Fig. 1: High-affinity receptor–cargo interactions impair selective autophagy.
From: Phase separation of initiation hubs on cargo is a trigger switch for selective autophagy
a, Schematic of END condensate solidification. Dashed grey arrows show low-affinity interactions. b, Cells expressing 2×GFP–Ede1 without (light grey) or with (orange) BFP–3×GBP or with BFP–3×GBPlow (dark grey) under the control of a copper-inducible promoter were grown to mid-log phase in the presence of 1 mM CuSO4 for 6 h. Autophagy was then induced by rapamycin addition. Quantification: cells with END condensates. Data are mean values (n > 150 cells per condition and replicate, three biological replicates). Circles show mean values of each replicate, bars show mean. Statistical analysis was carried out by two-tailed unpaired t-test. P values are as follows. Untreated: −3×GBP (--) versus +3×GBP, P = 0.0978; −3×GBP (--) versus +3×GBPlow, P = 0.5104; rapamycin: −3×GBP (--) versus +3×GBP, ****P < 0.0001; −3×GBP (--) versus +3×GBPlow, P = 0.1363. c, The −3×GBP (–), the +3×GBP and the +3×GBPlow strains were grown to mid-log phase as described in b. 2×GFP–Ede1 assemblies were photobleached and recovery of the signal was monitored. White arrowheads indicate the photobleached area. Scale bar, 1 µm. Quantification: recovery of the GFP signal. Data are mean values ± s.e.m. (n > 26 ENDs per condition across replicates, three biological replicates). d, A 2×GFP–Ede1 strain without (−) and with (+) 3×GBP and a 2×GFP–Ede1AIM (Atg8 binding mutant21) strain with +3×GBP coexpressing mCherry–Atg8 were grown to mid-log phase in the presence of 1 mM CuSO4. GFP/mCherry-positive structures were quantified. Data are mean values (n > 100 ENDs per condition and replicate, three biological replicates). Circles show mean values of each replicate, bars show mean. Statistical analysis was conducted by a two-tailed unpaired t-test. P values are WT(−) versus WT(+), P = 0.525; WT(+) versus AIM(+) ****P < 0.0001. AIM, Atg8 binding mutant. e, GST–BFP, GST–BFP–GBP, GST–BFP–GBPlow and GST–BFP–Ape11–45 were expressed in Escherichia coli and bound to GSH beads. Protein-bound beads were incubated with E. coli cell lysates containing 6×His–GFP or mCherry–Atg19, and bound GFP or mCherry–Atg19 was analysed before and after washing. Scale bar, 20 µm. Quantification: ratio of bead-bound protein to soluble protein in a box plot. Horizontal lines show the median, box shows the 25th to 75th percentiles, whiskers indicate the 5th and 95th percentiles, circles show mean value of each replicate, outliers show black dots (n > 25 beads per condition across replicates, three technical replicates). Statistical analysis was carried out by a one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. P values: GBP versus GBPlow, ****P < 0.0001; GBP versus Ape11–45, ****P < 0.0001. f, GST, GST–Ape11–45 and GST–Atg11685-1178 were expressed in E. coli and bound to GSH beads, incubated with purified recombinant Atg193D and analysed by immunoblotting. One experiment out of three technical replicates is shown. g, atg19∆ cells expressing GFP–Ape1 and Atg19, an empty control vector (−), Atg19–GBP, Atg19–GBPlow or Atg19–GBPmed were grown to mid-log phase. Cell extracts were analysed by immunoblotting. One out of three biological replicates is shown. Source numerical data and unprocessed blots are available in Source data.
