FIGURE 2.

Atg18-Atg2 complex formation is independent of PtdIns(3)P binding of Atg18. A, atg18Δ cells expressing Atg18 variants were treated with rapamycin. The postsolubilization 100,000 × g supernatants were subjected to immunoprecipitation (IP) with or without affinity-purified anti-Atg2 antibody (Ab). Precipitates were separated by SDS-PAGE, followed by immunoblotting (IB) with affinity-purified anti-Atg2 antibody and anti-Atg18 serum. Signals shown were obtained on the same membrane with the same exposure time and processed equally. B, postsolubilization, 100,000 × g supernatants were prepared and subjected to immunoprecipitation with affinity-purified anti-Atg2 antibody. The precipitates were separated by SDS-PAGE, followed by immunoblotting with affinity-purified anti-Atg2 antibody and anti-Atg18 serum. C, cells were treated with rapamycin, and the postsolubilization 100,000 × g supernatants were subjected to gel filtration analysis. D, the signal intensity of each fraction shown in C was measured, and the percentage of the total was calculated. For the lower graph, see Fig. S2.