1. Volume 23, Issue 12, Pages 3579-3590 (June 2018)
The PP2A-like Protein Phosphatase Ppg1 and the Far Complex Cooperatively Counteract CK2-Mediated Phosphorylation of Atg32 to Inhibit Mitophagy 
Kentaro Furukawa, Tomoyuki Fukuda, Shun-ichi Yamashita, Tetsu Saigusa, Yusuke Kurihara, Yutaka Yoshida, Hiromi Kirisako, Hitoshi Nakatogawa, Tomotake Kanki 
Cell Reports 
Volume 23, Issue 12, Pages (June 2018)
DOI: /j.celrep
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2. Cell Reports 2018 23, 3579-3590DOI: (10.1016/j.celrep.2018.05.064)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1
Accumulation of GFP-Atg32 as a Punctum on Mitochondria Is a Sensitive Indicator for Initiation of Mitophagy
(A–D) atg32Δ (Α), atg1Δ atg32Δ (Β), atg11Δ atg32Δ (C), and atg32Δ (D) cells expressing GFP-Atg32 (A–C) or GFP-Atg32-2SA(S114A/S119A) (D) under control of the CUP1 promoter were cultured in SMD-Ura medium until early log growth phase. These cells were then shifted to nitrogen starvation medium (SD-N) for the indicated hours. Cells were labeled with the vacuolar marker FM4-64 and analyzed by fluorescence microscopy. The arrowheads indicate puncta of GFP-Atg32. Scale bars, 2 μm.
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4. Figure 2
Deletion of PPG1 Causes Phosphorylation of Atg32 and Accelerates Mitophagy
(A) Wild-type, atg32Δ, and the indicated phosphatase mutant cells were cultured in YPL medium until mid-log growth phase. The phosphorylation status of Atg32 was analyzed by immunoblotting with anti-Atg32 antibodies. Pgk1 was detected as a loading control (throughout this study).
(B) Wild-type and ppg1Δ cells were transformed with control vector (pRS413) only, PPG1, or the catalytically inactive PPG1-H111N gene. Cells were cultured in SMD-His medium until mid-log growth phase. The phosphorylation status of Atg32 was detected as in (A).
(C) atg11Δ atg32Δ ppg1Δ cells expressing wild-type Atg32 or the indicated Atg32 mutant were cultured in SML-Ura medium until mid-log growth phase. The phosphorylation status of Atg32 was examined as in (A).
(D) Wild-type and ppg1Δ cells were cultured in YPL medium until early log growth phase. TBB was then added to the medium, and cells were further cultured for 4 hr. The phosphorylation status of Atg32 was detected as in (A).
(E) atg11Δ atg32Δ or atg11Δ atg32Δ ppg1Δ cells expressing HA-Atg11 and PA only or PA-Atg32 under control of the CUP1 promoter were cultured in SMD-Trp-Ura medium until early log growth phase. PA or PA-Atg32 was precipitated using immunoglobulin G (IgG)-Sepharose from cell lysates. Immunoblots of total cell lysates (left) and the IgG precipitates (right) were probed with anti-PA and anti-HA antibodies.
(F) The indicated cells expressing GFP-Atg32 under control of the CUP1 promoter were cultured in SMD-Ura medium until early log growth phase and analyzed as in Figure 1. The arrowheads indicate puncta of GFP-Atg32. Scale bars, 2 μm.
(G) Wild-type, ppg1Δ, PPG1 overexpression (O.E. PPG1), and atg1Δ cells expressing Idh1-GFP were continuously cultured in YPL medium for 20–40 hr. Atg32 status and Idh1-GFP processing were monitored by immunoblotting with anti-Atg32 and anti-GFP antibodies, respectively. The relative amount of processed GFP was calculated. The values represent the means and SD from three independent experiments.
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5. Figure 3
Two Independent Triggers, Atg32 Phosphorylation and Atg1 Complex Activation, Are Sufficient for Progression of Mitophagy
(A) Wild-type and atg1Δ cells expressing Pho8Δ60 were transformed with control vector (pRS416) only, ATG13, or the ATG13-8SA gene. Cells were cultured in SML-Ura medium until early log growth phase. Cells were collected, and protein extracts were assayed for Pho8Δ60 activity. The results represent the mean and SD of three experiments.
(B) Wild-type and atg1Δ cells expressing GFP-Atg8 were transformed with control vector (pRS416) only, ATG13, or the ATG13-8SA gene. Cells were cultured in SML-Ura medium until early log growth phase. GFP-Atg8 processing was monitored by immunoblotting with anti-GFP antibodies.
(C) atg13Δ and atg13Δ ppg1Δ cells were transformed with control vector (pRS416) only, ATG13, or the ATG13-8SA gene. Cells were cultured in SML-Ura medium until early log growth phase. Atg13 status and Idh1-GFP processing were monitored by immunoblotting with anti-Atg13 and anti-GFP antibodies, respectively. The relative amount of processed GFP was calculated as in Figure 2G.
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6. Figure 4
Deletion or Overexpression of PPG1 Does Not Affect Bulk Autophagy, the Cvt Pathway, and Pexophagy
(A) The indicated cells expressing Pho8Δ60 were cultured in YPD medium until early log growth phase and shifted to SD-N. Cells were collected at the indicated time points, and protein extracts were assayed for Pho8Δ60 activity. The results represent the mean and SD of three experiments.
(B) The indicated cells expressing GFP-Atg8 were cultured in YPD medium until early log growth phase and shifted to SD-N. Cells were collected at the indicated time points, and GFP-Atg8 processing was monitored by immunoblotting with anti-GFP antibodies.
(C) The indicated cells were cultured in YPD medium until early log growth phase and then shifted to SD-N for 1 hr. prApe1 maturation during vegetative growth and after starvation was analyzed by immunoblotting using anti-Ape1 antibodies. The positions of precursor and mature Ape1 are indicated.
(D) The indicated cells expressing Pex14-GFP were cultured in medium containing oleic acid for 20 hr and then shifted to SD-N. Cells were collected at the indicated time points, and Pex14-GFP processing was analyzed by immunoblotting with anti-GFP antibodies.
(E) Wild-type and ppg1Δ cells expressing Atg19-GFP were cultured in YPD medium until optical density 600 (OD600) = 2, 4, and 6. Cells were collected and analyzed to monitor phosphorylation of Atg19 by immunoblotting with anti-GFP antibodies. The positions of non-phosphorylated and phosphorylated Atg19 are indicated by a black and a white arrowhead, respectively.
(F) Wild-type and ppg1Δ cells expressing Atg36-GFP were cultured in medium containing oleic acid and shifted to SD-N for 0, 3, and 6 hr. Cells were collected and analyzed to monitor phosphorylation of Atg36 by immunoblot with anti-GFP antibodies. The positions of non-phosphorylated and phosphorylated Atg36 are indicated by a black and a white arrowhead, respectively.
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7. Figure 5
The Far Complex Is Essential for Ppg1 Function to Dephosphorylate Atg32
(A) The phosphorylation status of Atg32 in the indicated cells was analyzed as in Figure 2.
(B) Schematic of proteomic analysis. Ppg1 (no tag) or FLAG-His6-Ppg1 was expressed under control of the CUP1 promoter in ppg1Δ cells. Cell lysates were subjected to tandem affinity purification using an anti-FLAG M2 affinity gel and TALON metal affinity resin. The eluates were resolved on SDS-PAGE followed by silver staining.
(C) Mass spectrometric analysis of the proteins co-purified with FLAG-His6-Ppg1 identified Tpd3, Hsp60, and Far8 as candidates of Ppg1-associated proteins. Mass spectrometric profiles were identified using the Mascot search engine. Score, Mascot scores.
(D) The phosphorylation status of Atg32 in the indicated cells was analyzed as in Figure 2.
(E) The indicated cells expressing GFP-Atg32 under control of the CUP1 promoter were cultured and analyzed as in Figure 1. The arrowheads indicate puncta of GFP-Atg32. Scale bars, 2 μm.
(F) The phosphorylation status of Atg32 and Idh1-GFP processing in the indicated cells were monitored as in Figure 2G. The relative amount of processed GFP was calculated as in Figure 2G.
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8. Figure 6
A Cytosolic Region (aa Residues 151–200) of Atg32 Is Required for Suppressing Induction of Mitophagy
(A) Schematic diagram of Atg32 and its deletion derivatives. Atg11 BR, Atg11-binding region; TMD, transmembrane domain; S114 and S119, serine residues phosphorylated by CK2.
(B) atg32Δ cells expressing GFP-Atg32 or its deletion derivatives under control of the CUP1 promoter were cultured in SMD-Ura medium until early log growth phase and shifted to SD-N medium for 1 hr. The arrowheads indicate puncta of GFP-Atg32. Scale bars, 2 μm.
(C) atg32Δ (top and bottom) and atg11Δ atg32Δ (center) cells expressing GFP-Atg32 or GFP-Atg322SA were cultured as in (B).
(D) atg11Δ atg32Δ cells expressing HA-Atg11 and PA only or PA-Atg32 (or Δ151–200) under control of the CUP1 promoter were cultured in SMD-Trp-Ura medium until early log phase and then shifted to SD-N for 1 hr. PA or PA-Atg32 was precipitated using IgG-Sepharose from cell lysates. Immunoblots of total cell lysates (left) and the IgG precipitates (right) were probed with anti-PA and anti-HA antibodies.
(E) atg32Δ cells expressing wild-type Atg32 or the indicated Atg32 mutant under control of the ATG32 promoter were continuously cultured in SML medium for 1–3 days. Idh1-GFP processing was monitored by immunoblotting with anti-GFP antibodies.
(F) atg13Δ atg32Δ cells co-expressing Atg13/Atg13-8SA and Atg32/Atg32Δ151–200 were cultured in SML-Trp-Ura medium until early log growth phase. Atg13 status and Idh1-GFP processing were monitored by immunoblotting with anti-Atg13 and anti-GFP antibodies, respectively.
(E and F) The relative amount of processed GFP was calculated as in Figure 2G.
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9. Figure 7 Model for the Regulatory Mechanism of Mitophagy
Ppg1, together with the Far complex, dephosphorylate Atg32 by competing with CK2 and constitutively suppresses mitophagy when mitophagy should not be induced. The aa 151–200 region of Atg32 is required for dephosphorylation of Atg32 by the Ppg1-Far complex. Upon induction of mitophagy, Ppg1 activity is suppressed, and CK2 acts toward Atg32 to phosphorylate Atg32. Phosphorylation of Atg32 triggers punctum formation of Atg32 in an Atg11-dependent manner, and mitochondria are delivered to the PAS. The core autophagy machinery, including the activated Atg1 complex, are recruited to the PAS for further progression of mitophagy.
Cell Reports  , DOI: ( /j.celrep )
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