Figure 1.
Vacuolar lipase activity derived from Atg15 depends on Pep4/Prb1. (A) Schematic diagram of Atg15. The transmembrane domain and a lipase consensus motif are shown. (B) Schematic overview of the in vitro assay system used to measure vacuolar lipase activity. (C) Lipase activities of vacuolar lysates from WT, atg15Δ, and atg15Δ cells harboring ATG15 from expressed from either a single-copy plasmid (pRS316) or multicopy (OE) plasmid (pRS426). Lysates were incubated with NBD-PE (18:1–12:0) for 20 min. The amount of lysate used for each assay was normalized by the amount of the vacuolar membrane protein Pho8 (see Fig. S1 B). TLC plate data (upper panel) are representative results from three independent experiments. Relative intensity (lower panel) was calculated as follows: relative intensity (%) = (peak area for NBD-PE, NBD-LPE, or NBD-FFA) ×100/(peak area for NBD-PE + peak area for NBD-LPE + peak area for NBD-FFA). Error bars represent means ± SD of three independent experiments. (D) Lipase activity of vacuolar lysates from Atg15OE cells. The lysate was incubated with NBD-PE for 0, 10, 30, and 90 min. (E) Lipase activities of vacuolar lysate from Atg15OE cells. A vacuolar lysate preparation was added at three concentrations relative to standard assay conditions (1×, 5×, and 25×) and incubated with NBD-PE for 20 min. The amount of lysate used for this assay is shown in Fig. S1 D. (F) Lipase activities of vacuolar lysates from WT, prb1Δ, pep4Δ, and pep4Δ prb1Δ cells. A vacuolar lysate preparation was added at three concentrations relative to standard assay conditions (1×, 5×, and 25×) and incubated with NBD-PE for 20 min. The amount of lysates used for this assay is shown in Fig.  S1 E. Source data are available for this figure: SourceData F1.

Vacuolar lipase activity derived from Atg15 depends on Pep4/Prb1. (A) Schematic diagram of Atg15. The transmembrane domain and a lipase consensus motif are shown. (B) Schematic overview of the in vitro assay system used to measure vacuolar lipase activity. (C) Lipase activities of vacuolar lysates from WT, atg15Δ, and atg15Δ cells harboring ATG15 from expressed from either a single-copy plasmid (pRS316) or multicopy (OE) plasmid (pRS426). Lysates were incubated with NBD-PE (18:1–12:0) for 20 min. The amount of lysate used for each assay was normalized by the amount of the vacuolar membrane protein Pho8 (see Fig. S1 B). TLC plate data (upper panel) are representative results from three independent experiments. Relative intensity (lower panel) was calculated as follows: relative intensity (%) = (peak area for NBD-PE, NBD-LPE, or NBD-FFA) ×100/(peak area for NBD-PE + peak area for NBD-LPE + peak area for NBD-FFA). Error bars represent means ± SD of three independent experiments. (D) Lipase activity of vacuolar lysates from Atg15OE cells. The lysate was incubated with NBD-PE for 0, 10, 30, and 90 min. (E) Lipase activities of vacuolar lysate from Atg15OE cells. A vacuolar lysate preparation was added at three concentrations relative to standard assay conditions (1×, 5×, and 25×) and incubated with NBD-PE for 20 min. The amount of lysate used for this assay is shown in Fig. S1 D. (F) Lipase activities of vacuolar lysates from WT, prb1Δ, pep4Δ, and pep4Δ prb1Δ cells. A vacuolar lysate preparation was added at three concentrations relative to standard assay conditions (1×, 5×, and 25×) and incubated with NBD-PE for 20 min. The amount of lysates used for this assay is shown in Fig.  S1 E. Source data are available for this figure: SourceData F1.

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