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. 2018 Nov 2;37(21):e98899.
doi: 10.15252/embj.201798899. Epub 2018 Sep 20.

Syntaxin 17 Regulates the Localization and Function of PGAM5 in Mitochondrial Division and Mitophagy

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Free PMC article

Syntaxin 17 Regulates the Localization and Function of PGAM5 in Mitochondrial Division and Mitophagy

Masashi Sugo et al. EMBO J. .
Free PMC article

Abstract

PGAM5, a mitochondrial protein phosphatase that is genetically and biochemically linked to PINK1, facilitates mitochondrial division by dephosphorylating the mitochondrial fission factor Drp1. At the onset of mitophagy, PGAM5 is cleaved by PARL, a rhomboid protease that degrades PINK1 in healthy cells, and the cleaved form facilitates the engulfment of damaged mitochondria by autophagosomes by dephosphorylating the mitophagy receptor FUNDC1. Here, we show that the function and localization of PGAM5 are regulated by syntaxin 17 (Stx17), a mitochondria-associated membrane/mitochondria protein implicated in mitochondrial dynamics in fed cells and autophagy in starved cells. In healthy cells, loss of Stx17 causes PGAM5 aggregation within mitochondria and thereby failure of the dephosphorylation of Drp1, leading to mitochondrial elongation. In Parkin-mediated mitophagy, Stx17 is prerequisite for PGAM5 to interact with FUNDC1. Our results reveal that the Stx17-PGAM5 axis plays pivotal roles in mitochondrial division and PINK1/Parkin-mediated mitophagy.

Keywords: autophagy receptor; mitochondrial division; mitochondria‐associated membrane; mitophagy; protein phosphatase.

Figures

Figure 1
Figure 1. Stx17 binds to PGAM5

Schematic representation of Stx17 and PGAM5.

293T cells were transfected with a plasmid encoding FLAG‐Stx17 wild type (WT) or the indicated constructs. At 24 h after transfection, cell lysates were immunoprecipitated (IP) with anti‐FLAG M2 beads, and analyzed by IB using antibodies against PGAM5 and FLAG. Five percent of lysates was analyzed as input.

HeLa cells stably expressing FLAG‐Stx17 WT or the K254C mutant were fixed and subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P < 0.001 as compared with WT (paired Student's t‐test).

MBP or the MBP‐Stx17 constructs attached to amylose resin were mixed with GST‐PGAM5, and the proteins bound to the resin were separated by SDS–PAGE and blotted onto PVDF membranes. The blots were detected by an anti‐GST antibody (upper panels) or stained with Coomassie Brilliant Blue R‐250 (lower panels). Ten percent of the proteins used for each experiment was analyzed as input. Asterisks and double asterisk represent possible MBP dimers and degradation products, respectively.

293T cells were cotransfected with plasmids encoding FLAG‐Stx17 WT and the indicated PGAM5‐GFP constructs and analyzed as described in (B).

293T cells were cotransfected with plasmids encoding FLAG‐tagged Stx17 or Stx18 and the C‐terminally GFP‐tagged transmembrane domain of PGAM5 (amino acids 1–35) constructs and analyzed as described in (B).

Source data are available online for this figure.
Figure EV1
Figure EV1. Stx17 binds to Drp1 and PGAM5

293T cells were cotransfected with plasmids encoding GFP‐Drp1 K38A and FLAG‐Stx17 wild type (WT) or the indicated FLAG‐Stx17 constructs. At 24 h after transfection, cell lysates were immunoprecipitated (IP) with anti‐FLAG M2 beads and analyzed by IB using antibodies against GFP and FLAG. Five percent of lysates was analyzed as input.

MBP or the MBP‐Stx17 constructs attached to amylose resin were mixed with His6‐Drp1 K38A, and the proteins bound to the resin were separated by SDS–PAGE and blotted onto PVDF membranes. The blots were detected by an anti‐penta‐His tag antibody (upper panels) or stained with Coomassie Brilliant Blue R‐250 (lower panels). Ten percent of the proteins used for each experiment was analyzed as input. Asterisks and double asterisk represent possible MBP dimers and degradation products, respectively.

MBP‐Stx17 WT attached to amylose resin was mixed with the indicated GST‐PGAM5 constructs, and the proteins bound to the resin were separated by SDS–PAGE and blotted onto PVDF membranes. The blots were detected by an anti‐GST antibody (upper panels) or stained with Coomassie Brilliant Blue R‐250 (lower panels). Ten percent of the proteins used for each experiment was analyzed as input. PGAM5 ΔTMD (amino acids 30–289) and ΔTMD# (amino acids 25–289, corresponding to the PARL‐cleaved form) (Sekine et al, 2012).

Source data are available online for this figure.
Figure 2
Figure 2. PGAM5 is localized at the ER–mitochondria interface

HeLa cells were treated with DMSO (Vehicle) or 0.03 mg/ml digitonin (+Digitonin), fixed, and then double‐immunostained for PGAM5 and Tom20. Scale bar, 5 μm. The bar graph on the right shows the Manders’ coefficients for the colocalization of PGAM5 and Tom20. Values are means ± SEM (n = 3). ***P < 0.001 as compared with Vehicle (paired Student's t‐test).

HeLa cells stably expressing FLAG‐Stx17 wild type (WT) were transfected with a plasmid encoding Su9‐GFP (mitochondria) or Sec61β‐GFP (ER). At 24 h after transfection, the cells were subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. The bar graph on the right shows the Manders’ coefficients for the colocalization of PLA dots and Su9‐GFP or Sec61β‐GFP. Values are means ± SEM (n = 3). ***P < 0.001 (paired Student's t‐test).

HeLa cells were treated with DMSO (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed, and subjected to Percoll‐based fractionation. Equal amounts of proteins were analyzed by IB using the indicated antibodies. PNS, postnuclear supernatant; MS, microsomes; Mt, mitochondria. The amounts of proteins recovered on fractionation were as follows for vehicle and CCCP treatment, respectively: PNS (6.6 mg and 5.5 mg), cytosol (4.8 mg and 4.9 mg), MS (2.0 mg and 2.2 mg), MAM (0.55 mg and 0.46 mg), and Mt (0.30 mg and 0.28 mg).

Electron microscopic analysis of HeLa cells expressing PGAM5‐GFP and APEX2‐GFP‐binding peptide. Samples were prepared as described in Materials and Methods. Arrows indicate the position of 3,3′‐diaminobenzidine reaction at the ER–mitochondria interface. Scale bar, 500 nm.

HeLa cells stably expressing FLAG‐Stx17 WT were mock‐transfected or transfected with siRNA for Mfn1, Mfn2, or PACS‐2. At 72 h after transfection, the cells were subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P < 0.001 as compared with Mock (paired Student's t‐test).

HeLa cells were mock‐transfected or transfected with siRNA for Mfn1, Stx17, Mfn2, or PACS‐2. At 72 h after transfection, the cells were subjected to PLA using antibodies against Drp1 and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P < 0.001 as compared with Mock (paired Student's t‐test).

Source data are available online for this figure.
Figure EV2
Figure EV2. PGAM5 dephosphorylates and activates Drp1 in healthy cells

HeLa cells were incubated with DMSO (Vehicle), 5 mM MβCD for 1 h (MβCD), or 10 μg/ml nystatin (Nystatin) for 20 min and then double‐immunostained for PGAM5 (Alexa Fluor 488) and Tom20 (Alexa Fluor 594).

HeLa cells with mock treatment (Mock) or depleted of PGAM5 (PGAM5 KD) were fixed and then double‐immunostained for PGAM5 and Tom20.

HeLa cells with mock treatment or depleted of PGAM5 were fixed after treatment with 20 μM CCCP for 2 h and then double‐immunostained for PGAM5 and Tom20.

HeLa cells with mock treatment or depleted of PGAM5 or Stx17 were lysed and analyzed IB using the indicated antibodies.

HeLa cells were transfected with a plasmid encoding C‐terminally FLAG‐tagged PGAM5 or the H105A mutant. At 24 h after transfection, the cells were double‐immunostained for FLAG and Tom20.

Data information: Scale bars, 5 μm. Values are means ± SEM (n = 3). **P < 0.01 and as compared with Mock; ***P < 0.001 as compared with PGAM5 (WT) by paired Student's t‐test.Source data are available online for this figure.
Figure 3
Figure 3. Localization of PGAM5 is regulated by Stx17

HeLa cells with mock treatment (Mock) or depleted of Stx17 (Stx17 KD) were fixed and double‐immunostained for PGAM5 and Tom20. Scale bar, 5 μm.

293T cells with mock treatment or depleted of Stx17 were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed, and then analyzed by IB using the indicated antibodies.

293T cells transiently expressing FLAG‐Stx17 wild type (WT) or the K254C mutant were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed, immunoprecipitated (IP) with anti‐FLAG M2 beads, and then analyzed by IB using the indicated antibodies.

HeLa cells stably expressing FLAG‐Stx17 WT were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, and subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P < 0.001 as compared with Vehicle (paired Student's t‐test).

Source data are available online for this figure.
Figure 4
Figure 4. Mitophagy is inhibited upon depletion of Stx17 or PGAM5

HeLa cells stably expressing GFP‐Parkin with mock treatment (Control) or depleted (KD) of Stx17, PGAM5 or Drp1 were incubated with ethanol (Vehicle) or 10 μM CCCP (+CCCP) for 16 h and analyzed by IB using the indicated antibodies.

HeLa cells stably expressing GFP‐Parkin with mock treatment (Mock) or depleted of Stx17, PGAM5, or Drp1 were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h and analyzed by immunofluorescence microscopy. Scale bar, 5 μm. The bar graph on the right shows the Manders’ coefficients for the colocalization of GFP‐Parkin and Tom20. Values are means ± SEM (n = 3). ***P < 0.001 as compared with Mock (paired Student's t‐test).

PINK1‐FLAG and GFP‐Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence (ethanol) or presence of 20 μM CCCP for 2 h and analyzed by IB using the indicated antibodies. F, full‐length PINK1; C, cleaved PINK1.

Source data are available online for this figure.
Figure EV3
Figure EV3. FLAG‐Stx17 wild type, but not the K254C mutant, can compensate for Stx17 depletion in mitophagy

GFP‐Parkin stably expressing HeLa cells with mock treatment (Mock) or treated with siRNA (KD) for Stx17, PGAM5, or FUNDC1 for 72 h were untreated (Fed) or starved for 2 h (SV), lysed and then analyzed by IB using the indicated antibodies.

HeLa cells stably expressing FLAG‐Stx17 wild type (WT) or the K254C mutant were transfected with siRNA for Stx17. At 48 h after transfection, the cells were transfected with a plasmid encoding GFP‐Parkin and incubated for 24 h. They were then incubated with 10 μM CCCP (+CCCP) for 16 h, lysed, and analyzed by IB using the indicated antibodies.

HeLa cells stably expressing FLAG‐Stx17 WT or the K254C mutant were transfected with siRNA for Stx17. At 48 h after transfection, the cells were transfected with a plasmid encoding GFP‐Parkin and incubated for 24 h. They were then incubated with 20 μM CCCP for 2 h and then immunostained for Tom20. Scale bar, 5 μm.

GFP‐Parkin stably expressing HeLa cells incubated with siRNA for PGAM5 for 48 h were transfected with a plasmid encoding C‐terminally FLAG‐tagged PGAM5 or the H105A mutant without a mutation in the siRNA targeting sequence. After 24 h, the cells were incubated with 20 μM CCCP for 2 h and then immunostained for FLAG. PGAM5 proteins were found to be expressed likely due to the overexpression of their mRNAs. Scale bar, 5 μm.

GFP‐Parkin stably expressing HeLa cells, or ones depleted of PINK1 (PINK1 KD) or depleted of both PINK1 and Stx17 or PGAM5 (DKD) were incubated with 20 μM CCCP for 2 h and immunostained for Tom20. Scale bar, 5 μm.

GFP‐Parkin stably expressing HeLa cells or ones depleted of Stx17 or PGAM5 were incubated with 20 μM CCCP for the indicated times and immunostained for Tom20. Scale bar, 5 μm.

Source data are available online for this figure.
Figure EV4
Figure EV4. GFP‐Parkin‐associated structures are sensitive to digitonin

GFP‐Parkin stably expressing HeLa cells were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, treated with 0.02% Triton X‐100 (A) or 0.03 mg/ml digitonin (B), and then immunostained for Tom20. Scale bars, 5 μm.

GFP‐Parkin stably expressing HeLa cells depleted of Stx17 were incubated with 20 μM CCCP (+CCCP) for 2 h, untreated or treated with 0.03 mg/ml digitonin, and then immunostained for Tom20. Scale bar, 5 μm.

Figure 5
Figure 5. PGAM5 depletion abrogates the proximity of GFP‐Parkin‐associated mitochondria to omegasomes

GFP‐Parkin stably expressing HeLa cells were mock‐transfected (Mock) or transfected with siRNA (KD) for Stx17 or PGAM5. At 48 h after siRNA transfection, the cells were transfected with a plasmid encoding FLAG‐DFCP1, incubated for 24 h, treated with 20 μM CCCP for 2 h, and then immunostained for FLAG. Scale bars, 5 μm. The bar graph below shows the Manders’ coefficients for the colocalization of GFP‐Parkin and FLAG‐DFCP1. Values are means ± SEM (n = 3). **P < 0.01 as compared with Mock (paired Student's t‐test).

GFP‐Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated with 20 μM CCCP for 2 h and immunostained for LC3. Scale bar, 5 μm. The bar graph below shows the Manders’ coefficients for the colocalization of GFP‐Parkin and LC3. Values are means ± SEM (n = 3). **P < 0.01 as compared with Mock (paired Student's t‐test).

GFP‐Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h and analyzed by IB using the indicated antibodies.

Source data are available online for this figure.
Figure EV5
Figure EV5. Ubiquitination occurs at the sites to which GFP‐Parkin has translocated even in the absence of Stx17 or PGAM5

GFP‐Parkin stably expressing cells with mock treatment (Mock) or depleted of PGAM5 (PGAM5 KD) or Stx17 (Stx17 KD) were incubated with 20 μM CCCP for 2 h and immunostained for LC3 and ubiquitin (A), ubiquitin (B), or p62 (C). Scale bars, 5 μm.

Figure 6
Figure 6. Stx17 is required for the link between PGAM5 and FUNDC1

HeLa cells stably expressing GFP‐Parkin with mock treatment (Control) or depleted of FUNDC1 (FUNDC1 KD) were incubated for 16 h with ethanol (Vehicle) or 10 μM CCCP (+CCCP), and analyzed by IB using the indicated antibodies.

GFP‐Parkin stably expressing HeLa cells with mock treatment (Mock) or depleted of FUNDC1 (FUNDC1 KD) were incubated with 20 μM CCCP for 2 h and immunostained for Tom20. Scale bar, 5 μm.

293T cells with mock treatment (Mock) or depleted of Stx17 (Stx17 KD) were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h, immunoprecipitated with anti‐FLAG M2 beads and then analyzed by IB using the indicated antibodies. Three percent of lysates was analyzed as input. During Stx17 knockdown, cells were transfected with a plasmid encoding FLAG‐FUNDC1.

GFP‐Parkin stably expressing HeLa cells were treated as described in (C) and then subjected to PLA using antibodies against FLAG and PGAM5. During Stx17 knockdown, cells were transfected with a plasmid encoding FLAG‐FUNDC1. Scale bar, 5 μm. Values are means ± SEM (n = 3). *P < 0.05 and **P < 0.01 as compared with Mock (+CCCP) by paired Student's t‐test.

Source data are available online for this figure.
Figure 7
Figure 7. PGAM5 modulates the mitochondrial phenotypes caused by Stx17 inhibition in Drosophila

PGAM5 overexpression partially rescues the mitochondrial defects by Stx17 loss. Transmission electron microscope images of the indirect flight muscle in the indicated genotypes (a, Control; b, Stx17−/−; c, Stx17−/− and Stx17 overexpression; d, Stx17−/− and PGAM5 overexpression) of 7‐day‐old adult flies are shown. The bar graph on the right shows frequency of healthy and abnormal mitochondria presented as percentages (mean ± SEM) using the scoring system: Class 0, normal; Class 1, fuzzy or dilated cristae; Class 2, fragmented cristae and loss of electron density. *< 0.05, # < 0.001 vs. the same class of control (Dunnett's test). n = 50–119 mitochondria from three or four independent samples. Genotypes used were as follows: +/y; Act5c‐GAL4/+ (a), +/y; Act5c‐GAL4/+; Stx17 LL06330 /Stx17 LL06330 (b), +/y; Act5c‐GAL4/UAS‐FLAG‐Stx17; Stx17 LL06330 /Stx17 LL06330 (c), +/y; Act5c‐GAL4/UAS‐PGAM5; Stx17 LL06330 /Stx17 LL06330 (d). Scale bars, 200 nm.

Simultaneous reduction in Stx17 and PGAM5 results in mitochondrial degeneration. a, Stx17+/−; b, PGAM5−/−; c, Stx17+/−, PGAM5−/−. The bar graph on the right represents the mitochondrial phenotypes classified as described in (A). *< 0.05, **< 0.01 vs. the same class of Stx17+/− (Dunnett's test). n = 62–195 mitochondria from three independent samples. Scale bars = 1 μm. Genotypes used were as follows: +/y; Act5c‐GAL4/+; Stx17 LL06330 /+ (a), PGAM5 1 /y; Act5c‐GAL4/+ (b), PGAM5 1 /y; Act5c‐GAL4/UAS‐Stx17‐FLAG; Stx17 LL06330 /+ (c).

Source data are available online for this figure.
Figure 8
Figure 8. Reciprocal relationship between Stx17 and FUNDC1 with respect to Drp1 binding
In healthy cells, Stx17 facilitates normal division of mitochondria (Mt) by interacting with Drp1. Upon mitophagy stimulation, Stx17 dissociates from Drp1 and interacts with Atg14L for autophagosome formation. In the case of autophagy, mitochondria elongate due to inactivation of Drp1, whereas in mitophagy, FUNDC1 (FDC1) is dephosphorylated by cleaved PGAM5 (PGAM5*), which facilitates excessive division of mitochondria (Mt*) by interacting with Drp1. Stx17 supports this process by releasing PGAM5. Under normal division conditions, FUNDC1 associates with CNX through unknown protein(s).

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