Fig. 5

In vivo condensation of Aip5 under stress conditions is Spa2 dependent. a Representative maximum Z-projection images of Aip5-GFP, Aip5-C-GFP, and Aip5-N-GFP protein localization in yeast. S small-budded cell, M medium size-budded cell, BN bud neck. Scale bar represents 3 µm. b Representative maximum Z-projection images of in vivo condensates of Aip5-GFP variants in WT and spa2Δ after 1 h of energy depletion. Scale bar represents 3 µm. c Quantification of the condensate number of Aip5-GFP imaged in b. Aip5-GFP, n = 108 cells; spa2Δ Aip5-GFP, n = 126 cells; Aip5-N-GFP, n = 80 cells; spa2Δ Aip5-N-GFP, n = 154 cells; Aip5-C-GFP, n = 86 cells; spa2Δ Aip5-C-GFP, n = 82 cells. d Representative deconvoluted confocal images of Aip5-GFP in FRAP assay. The scale bar represents 5 μm. e Quantification of fluorescent signals of FRAP experiments, as in d. (n = 11 condensates for Aip5-GFP control; n = 11 for Aip5-GFP under 1 h energy depletion; n = 8 for spa2Δ Aip5-GFP under 1 h energy depletion.) f Representative maximum Z-projection images of Aip5-3mCherry and Spa2-GFP localization after 1 h energy-depletion treatment; the white arrow indicates condensate formation. The scale bar represents 3 μm. g Representative deconvoluted confocal images of Spa2-GFP condensates (indicated by the white arrow) monitored in FRAP when cells were stressed by energy depletion for 1 h. The scale bar represents 3 μm. h Quantification of fluorescent signals in FRAP experiments, as in g. n = 15. i Quantification of in vitro formation of Aip5 assemblies (250 nM, 10% Aip5-GFP) under various pH conditions in the presence of 10% PEG3,350, as shown in Supplementary Fig. 8b. (pH4.5, n = 95; pH5.0, n = 175; pH5.5, n = 412; pH6.0, n = 123; pH6.6, n = 216; pH7.0, n = 249; pH7.5, n = 341). j Representative maximum Z-projection images of Aip5-GFP condensates formed in wild-type and spa2Δ under the indicated pH conditions. Scale bar represents 3 µm. k Quantification of the Aip5 protein condensates imaged in j. (pH5.5 Aip5-GFP: n = 75 cells; pH5.5 spa2Δ Aip5-GFP: n = 55 cells; pH6.5 Aip5-GFP: n = 52 cells; pH6.5 spa2Δ Aip5-GFP: n = 70 cells; pH7.5 Aip5-GFP: n = 92 cells; pH7.5 spa2Δ Aip5-GFP: n = 81 cells). The color key indicates signal intensity. p Values were determined by the one-way ANOVA, ****p < 0.0001, ***p < 0.001, and ns not significant. All the bar graphs represent mean with an error bar, S.D. Source data are provided as a Source Data file