydrophobic residues in BuGZ. (Organism: Xenopus laevis; Cell line: HeLa cells,X. laevis oocytes) |
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| Material state | liquid : We purified His-, GST-, GFP-, or YFP-tagged xBuGZ or mBuGZ expressed in either Sf9 cells or bacteria. Upon warming, each protein formed droplets of varying sizes in physiologically relevant buffers and the droplet size increased over time. When the solutions were cooled on ice, the droplets disintegrated over time, as judged by the disappearance of fluorescence in the droplets. | ||
| LLPS region | 111-187,258-334,376-452 | Key domains | xBuGZ-A,xBuGZ-B,xBuGZ-C,MT-binding sequence : We found that a fragment of xBuGZ corresponding to amino acids 258–334 (xBuGZ-B), which did not form droplets on its own, inhibited the phase transition of YFP-xBuGZ when used at high concentrations. We also created two additional GFP-tagged fragments, xBuGZ-A and -C, corresponding to 111–187 aa and 376–452 aa in the DLC region. GFP-xBuGZ-A, -B, and -C, but not their mutants, exchanged into and disrupted the His-BuGZ droplets. YFP-xBuGZΔN exhibited similar coacervation properties as YFP-xBuGZ, especially at higher protein concentrations, indicating that the MT-binding sequence is dispensable for phase transition in vitro. |
| Protein partner | _ ( _ ) |
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| RNA partner | _ |
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| Others | _ |
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| PTM | _ |
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| Mutation | ΔN,p.Y402S/Y412S/F420S/F430S/Y452S,p.Y117S/F129S/F237S/F257S/F275S/F292S/F295S/Y352S/Y402S/Y412S/F420S/F430S/Y452S |
YFP-xBuGZΔN exhibited similar coacervation properties as YFP-xBuGZ, especially at higher protein concentrations, indicating that the MT-binding sequence is dispensable for phase transition in vitro. We mutated the last 5 or all 13 conserved Fs and Ys in the predicted DLC region of xBuGZ to serine (S) to create YFP-xBuGZ5S(p.Y402S/Y412S/F420S/F430S/Y452S) or YFP-xBuGZ13S(p.Y117S/F129S/F237S/F257S/F275S/F292S/F295S/Y352S/Y402S/Y412S/F420S/F430S/Y452S). Coacervation of YFP-xBuGZ5S and YFP-xBuGZ13S required increasingly higher concentration and temperature. Thus conserved Fs and Ys are required for BuGZ phase transition. |
| Alternative splicing | _ | |
| Repeat | _ | |
| Oligomerization | _ | |