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© The Author(s), under exclusive licence to Springer Nature Limited 2020020406080100Dots near the vacuole (%)WT Mutant05101520No.ofdropletstetheredtoGUV****Vac8+ Vac8–051015No.ofdropletstetheredtoGUV ****bceghVac8 Atg1 Atg13Atg17Vac8 Atg13Atg17 MergeGFP–Atg1 DICMergeDICMerge100 316Atg13Atg17DICTime (s)VAC8vac8ΔfaAtg13DICTime (s) 605 607 609d100200 30005101551015Time (s)No.ofdropletsonGUVAverage droplet area (AU)No. of droplets
Average
droplet areaVAC8vac8ΔFig. 5 | The liquid-like PAS is tethered to the vacuolar membrane via specif ic
protein–protein interactions. a, Observation of the PAS by monitoring GFP–
Atg1 in wild-type and vac8Δ cells treated with rapamycin for 3 h. Broken circles
indicate the vacuole. Scale bar, 5 μm. b, Proportion of GFP–Atg1 dots observed
adjacent to the vacuole. Data are mean ± s.d. (n = 3 (b), 20 (d) and 30 (f)
independent experiments). *P = 0.0126, two-sided t-test. c, Tethering of Atg1-
complex droplets to a Vac8-anchored GUV. d, Quantification of the number of
Atg1-complex droplets tethered to GUVs after equilibrium. ****P = 1. 3 × 10−9,
two-sided t-test. e, Tethering of scaffold droplets to a Vac8-anchored GUV.
f, Quantification of the number of scaffold droplets tethered to GUVs after
equilibration. ****P = 3.6 × 10−1 3, two-sided t-test. g, Top, time-dependent
change in the number and size of scaffold droplets on a Vac8 GUV. Bottom,
graph shows the number and mean area of droplets ± s.d. (n = droplet numbers).
h, Coalescence between droplets observed in g. Scale bars, 10 μm unless
otherwise indicated. Experiments were repeated independently 3 (a), 20 (c), 30
(e) or 5 (g, h) times with similar results.