a, The IDRs of WAC and LAF1 promote LLPS. Phase-separation assay of recombinant 6×His–Lge1 and the fusion constructs 6×His–WAC(1–318)–Lge1(CC) and 6×His–LAF1(1–169)–Lge1(CC) (both proteins, 5 μM; buffer, 20 mM Tris pH 7.5, 100 mM NaCl and 1 mM DTT, 20 °C). Scale bar, 10 μm. Protein inputs are shown on the right (black arrows). b, Quantification of condensate sizes (in μm2) in a. n, number of condensates. Dot plot showing median and interquartile range. **P = 0.004, ****P < 0.0001, determined by two-sided Mann–Whitney test. c, A synthetic genetic approach was used to investigate the functionality of Lge1 LLPS in vivo. Cells were inviable when LGE1 and HTZ1 were deleted together, indicating a functional relationship. Double-deletion strains containing a wild-type LGE1 cover plasmid (Ura marker) were cotransformed with the indicated plasmids (His marker). Growth was followed on SDC–His (loading control) and on SDC + 5-FOA, which shuffles out the Ura cover plasmid. Cells were spotted in tenfold serial dilutions and incubated for two (SDC–His) or three days (5-FOA) at 30 °C. d, Live imaging of bre1∆ lge1∆ cells expressing mGFP–Bre1 and WAC(1–318)–Lge1(CC)–mCherry or LAF1(1–169)–Lge1(CC)–mCherry shows protein import into the nucleus. Dashed white line indicates the cell contour. Fluorescence intensity of the mCherry construct was quantified across a line spanning the nucleus. For comparison, the arbitrary fluorescence unit value = 1 is marked with a horizontal dashed line. n, number of cells. Scale bar, 2 μm. e, Cell lysates of strains in c and d were analysed by SDS–PAGE and immunoblotting with anti-mCherry antibody. Anti-Pgk1 serves as a loading control; asterisks indicate degradation products. Red arrowheads indicate Lge1 constructs according to their predicted sizes. f, Live imaging of bre1∆ lge1∆ cells expressing VC–Bre1 and WAC (1–318)–Lge1(CC)–VN or LAF1(1–169)–Lge1(CC)–VN constructs from LGE1 endogenous promoter. Arrowheads label nuclear BiFC puncta, Nup188–mCherry labels the nuclear envelope and dashed lines indicate the cell contours. Histograms represent pixel frequency of fluorescent intensity values. Scale bar, 2 μm. g, Coefficient of variation of the fluorescence-intensity distribution of BiFC signals in f and Fig. 3e. The higher the coefficient of variation, the greater the heterogeneity of the BiFC signal. A propensity for LLPS is suggested by an increased coefficient of variation of the WAC(1–318)–Lge1(CC)–VN construct. Dot plot showing median and interquartile range. n, number of cells. **P = 0.0024, ***P < 0.001, determined by two-sided Mann–Whitney test. h, Expression levels of Lge1–mCherry constructs used in i. Cells lysates were analysed by SDS–PAGE and immunoblotting with anti-mCherry antibody. Anti-Pgk1 serves as a loading control. Asterisk indicates degradation products. Red arrowhead indicates Lge1 constructs. i, Genetic interaction analysis, set up as in c with the indicated plasmids. See Supplementary Fig. 2 for uncropped gels and western blots.