(A) Diagram of the lipid transfer assay with PI3P-containing donor LUVs (extruded through a 100 nm filter, the lipid compositions; 5% PI3P, 46% DOPC, 25% DOPE, 20% DOPS, 2% NBD-PE, and 2% Rh-PE) and PI3P-free acceptor LUVs (extruded through a 100 nm filter, the lipid composition; 50% DOPC, 25% DOPE, and 25% DOPS). (B) Titration of WIPI4 into 100 nM ATG2A. The concentrations of WIPI4 are indicated. A representative data set is shown. (C) Plot of the initial NBD-PE transfer rates versus the molar ratios of WIPI4:ATG2A. The SD of each time point was obtained from three repeats. (D, E) Diagram of (D) and data from (E) the lipid transfer assay with 25 µM PI3P-free donor LUVs (extruded through a 100 nm filter, 46% DOPC, 25% DOPE, 25% DOPS, 2% NBD-PE, and 2% Rh-PE) and PI3P-containing acceptor LUVs (extruded through a 100 nm filter, 5% PI3P, 50% DOPC, 25% DOPE, and 20% DOPS). Note that the PI3P is in the acceptor. (F) Comparisons of the initial NBD-PE transfer rates between the experiments performed in (A, B) and (D, E). Data obtained with 100 nM ATG2A in the presence or absence of 100 nM WIPI4 are compared. (G) Lipid transfer assays with 100 nm filter-extruded LUVs with different lipid compositions. Both WIPI4 and ATG2A concentrations were fixed at 100 nM. All donor LUVs contained 5% PI3P, 2% NBD-PE, 2% Rh-PE, and 25% DOPE or POPE, for DO or PO lipids-based LUVs, respectively. The rest of the PS and PC compositions in each liposome was 20% DOPS/46% DOPC, 5% DOPS/61% DOPC, 25% POPS/41% POPC, and 5% POPS/61% POPC. All acceptors contained 25% DOPE or POPE, and the PS and PC compositions in each liposome was 25% DOPS/50% DOPC, 5% DOPS/70% DOPC, 25% POPS/50% POPC, or 5% POPS/70% POPC. Each donor was paired with the acceptor that contained the same or similar percentage of PS, as indicated. (H) Lipid transfer assays with various LUV50s. Both donor and acceptor LUV50s were prepared by extrusion through a 50 nm filter. The donor LUV50 was composed of 5% PI3P, 2% NBD-PE, 2% Rh-PE, 25% POPE, 5% POPS, and 61% POPC. This donor was used in all four experiments with different acceptor LUV50s. The lipid composition of each acceptor was identical to that of the acceptor with the same name (based on DOPS or POPS percentages) in (G). 100 nM each of WIPI4 and ATG2A were used. (I) Lipid transfer assays with 50 nm filter-extruded donors (LUV50s) and sonicated acceptors (SUVs). The same donor as in (H) (5% POPS/5% PI3P-containing LUV50) was used, and the composition of the acceptor SUV was 5% POPS, 25% POPE, and 70% POPC (the same as that of 5% POPS acceptor LUV50 in (H)). The concentration of ATG2A was 100 nM, and those of the WIPI4 used are indicated. (J) Titration of WIPI1 into 100 nM ATG2A. The experiment was performed as in (A). (K) Initial NBD-PE transfer rates in the experiment shown in (J). (L) The same experiment as (I) except that WIPI1 was tested. All experiments were performed at 25 °C.