Figure 6.
Atg9 contribution to tethering and lipid transfer activities of the Atg2-Atg18 complex. (A) Schematic representation of the liposome-based tethering assay. SUVs containing the fluorescently labeled lipid NBD-PE constitute the donor vesicles (D). Heavy liposomes reconstituted in buffer containing 5% sucrose are not labeled and constitute the acceptor vesicles (A). Tethering was measured by detecting the NBD fluorescence intensity that is present in the fraction of heavy liposomes after centrifugation. (B) For the tethering assay, SUVs and heavy liposomes were incubated in the presence of the Atg2-Atg18 complex or buffer as control. Atg9 or Atg9F627A were incorporated in donor vesicles (D), acceptor vesicles (A) or both. After incubation, half of the sample was incubated with 1 mg/ml proteinase K (ProtK). Heavy liposomes and the tethered donor vesicles were separated from non-attached SUVs by centrifugation and the fluorescence intensity of NBD was measured in the pellet fraction. The plot shows the fluorescence intensity (FI) of NBD normalized to the fluorescence intensity in the control condition without Atg9 or Atg9F672A (protein free vesicles) and incubated in the presence of the Atg2-Atg18 complex. The data corresponds to the mean ±SE of measurements from four independent experiments (C) Schematic representation of the FRET-based in vitro lipid transfer assay. The NBD fluorescence is quenched by rhodamine in donor vesicles. After lipid transfer to non-labeled acceptor vesicles, the fluorophores are diluted and the signal of NBD can be detected. (D) For the lipid transfer assay, donor vesicles and acceptor vesicles containing Atg9, Atg9F627A or protein-free, were mixed and incubated with 50 nM of the purified Atg2-Atg18 complex (+Atg2-Atg18) or buffer (-Atg2-Atg18) as control. The NBD fluorescence intensity was measured every 1 min over a period 2 h. The plot shows the fluorescence intensity (FI) of NBD at each point relative to the initial fluorescence signal (FI0). The data corresponds to the mean ±SE of measurements from three independent experiments. The data were analyzed using a Kruskal-Wallis test followed by a Dunn’s Multiple Comparison test with a significance level = 0.01. The asterisks indicate that the means of curves corresponding to Atg9 or Atg9F627A vary significantly in respect to the control condition (+Atg2-Atg18). The difference between Atg9 and Atg9F627A, however, is not significant.