Figure S5.
Two PxL motifs in Atg13 are critical for the function of Atg13 in autophagy. (A) A PyMOL cartoon representation of the homology model of Atg13 (S. cerevisiae) residues 7–267 (green) made by SWISS-MODEL homology modeling (Waterhouse et al., 2018) superimposed on the cartoon of the crystal structure of Lachancea thermotolerans Atg13 HORMA domain (cyan; Protein Data Bank accession no. 4J2G). PxL1 and PxL2 sites mutated in this study are indicated as sticks in red. (B) The effects of rapamycin (RAP) or Cdc14(IN) plus rapamycin on cytosolic puncta formation of mScarlet-tagged Atg13 (variants) during synchronized meiosis. Shown are the percentage of cells with Atg13 (variants) puncta counted at t = 6 h after NDT80 induction (n ≥ 300, t test), by FM analysis. If applied, rapamycin was added to SPM 2 h before NDT80 induction; Cdc14(IN) (PZEV-CDC14-GFP) was induced simultaneously with NDT80. Colors of bars indicate the experimental conditions: no treatment (green); rapamycin (yellow); and rapamycin plus Cdc14(IN) (gray). (C) IB of cell extracts from synchronized meiotic cells expressing ATG13 (variants) with indicated antibodies. The mCherry-GFP (pZD: mCherry-GFP) and NDT80 were simultaneously induced by 1 µM β-estradiol. ATG13 variants carried by pRS303 or empty vector were introduced into atg13Δ cells. Note that mCherry-GFP processing was reduced by PxL1m (A76-PG) and by PxL3m (A476-DG), but not by PxL2m (A207-IG). (D) IB of extracts from cells expressing indicated ATG13 variants during log-phase (SD). ATG13 variants carried by pRS303 or empty vector were introduced into atg13Δ cells, in which GFP-Atg8 (pZD: GFP-ATG8) was induced by β-estradiol for 4 h before collection of cells. 2SA, Atg13-S129A-S454A; 6SA, Atg13-S129A-S348 A-S454A-S535 A-S541A-S646A. Note that the GFP-Atg8 processing levels in 2SA and 6SA cells increased, indicated by increased free GFP generation normalized by GFP-Atg8. (E) IB of cell extracts from nonsynchronized meiotic ATG13 and Atg13-6SA cells with indicated antibodies. ATG13 or Atg13-6SA carried by pRS303 was introduced into atg13Δ cells, which harbor inducible GFP-Atg8 (pZD: GFP-ATG8). After initiating sporulation for 6 h in SPM, GFP-Atg8 expression was induced; next, cells were harvested at indicated time points. Note that free GFP generation increased in Atg13-6SA cells. (F) Percentage of cells tri-/tetranucleated (three or four DAPI dots) at 16 h in SPM (nonsynchronized meiosis). From the experiment of Fig. 8 B. Cells collected at indicated time points were fixed and subjected to DAPI staining as described in Materials and methods. Note that 6SA restored the rate of tri-/tetranucleation in PxL3m cells to WT level. Source data are available for this figure: SourceData FS5.

Two PxL motifs in Atg13 are critical for the function of Atg13 in autophagy. (A) A PyMOL cartoon representation of the homology model of Atg13 (S. cerevisiae) residues 7–267 (green) made by SWISS-MODEL homology modeling (Waterhouse et al., 2018) superimposed on the cartoon of the crystal structure of Lachancea thermotolerans Atg13 HORMA domain (cyan; Protein Data Bank accession no. 4J2G). PxL1 and PxL2 sites mutated in this study are indicated as sticks in red. (B) The effects of rapamycin (RAP) or Cdc14(IN) plus rapamycin on cytosolic puncta formation of mScarlet-tagged Atg13 (variants) during synchronized meiosis. Shown are the percentage of cells with Atg13 (variants) puncta counted at t = 6 h after NDT80 induction (n ≥ 300, t test), by FM analysis. If applied, rapamycin was added to SPM 2 h before NDT80 induction; Cdc14(IN) (PZEV-CDC14-GFP) was induced simultaneously with NDT80. Colors of bars indicate the experimental conditions: no treatment (green); rapamycin (yellow); and rapamycin plus Cdc14(IN) (gray). (C) IB of cell extracts from synchronized meiotic cells expressing ATG13 (variants) with indicated antibodies. The mCherry-GFP (pZD: mCherry-GFP) and NDT80 were simultaneously induced by 1 µM β-estradiol. ATG13 variants carried by pRS303 or empty vector were introduced into atg13Δ cells. Note that mCherry-GFP processing was reduced by PxL1m (A76-PG) and by PxL3m (A476-DG), but not by PxL2m (A207-IG). (D) IB of extracts from cells expressing indicated ATG13 variants during log-phase (SD). ATG13 variants carried by pRS303 or empty vector were introduced into atg13Δ cells, in which GFP-Atg8 (pZD: GFP-ATG8) was induced by β-estradiol for 4 h before collection of cells. 2SA, Atg13-S129A-S454A; 6SA, Atg13-S129A-S348 A-S454A-S535 A-S541A-S646A. Note that the GFP-Atg8 processing levels in 2SA and 6SA cells increased, indicated by increased free GFP generation normalized by GFP-Atg8. (E) IB of cell extracts from nonsynchronized meiotic ATG13 and Atg13-6SA cells with indicated antibodies. ATG13 or Atg13-6SA carried by pRS303 was introduced into atg13Δ cells, which harbor inducible GFP-Atg8 (pZD: GFP-ATG8). After initiating sporulation for 6 h in SPM, GFP-Atg8 expression was induced; next, cells were harvested at indicated time points. Note that free GFP generation increased in Atg13-6SA cells. (F) Percentage of cells tri-/tetranucleated (three or four DAPI dots) at 16 h in SPM (nonsynchronized meiosis). From the experiment of Fig. 8 B. Cells collected at indicated time points were fixed and subjected to DAPI staining as described in Materials and methods. Note that 6SA restored the rate of tri-/tetranucleation in PxL3m cells to WT level. Source data are available for this figure: SourceData FS5.

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