Figure 4.
Isolation of active Atg15 from pellet fraction of the vacuole. (A) Maturation of prApe1 and expression of Atg15iFLAG, Atg15S332A,iFLAG, CPYN50-Atg15ΔN35,iFLAG, or CPYN50-Atg15ΔN35,S332A,iFLAG. Cells were grown in SD/CA medium and treated with rapamycin for 2 h. *, nonspecific band. (B) Overview of the strategy employed for the fractionation of prcAtg15s. Total vacuolar lysate (T) was fractionated to supernatant (S) and pellet (P) fractions by centrifugation. The pellet (P = T2) was then resuspended in buffer containing 2% Tx, 0.1 M Na2CO3, 2.5 M urea, or 1 M KCl and subjected to a second round of centrifugation to yield a pellet fraction (P2) and supernatant (S2) fraction. (C) Western blotting of prcAtg15 and vacuolar membrane proteins. Vacuolar lysates from Atg15iFLAG-expressing cells were subjected to fractionation as in B. Equivalent amounts (relative to starting materials) of each fraction were analyzed. (D) Lipase activities of T, S, and P fractions (panel C, lane 1–3). Each fraction was incubated with NBD-PE for 20 min. TLC analysis (upper panel) shows representative data from three independent experiments. The bar graph (lower panel) represents the relative intensity calculated as described in Fig. 1 C. Error bars represent means ± SD of three independent experiments. (E) Western blotting of the processed forms of CPYN50-Atg15ΔN35,iFLAG (prcAtg15ΔN35s) and vacuolar membrane proteins (as in C). (F) Western blotting of prcAtg15s obtained by immunoprecipitation. Untreated vacuolar pellet fraction (P), vacuolar pellet fraction treated with 0.5% DDM (P+DDM), prcAtg15s pulled down by α-FLAG antibody-conjugated agarose beads (Bound), and prcAtg15s eluted from bound by an excess amount of 3xFLAG peptides (Eluate) were analyzed. Samples equivalent to 0.2% of P and P+DDM, and 5% of Bound and Eluate were subjected to Ponceau S staining (left) and western blotting using α-FLAG antibodies (right). *, nonspecific band; **, mouse-IgG heavy chain; ⁂, mouse-IgG light chain. Source data are available for this figure: SourceData F4.

Isolation of active Atg15 from pellet fraction of the vacuole. (A) Maturation of prApe1 and expression of Atg15iFLAG, Atg15S332A,iFLAG, CPYN50-Atg15ΔN35,iFLAG, or CPYN50-Atg15ΔN35,S332A,iFLAG. Cells were grown in SD/CA medium and treated with rapamycin for 2 h. *, nonspecific band. (B) Overview of the strategy employed for the fractionation of prcAtg15s. Total vacuolar lysate (T) was fractionated to supernatant (S) and pellet (P) fractions by centrifugation. The pellet (P = T2) was then resuspended in buffer containing 2% Tx, 0.1 M Na2CO3, 2.5 M urea, or 1 M KCl and subjected to a second round of centrifugation to yield a pellet fraction (P2) and supernatant (S2) fraction. (C) Western blotting of prcAtg15 and vacuolar membrane proteins. Vacuolar lysates from Atg15iFLAG-expressing cells were subjected to fractionation as in B. Equivalent amounts (relative to starting materials) of each fraction were analyzed. (D) Lipase activities of T, S, and P fractions (panel C, lane 1–3). Each fraction was incubated with NBD-PE for 20 min. TLC analysis (upper panel) shows representative data from three independent experiments. The bar graph (lower panel) represents the relative intensity calculated as described in Fig. 1 C. Error bars represent means ± SD of three independent experiments. (E) Western blotting of the processed forms of CPYN50-Atg15ΔN35,iFLAG (prcAtg15ΔN35s) and vacuolar membrane proteins (as in C). (F) Western blotting of prcAtg15s obtained by immunoprecipitation. Untreated vacuolar pellet fraction (P), vacuolar pellet fraction treated with 0.5% DDM (P+DDM), prcAtg15s pulled down by α-FLAG antibody-conjugated agarose beads (Bound), and prcAtg15s eluted from bound by an excess amount of 3xFLAG peptides (Eluate) were analyzed. Samples equivalent to 0.2% of P and P+DDM, and 5% of Bound and Eluate were subjected to Ponceau S staining (left) and western blotting using α-FLAG antibodies (right). *, nonspecific band; **, mouse-IgG heavy chain; ⁂, mouse-IgG light chain. Source data are available for this figure: SourceData F4.

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