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. 2022 Nov 10;19(5):1459–1478. doi: 10.1080/15548627.2022.2136340

Figure 5.

Figure 5.

Atg2 and Atg18 are recruited to the PAS and Atg9-Atg2-Atg18 complex is formed in cells expressing Atg9F627A. (A) The atg9∆ strain carrying endogenous Atg2-GFP, mCherry-Atg8 and either an empty vector (RGY742, atg9∆) or an integrative plasmid expressing Atg9-13xMYC (RGY748, Atg9) or Atg9F627A-13xMYC (RGY875, Atg9F627A), was imaged as in Figure 2C. DIC, differential interference contrast. Scale bar: 2 μm. (B) Percentage of the mCherry-Atg8-positive PAS that are also positive for Atg2-GFP in the experiment shown in panel A. Asterisks highlight significant differences with the atg9∆ mutant transformed with the plasmid expressing WT Atg9. (C) The atg9∆ mutant expressing endogenous Atg18-GFP, mCherry-Atg8 and an empty vector (RGY877, atg9∆) or an integrative plasmid expressing Atg9-13xMYC (RGY878, Atg9) or Atg9F627A-13xMYC (RGY879, Atg9F627A), was examined as in Figure 2C. DIC, differential interference contrast. Scale bar: 2 μm. (D) Quantification of the percentage of mCherry-Atg8-positive PAS that are also positive for Atg18-GFP in the experiment depicted in panel H. Asterisks indicate significant differences with atg9∆ cells transformed with the plasmid expressing WT Atg9. (E and F) The atg9∆ strain expressing Atg2-TAP, Atg18-13xMYC and Atg9-GFP (RGY885), or Atg9F627A-GFP (RGY886), or WT cells containing Atg2-TAP and Atg18-13xMYC (RSGY012) were grown in YPD to a log phase and nitrogen starved in SD-N for 1 h. Cell lysates in a buffer containing either 1% Triton X-100 (E) or 1% CHAPS (F) were subsequently immunoprecipitated using GFP-trap beads (IP:GFP). Immunoprecipitates were analyzed by western blot using antibodies against GFP, TAP and MYC. (G and H) Quantifications of the experiments shown in panels E and F, respectively. Ratios between Atg2-TAP:Atg9-GFP and Atg18-13xMYC:Atg9-GFP were determined by quantifying the intensity of the corresponding bands and subsequently expressing the values relative to the cells carrying Atg9-GFP. Asterisks highlight significant differences with the cells expressing WT Atg9-GFP.