Extended Data Fig. 4: Initiation hubs coalesce at the vacuolar contact site to trigger phagophore initiation.

a, Vac8-mCherry GFP–Atg11 cells expressing endogenous BFP–Ape1 and copper-inducible untagged Ape1 were grown to mid-log phase in the presence of 50 µM CuSO₄. The dynamics of GFP–Atg11 foci were monitored. Scale bar: 2 µm. b, Kymograph corresponding to Extended Data Fig. 3a (i) and additional representative examples of kymographs (ii,iii). Scale bar: 2 µm. c, GST–BFP or GST–BFP–Vac8 were expressed in E. coli and bound to Glutathione Sepharose (GSH) beads. Protein-bound beads were incubated with Sf9 insect cell lysates containing overexpressed GFP–Atg11, and bound GFP–Atg11 was analysed before and after eight washing steps. Scale bar: 20 µm. Data are mean values (n > 65 condensates per condition and replicate, three technical replicates). Circles: mean values of each replicate, bars: mean. Statistical analysis: two-tailed unpaired t-test. P values: P = 0.7. d, Purified vacuoles were incubated with recombinant GFP–Atg11 droplets. Scale bar: 1 µm. Quantification: Atg11 condensates bound to vacuoles in a box plot. Horizontal lines: median, box: 25th to 75th percentiles, whiskers: expand to 5th and 95th percentiles, circles: mean value of each replicate, outliers: black dots (n > 599 condensates per condition and replicate, three technical replicates). Statistical analysis: two-tailed unpaired t-test. P value: vac8∆:WT P < 0.0001. Source numerical data are available in source data, not significant (n.s.), arbitrary units (a.u.).