Figure 5.

Reconstitution of Atg8 and Atg19 dependent cargo recruitment to the membrane

(a) prApe1 propeptide recruitment to Atg8 harbouring GUVs in the presence and absence of Atg19. The experimental setup is shown on the right. (b) Atg19 dependent envelopment of prApe1-coated beads with Atg8 positive membranes. The experimental setup is shown on the right. The membrane was labelled by incorporation of a rhodamine labelled lipid. (c) Quantification of the percentage of GUV-associated eGFP-Ape1 coated beads showing detectable membrane bending. (N = 3; total GUVs per condition = 40). The experimental setup is shown in (b). The density of Atg8-6xHis on the membrane was titrated by different amounts of Ni-lipids in the GUV membrane. The graph is based on at least 3 independent experiments. Shown are the averages and standard deviations. (d) Quantification of the percentage of GUV-associated eGFP-Ape1 coated beads showing detectable membrane bending. The quantification is based on 3 independent experiments for the mutant Atg19 versions and 16 independent experiments for wild type Atg19 and the no Atg19 control (Atg19: N = 16, total GUVs = 382; Atg19 W412A: N = 3, total GUVs = 55; Atg19 F376A, F379A: N = 3, total 66 GUVs; Atg19 F376A, F379A, W412A: N = 3, total GUVs = 84; Atg19 F376A, F379A, P385A, E386E, W412A: N = 3, total GUVs = 60; Atg19-34: N = 4, total GUVs = 110; no Atg19: N = 16, total GUVs = 382). Shown are the averages and standard deviations. The experimental setup is as shown in (b) the p values in (c) and (d) were calculated using a 2-tailed Student’s T-test. (e) Visualization of Atg19 during the envelopment of prApe1-coated beads with Atg8 containing membranes. Note that Atg19 directly binds the prApe1-coated beads (arrows). The experimental setup is shown on the right. Scale bars: 5μM. Abbreviations: 8: Atg8; 19: Atg19; DIC: Differential interference contrast. All experiments have been conducted at least three times.