Figure 2. Processing is necessary for... | Rockefeller University Press

Figure 2.

$Processing is necessary for activation of the vacuolar luminal region of Atg15. (A) Schematic illustration of the transport of Atg15 from the ER/Golgi to the vacuole via the MVB pathway. (B) Western blotting of Atg15-FLAG in the vacuole. Vacuolar fractions were obtained from atg15Δ or pep4Δ prb1Δ cells expressing Atg15-FLAG. The samples were treated with or without Endo H and analyzed using α-FLAG antibody. Red arrowhead, 75 kD band of glycosylated whole Atg15-FLAG; blue arrowhead, unglycosylated whole Atg15-FLAG. The amount of lysates used for this analysis is shown in Fig. S2 D. (C) Schematic diagram of Atg15 and CPYN50-Atg15ΔN35. TMD, transmembrane domain. (D) Fluorescence microscopy image of atg15∆ cells expressing CPYN50-Atg15ΔN35-mNG under the control of the endogenous ATG15 promoter from a multicopy plasmid (pRS426). Cells grown in SD/CA medium were treated with rapamycin for 3 h. Scale bar, 1 µm. Dashed lines indicate vacuole boundaries. DIC, differential interference contrast. (E) Analysis of the maturation of prApe1 in atg15Δ cells expressing Atg15 or CPYN50-Atg15ΔN35 under the control of the endogenous ATG15 promoter from a multicopy plasmid (pRS426). Cells grown in SD/CA medium were treated with rapamycin for 2 h. prApe1 and mApe1 were detected using α-Ape1 antibodies. (F) Localization of mCherry-Atg8 in atg15Δ cells or pep4Δ prb1Δ cells, each expressing CPYN50-Atg15ΔN35. Cells grown in YPD medium were treated with rapamycin for 3 h. Scale bar, 1 µm. See also Video 1. Dashed lines indicate vacuole boundaries. (G) Lipase activities of CPYN50-Atg15∆N35–expressing atg15Δ cells and pep4Δ prb1Δ cells. A vacuolar lysate preparation was added at three concentrations relative to standard assay conditions (1×, 2×, and 4×) and incubated with NBD-PE for 20 min. The amount of lysates used for these analyses is shown in Fig. S2 G. Source data are available for this figure: SourceData F2.$

Processing is necessary for activation of the vacuolar luminal region of Atg15. (A) Schematic illustration of the transport of Atg15 from the ER/Golgi to the vacuole via the MVB pathway. (B) Western blotting of Atg15-FLAG in the vacuole. Vacuolar fractions were obtained from atg15Δ or pep4Δ prb1Δ cells expressing Atg15-FLAG. The samples were treated with or without Endo H and analyzed using α-FLAG antibody. Red arrowhead, 75 kD band of glycosylated whole Atg15-FLAG; blue arrowhead, unglycosylated whole Atg15-FLAG. The amount of lysates used for this analysis is shown in Fig. S2 D. (C) Schematic diagram of Atg15 and CPY^N50-Atg15^ΔN35. TMD, transmembrane domain. (D) Fluorescence microscopy image of atg15∆ cells expressing CPY^N50-Atg15^ΔN35-mNG under the control of the endogenous ATG15 promoter from a multicopy plasmid (pRS426). Cells grown in SD/CA medium were treated with rapamycin for 3 h. Scale bar, 1 µm. Dashed lines indicate vacuole boundaries. DIC, differential interference contrast. (E) Analysis of the maturation of prApe1 in atg15Δ cells expressing Atg15 or CPY^N50-Atg15^ΔN35 under the control of the endogenous ATG15 promoter from a multicopy plasmid (pRS426). Cells grown in SD/CA medium were treated with rapamycin for 2 h. prApe1 and mApe1 were detected using α-Ape1 antibodies. (F) Localization of mCherry-Atg8 in atg15Δ cells or pep4Δ prb1Δ cells, each expressing CPY^N50-Atg15^ΔN35. Cells grown in YPD medium were treated with rapamycin for 3 h. Scale bar, 1 µm. See also Video 1. Dashed lines indicate vacuole boundaries. (G) Lipase activities of CPY^N50-Atg15^∆N35–expressing atg15Δ cells and pep4Δ prb1Δ cells. A vacuolar lysate preparation was added at three concentrations relative to standard assay conditions (1×, 2×, and 4×) and incubated with NBD-PE for 20 min. The amount of lysates used for these analyses is shown in Fig. S2 G. Source data are available for this figure: SourceData F2.