Figure 4.
The F627A mutation does not have a major effect on Atg9 oligomerization and scramblase activity. (A-D) The atg9∆ strain expressing Atg9-GFP and Atg9-13xMYC (RGY881), Atg9F627A-GFP and Atg9F627A-13xMYC (RGY882) or Atg9-13xMYC and Atg9-mScarlet (RGY895) were grown in YPD to a log phase and nitrogen starved in SD-N for 1 h. Cell lysates generated in a buffer containing 1% Triton X-100 (A), 0.5% Tween 20 (B) or 1% CHAPS (C) were subsequently immunoprecipitated using GFP-trap beads (IP:GFP). Immunoprecipitates were analyzed by western blot using antibodies against GFP and MYC. (D) Quantification of the experiments shown in panels A, B and C. The ratio between Atg9-13xMYC:Atg9-GFP was determined by quantifying the intensity of the corresponding bands and subsequently expressing it relative to the cells carrying Atg9-GFP. Asterisks highlight significant differences with cells expressing WT Atg9-GFP. (C and D) Stoichiometric analysis of Atg9 oligomers by single-molecule fluorescence microscopy. The oligomeric state of Atg9 in membranes was determined by TIRF imaging of a SLBs containing Atg9-GFP or Atg9F627A-GFP (see Figure S4A-D). The occurrence percentage of Atg9-GFP (C) and Atg9F627A-GFP (D) oligomer species calculated as the averaged values from 3 different experiments ±SD. Data are provided after correction for partial labelling. (E) Schematic representation of the fluorescence-based lipid scramblase assay. NBD-PE is incorporated into protein-free or Atg9-containing liposomes. The fluorescence of NBD in the outer leaflet of the lipid bilayer can be quenched using the non-permeable reducing agent dithionite. As a result, the fluorescence of protein-free liposomes is reduced to 50% of the initial intensity. A scramblase activity leads to a quenching of fluorescence greater than 50%, as it exposes the phospholipids from the inner to the outer leaflet. (F) Scramblase activity was analyzed by measuring the fluorescence signal of NBD over time, in liposomes containing Atg9, Atg9F627A (protein: phospholipid ratio = 1:1500) or protein-free, before and after the addition of 30 mM dithionite. The fluorescence intensity (FI) was recorded every 5 s over a total period of 800 s. The plot shows the FI relative to the average initial intensity recorded before the addition of dithionite, followed by the addition of 0.1% Triton X-100 (TX-100). The data correspond to the mean with the standard error (±SE) of measurements from three independent experiments. The data were analyzed using a Kruskal-Wallis test followed by Dunn’s Multiple Comparison test with a significance level = 0.01. Asterisks indicate significant differences between the control (protein-free) and protein containing conditions (Atg9 and Atg9F627A). There are no significant differences between Atg9 and Atg9F627A conditions. The fact that the fluorescence decay is extremely fast and stabilizes after a few seconds, suggests that the remaining intensity is probably due to the presence of protein-free vesicles.