Sec24 phosphorylation regulates autophagosome abundance during nutrient deprivation
- University of California, San Diego, United States
- Columbia University, United States
- University of Pittsburgh School of Medicine, United States
- MRC Laboratory of Molecular Biology, United Kingdom
Figures
Figure 1 with 2 supplements
Identification of Sec24 phosphorylation sites required for autophagy.
(A) Ribbon diagram of Sec23 (lime) and Sec24 (lavender) with groups of key Sec24 phosphorylation sites (green, pink, and teal). The mSec24 membrin-binding site and conserved cargo binding B-site are …
Figure 1—figure supplement 1
Phosphorylation of Sec24 S730/S735 disrupts Sed5 packaging.
(A) Two different views of a ribbon diagram of Sec24 (lavender) bound to a Sed5 peptide (green) with S730 and S735 highlighted (pink). (B) Total membranes (T) and budded vesicles were collected from …
Figure 1—figure supplement 2
Sec24 S645/S678 does not affect cargo packaging.
Vesicles were collected from in vitro budding reactions performed with WT (lane 1), no Sec24 (lane 2) or mutant Sec24 proteins (lanes 3–4). The incorporation of a panel of cargo was determined by …
Figure 2 with 2 supplements
Phosphorylation of T324/T325/T328 in Sec24 is required for autophagy, but not ER-Golgi transport.
(A) Vacuolar alkaline phosphatase activity was assayed in lysates prepared from a sec24∆iss1∆ deletion strain harboring sec24 alanine mutations. The activity of wild-type (WT) 2 hr after starvation …
Figure 2—figure supplement 1
ER-Golgi transport is delayed during autophagy.
(A) WT cells were grown in nutrient rich media (SMD) (lanes 1–4) or starved for nitrogen (SD-N) for 1 hr at 25°C (lanes 5–8). Cells were pulse-labeled for 4 min and chased at the indicated times as …
Figure 2—figure supplement 2
Phosphorylation of the Sec23 membrane distal sites is not required for autophagy.
(A) Ribbon diagram of Sec23 (lime) and Sec24 (lavender) with membrane distal phosphorylation sites highlighted (red). (B) Plasmids encoding SEC23 (WT) or mutant sec23 were expressed in SFNY1948 and …
Figure 3 with 1 supplement
Phosphorylation of Sec24 regulates autophagosome frequency during starvation.
(A) Representative images from WT cells expressing GFP-Atg8 treated with 400 ng/ml rapamycin for 1 hr at 25°C (left top) or untreated (left bottom). Deconvolved images are shown. Scale bar, 1 µm. …
Figure 3—figure supplement 1
GFP-Atg8 puncta formation is affected in the sec12-4 mutant during autophagy induction.
(A) Cells with (left) and without (right) Iss1 were lysed as described in the Materials and methods and Ape1 processing was assayed by western blot analysis. Ape1 is processed in the sec24 alanine …
Figure 4 with 1 supplement
The Sec23/Sec24 complex binds the C-terminus of Atg9.
(A) Structure of yeast Sec24 and mSec24a with conserved residues in membrane distal sites (red). (B) ypt7∆ cells expressing Atg9-13myc were grown in nutrient rich (SMD) or starvation (SD-N) media …
Figure 4—figure supplement 1
Ponceau staining of in vitro bindings in Figure 4D, E and F.
(A) Ponceau staining of Figure 4D. Equimolar amounts (200 nM) of purified GST, GST-Sec31 (aa878-1114) or GST-Atg9 fragments were incubated with 50 or 100 nM of Sec23/Sec24-His6. Asterisks denote GST …
Figure 5 with 5 supplements
Phosphorylation of the Sec24 membrane distal sites regulates the Sec24-Atg9 interaction.
(A) GST-Atg9C (200 nM) was incubated with 37.5 nM of WT Sec23/Sec24-His6, Sec23/Sec24-T325E-His6 or Sec23/Sec24-T324E/T328E-His6 (left). Ratio of Sec24 bound to GST-Atg9C was quantified from three …
Figure 5—figure supplement 1
GST negative control for in vitro binding in Figure 5A.
(A) Equimolar amounts of GST or GST-Atg9C (200 nM) were incubated with 37.5 nM of WT Sec23/Sec24-His6, Sec23/Sec24-T325E-His6 or Sec23/Sec24-T324E/T328E-His6. (B) Sec24 T324E/T325E does not disrupt …
Figure 5—figure supplement 2
Sec24-3A does not affect Atg assembly at the PAS.
(A) Sec24 and Sec24-3A cells expressing Ape1-RFP and GFP tagged Atgs were treated with 400 ng/mL rapamycin for 1 hr at 25°C and the percent of Ape1-RFP colocalized with Atgs was determined in 300 …
Figure 5—figure supplement 3
Sec24-3A does not affect ERES formation.
(A) Cells with Sec13-GFP and expressing either Sec24 or Sec24-3A were grown in nutrient rich medium (SMD) or starved for nitrogen (SD-N) for 2 hr at 25°C. Scale bar 2 µm. (B) The number of Sec13-GFP …
Figure 5—figure supplement 4
Representative blots for quantitation shown in Figure 5E.
Sec24 was immunoprecipitated from lysates prepared from WT and hrr25-5 cells expressing Atg9-13myc. Cropped western blot (top). Uncropped western blot (bottom) from top showing pre-immune controls …
Figure 5—figure supplement 5
Autophagic body number is reduced in the hrr25-5 mutant.
(A) Representative images of autophagic bodies in pep4Δ (left) or hrr25-5pep4Δ (right) cells 1.5 hr after nitrogen starvation at 37°C. Scale bar represents 500 nm. (B) Histogram showing the …
Figure 6 with 4 supplements
Hrr25 regulates autophagy via phosphorylation of the Sec24 membrane distal sites.
(A) Sec24 was immunoprecipitated from WT or hrr25-5 cells expressing Atg9-13myc and either WT Sec24 or Sec24 T325A/T328A or Sec24 T325E/T328E. Precipitated Atg9-13myc was quantitated and normalized …
Figure 6—figure supplement 1
Nutrient rich controls for GFP-Atg8 cleavage in Figure 6C.
https://doi.org/10.7554/eLife.21167.020
Figure 6—figure supplement 2
Hrr25 is not regulated during starvation.
(A) Lysates were prepared from WT cells starved for nitrogen for the indicated time periods by incubating 2.5 OD600 units of cells with 200 µl 0.1 M NaOH for 5 min at room temperature. The …
Figure 6—figure supplement 3
The hrr25-5 mutant does not affect Atg assembly at the PAS.
(A) WT and hrr25-5 cells expressing Ape1-RFP and GFP tagged Atgs were starved for nitrogen for 2 hr at 37°C and the percent of Ape1-RFP colocalized with Atgs was determined in 300 cells. Arrowheads …
Figure 6—figure supplement 4
The sec12-4 mutant does not affect Atg2 or Atg14 PAS localization.
(A) WT and sec12-4 cells expressing Ape1-RFP and Atg2-GFP or Atg14-GFP were starved for nitrogen for 2 hr at 37°C and the percent of Ape1-RFP colocalized with Atg2 or Atg14 was determined in 300 …
Figure 7 with 1 supplement
Phosphorylation of a conserved regulatory surface of Sec24 enhances the ability of the COPII coat to recognize the autophagy machinery.
Phosphorylation of the Sec24 membrane distal sites regulates the interaction of Sec24 with the C-terminus of Atg9. During starvation, the Sec24-Atg9 interaction is needed to increase autophagosome …
Figure 7—figure supplement 1
Sec13-GFP accumulates at the PAS in the atg9∆ mutant.
(A) WT and atg9∆ cells expressing Sec13-GFP and Ape1-RFP were starved for nitrogen for 2 hr at 30°C and the percent of Ape1-RFP that colocalized with (left) or was adjacent to (right) Sec13-GFP was …
Additional files
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Supplementary file 1
Identification of Sec24 phosphorylation sites.
Sec24-His6 was purified from yeast and subjected to tandem mass spectrometry as described in the Materials and Methods. Phosphorylation sites with a confidence level of 40% or greater were further analyzed. Phosphorylated residues were mutated to either aspartic or glutamic acid and introduced into sec24∆ and sec24∆iss1∆ deletion strains as described in the Materials and Methods. Sec24 and Iss1 were aligned using MAFFT alignment program and residues were considered conserved if either serine or threonine.
- https://doi.org/10.7554/eLife.21167.026
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Supplementary file 2
Key yeast strains used in this study.
- https://doi.org/10.7554/eLife.21167.027
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Supplementary file 3
High confidence Sec24 phosphorylated peptides.
Compilation of mass spectrometry data of Sec24 phosphorylated peptides summarized in Figure 1B and Supplementary file 1.
- https://doi.org/10.7554/eLife.21167.028
