Figure S1. Supplemental data of Fig. 1 .... | Rockefeller University Press

Figure S1.

$Supplemental data ofFig. 1. (A) Purification of vacuole from WT cells. The whole-cell lysate (T) and vacuolar fraction (V) samples were subjected to western blot analysis using antibodies against Pho8 (vacuole membrane), Ape1 (autophagy cargo), Pep4, Prb1 (vacuole lumen), Pgk1, Hsp90 (cytosolic proteins), Dpm1 (ER membrane), Cox2 (mitochondrial matrix), Por1 (outer mitochondrial membrane), Van1 (Golgi), and Gsp1 (nucleus). Samples equivalent to 1/3,000 of total lysate and 1/160 of vacuolar fraction were loaded. (B) Western blotting of Pho8 in vacuolar lysates from WT, atg15Δ, and atg15Δ cells harboring ATG15 from either single plasmid (pRS316) or multicopy (OE) plasmid (pRS426). The relative band intensity of Pho8 for each strain is shown under the panel. (C) Lipase activities of vacuolar lysates from WT, atg15Δ, and atg15Δ cells harboring ATG15 from either single or multicopy (OE) plasmids. Each lysate was incubated with NBD-PS (18:1–12:0), NBD-PC (18:1–12:0), and NBD-PG (18:1–12:0) for 20 min. The amount of lysate used for the assay was the same as Fig. 1 C. TLC analysis (left panels) shows representative results from three independent experiments. The bar graphs (right panels) represent the relative intensity calculated as described in Fig. 1 C. Error bars represent means ± SD of three independent experiments. (D) Western blotting of Pho8 in vacuolar lysates from atg15Δ cells expressing Atg15 or Atg15S332A from multicopy plasmids. The experimental procedure was the same as in B. (E) Western blotting of Pho8 in vacuolar lysates from WT, prb1Δ, pep4Δ, and pep4Δ prb1Δ cells. The experimental procedure was the same as in B. (F) Lipase activities of vacuolar lysates from WT, pep4Δ prb1Δ, and pep4Δ prb1Δ cells expressing Atg15 from a multicopy plasmid (pRS426). A vacuolar lysate preparation was added at three concentrations relative to standard assay conditions (1×, 5×, and 25×) and incubated with NBD-PE (18:1–12:0) for 20 min. The amount of lysates used for this assay is shown in G. (G) Western blot of Pho8 in vacuolar lysates from WT, atg15Δ, and pep4Δ prb1Δ cells expressing Atg15 from multicopy plasmids. The experimental procedure was the same as in B. Source data are available for this figure: SourceData FS1.$

Supplemental data of Fig. 1 . (A) Purification of vacuole from WT cells. The whole-cell lysate (T) and vacuolar fraction (V) samples were subjected to western blot analysis using antibodies against Pho8 (vacuole membrane), Ape1 (autophagy cargo), Pep4, Prb1 (vacuole lumen), Pgk1, Hsp90 (cytosolic proteins), Dpm1 (ER membrane), Cox2 (mitochondrial matrix), Por1 (outer mitochondrial membrane), Van1 (Golgi), and Gsp1 (nucleus). Samples equivalent to 1/3,000 of total lysate and 1/160 of vacuolar fraction were loaded. (B) Western blotting of Pho8 in vacuolar lysates from WT, atg15Δ, and atg15Δ cells harboring ATG15 from either single plasmid (pRS316) or multicopy (OE) plasmid (pRS426). The relative band intensity of Pho8 for each strain is shown under the panel. (C) Lipase activities of vacuolar lysates from WT, atg15Δ, and atg15Δ cells harboring ATG15 from either single or multicopy (OE) plasmids. Each lysate was incubated with NBD-PS (18:1–12:0), NBD-PC (18:1–12:0), and NBD-PG (18:1–12:0) for 20 min. The amount of lysate used for the assay was the same as Fig. 1 C. TLC analysis (left panels) shows representative results from three independent experiments. The bar graphs (right panels) represent the relative intensity calculated as described in Fig. 1 C. Error bars represent means ± SD of three independent experiments. (D) Western blotting of Pho8 in vacuolar lysates from atg15Δ cells expressing Atg15 or Atg15^S332A from multicopy plasmids. The experimental procedure was the same as in B. (E) Western blotting of Pho8 in vacuolar lysates from WT, prb1Δ, pep4Δ, and pep4Δ prb1Δ cells. The experimental procedure was the same as in B. (F) Lipase activities of vacuolar lysates from WT, pep4Δ prb1Δ, and pep4Δ prb1Δ cells expressing Atg15 from a multicopy plasmid (pRS426). A vacuolar lysate preparation was added at three concentrations relative to standard assay conditions (1×, 5×, and 25×) and incubated with NBD-PE (18:1–12:0) for 20 min. The amount of lysates used for this assay is shown in G. (G) Western blot of Pho8 in vacuolar lysates from WT, atg15Δ, and pep4Δ prb1Δ cells expressing Atg15 from multicopy plasmids. The experimental procedure was the same as in B. Source data are available for this figure: SourceData FS1.