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. 2021 Nov 12;10:e71982. doi: 10.7554/eLife.71982

Figure 3. YBX1 phase separation is governed by association of aromatic and basic amino acids in C-terminal IDR.

(A) Structural organization of YBX1. Top, IUPred, prediction of disordered protein regions; Middle, NCPR, net charge per residue with a sliding window of five residues; Net positive, blue, net negative, red; Bottom, visualization outputs for residue plots. (B) Schematic diagrams of different YBX1 mutants with the distribution of mutated amino acids. (C) Truncation mapping and identification of residues in YBX1 C-terminal IDR that are required for YBX1 condensation formation. YFP fused YBX1 wild type and mutants were introduced in ΔYBX1 U2OS cells by transient transfection and visualized by fluorescence microscopy. (D) Phase separation of YBX1 wild type and variants at the indicated concentrations. 6xHis-MBP-mGFP fused YBX1 wild type and variant proteins were purified from insect cells. Phase separation was induced by diluting the salt concentration from 500 mM to 150 mM in this assay. Scale bars, 3 µm.

Figure 3.

Figure 3—figure supplement 1. YBX1 amino acid sequences and secondary structure prediction.

Figure 3—figure supplement 1.

(A) Secondary structure of YBX1 was predicted using PSIPRED 4.0 based on amino acid sequences. (B) Secondary structure of YBX1 was predicted using D2P2 (Database of Disordered Protein Prediction, http://d2p2.pro) based on amino acid sequences.
Figure 3—figure supplement 2. The ability of YBX1 to form LLPS requires C-terminal IDR, likely depending on tyrosine and basic amino acids arginine and lysine.

Figure 3—figure supplement 2.

(A) Schematic diagrams of YBX1 truncation analysis. (B) Analysis of condensation formation for different YBX1 truncations in U2OS cells. YFP- fused YBX1 mutants were introduced in ΔYBX1 U2OS cells by transient transfection and visualized by fluorescence microscopy. (C) Identification of residues in YBX1 C-terminal IDR that are involved in YBX1 condensation formation. (D) SDS-PAGE of YBX1 wild-type and variants tagged with 6xHis-MBP-mGFP. (E) Phase separation of YBX1 N-terminal at the indicated concentrations. (F) Immunoblot analysis of YFP-tagged WT YBX1 and variants transiently expressed in U2OS cells. The blots were probed with antibodies directed against YFP or actin. Scale bars, 3 µm.
Figure 3—figure supplement 2—source data 1. Uncropped SDS-PAGE corresponding to Figure 3—figure supplement 2.
Figure 3—figure supplement 3. A F85A mutation did not affect YBX1 liquid droplet formation in vitro.

Figure 3—figure supplement 3.

(A) Mutation of F85A caused YBX1 to translocate into nucleus. YFP-fused YBX1 wild type and F85A were introduced in ΔYBX1 U2OS cells by stable transfection and visualized by fluorescence microscopy. (B) YBX1-F85 is not deficient for YBX1 liquid droplet formation. 6xHis-MBP-mGFP fused YBX1 wild type and F85A protein were purified from insect cells. Phase separation was induced by diluting the salt concentration from 500 mM to 150 mM in this assay. (C, D) Images (C) and quantification (D) of recovery of YBX1-F85A signal after photobleaching. A representative result of three independent experiments is shown. Error bars represent standard errors. Scale bars, 3 µm.