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• W2890597757 abstract "Article20 September 2018free access Source DataTransparent process Syntaxin 17 regulates the localization and function of PGAM5 in mitochondrial division and mitophagy Masashi Sugo School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan Search for more papers by this author Hana Kimura School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan Search for more papers by this author Kohei Arasaki Corresponding Author [email protected] orcid.org/0000-0003-0647-3565 School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan Search for more papers by this author Toshiki Amemiya School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan Search for more papers by this author Naohiko Hirota School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan Search for more papers by this author Naoshi Dohmae Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science, Wako, Japan Search for more papers by this author Yuzuru Imai Department of Research for Parkinson's Disease, Juntendo University Graduate School of Medicine, Tokyo, Japan Department of Neurology, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Tsuyoshi Inoshita Department of Treatment and Research in Multiple Sclerosis and Neuro-intractable Disease, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Kahori Shiba-Fukushima Department of Treatment and Research in Multiple Sclerosis and Neuro-intractable Disease, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Nobutaka Hattori Department of Research for Parkinson's Disease, Juntendo University Graduate School of Medicine, Tokyo, Japan Department of Neurology, Juntendo University Graduate School of Medicine, Tokyo, Japan Department of Treatment and Research in Multiple Sclerosis and Neuro-intractable Disease, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Jinglei Cheng Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya, Japan Search for more papers by this author Toyoshi Fujimoto Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya, Japan Search for more papers by this author Yuichi Wakana School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan Search for more papers by this author Hiroki Inoue School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan Search for more papers by this author Mitsuo Tagaya Corresponding Author [email protected] orcid.org/0000-0001-9137-7142 School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan Search for more papers by this author Masashi Sugo School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan Search for more papers by this author Hana Kimura School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan Search for more papers by this author Kohei Arasaki Corresponding Author [email protected] orcid.org/0000-0003-0647-3565 School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan Search for more papers by this author Toshiki Amemiya School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan Search for more papers by this author Naohiko Hirota School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan Search for more papers by this author Naoshi Dohmae Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science, Wako, Japan Search for more papers by this author Yuzuru Imai Department of Research for Parkinson's Disease, Juntendo University Graduate School of Medicine, Tokyo, Japan Department of Neurology, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Tsuyoshi Inoshita Department of Treatment and Research in Multiple Sclerosis and Neuro-intractable Disease, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Kahori Shiba-Fukushima Department of Treatment and Research in Multiple Sclerosis and Neuro-intractable Disease, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Nobutaka Hattori Department of Research for Parkinson's Disease, Juntendo University Graduate School of Medicine, Tokyo, Japan Department of Neurology, Juntendo University Graduate School of Medicine, Tokyo, Japan Department of Treatment and Research in Multiple Sclerosis and Neuro-intractable Disease, Juntendo University Graduate School of Medicine, Tokyo, Japan Search for more papers by this author Jinglei Cheng Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya, Japan Search for more papers by this author Toyoshi Fujimoto Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya, Japan Search for more papers by this author Yuichi Wakana School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan Search for more papers by this author Hiroki Inoue School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan Search for more papers by this author Mitsuo Tagaya Corresponding Author [email protected] orcid.org/0000-0001-9137-7142 School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan Search for more papers by this author Author Information Masashi Sugo1, Hana Kimura1, Kohei Arasaki *,1, Toshiki Amemiya1, Naohiko Hirota1, Naoshi Dohmae2, Yuzuru Imai3,4, Tsuyoshi Inoshita5, Kahori Shiba-Fukushima5, Nobutaka Hattori3,4,5, Jinglei Cheng6, Toyoshi Fujimoto6, Yuichi Wakana1, Hiroki Inoue1 and Mitsuo Tagaya *,1 1School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan 2Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science, Wako, Japan 3Department of Research for Parkinson's Disease, Juntendo University Graduate School of Medicine, Tokyo, Japan 4Department of Neurology, Juntendo University Graduate School of Medicine, Tokyo, Japan 5Department of Treatment and Research in Multiple Sclerosis and Neuro-intractable Disease, Juntendo University Graduate School of Medicine, Tokyo, Japan 6Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya, Japan *Corresponding author. Tel: +81 42676 7116; E-mail: [email protected] *Corresponding author. Tel: +81 42 676 5419; E-mail: [email protected] EMBO J (2018)37:e98899https://doi.org/10.15252/embj.201798899 PDFDownload PDF of article text and main figures.AM PDF Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Synopsis Mitochondrial phosphatase PGAM5 has been shown to regulate mitophagy by dephosphorylating the mitophagy receptor FUNDC1. New data show that syntaxin 17 associates with PGAM5 and regulates its localization and Drp1 and FUNDC1 activation during normal and mitophagic division, respectively. Syntaxin 17 binds PGAM5 and regulates its localization and activity. Syntaxin 17 promotes Drp1 activity by facilitating its PGAM5-mediated dephosphorylation. Syntaxin 17 is required for the association of PGAM5 with the mitophagy receptor FUNDC1 in mitophagy. Syntaxin 17 and PGAM5 maintain mitochondrial architecture in Drosophila. Introduction Mitochondria are double membrane-bound and highly dynamic organelles that fuse, divide, and move along the cytoskeleton to form a mitochondrial network in response to cellular energy status (Labbé et al, 2014; Mishra & Chan, 2016; Wai & Langer, 2016). Mitochondrial dynamics is mediated by GTPases: Drp1 promotes mitochondrial fission, and Opa1 and mitofusins (Mfns) catalyze the fusion of the inner and outer membranes, respectively (Labbé et al, 2014; Mishra & Chan, 2016; Wai & Langer, 2016). Mitochondria in coordination with the endoplasmic reticulum (ER) play key roles in cellular metabolism and cell survival/death. The close proximity between mitochondria and a subdomain of the ER, termed the mitochondria-associated membrane (MAM; Paillusson et al, 2016; Herrera-Cruz & Simmen, 2017), allows efficient Ca2+ transfer from the ER to mitochondria. Uptake of Ca2+ into the matrix of mitochondria activates the tricarboxylic acid cycle, leading to the production of ATP, whereas prolonged excess Ca2+ uptake results in the release of apoptosis-inducing factors from mitochondria (Herrera-Cruz & Simmen, 2017; Marchi et al, 2017). Mitochondria generate ATP via the proton gradient across the mitochondrial inner membrane that is formed as a consequence of the passage of electrons through the electron transport chain. Electrons that have leaked from the electron transport chain produce reactive oxygen species (ROS; Murphy, 2009). While low levels of ROS function as signaling molecules (Holmström & Finkel, 2014), excess ROS production causes damage to cellular components, including the mitochondria themselves. Damaged parts of mitochondria are segregated from healthy parts by fission and cleared through a specific mode of autophagy, called mitophagy (Hamacher-Brady & Brady, 2016). PGAM5 is a member of the phosphoglycerate mutase family, but exhibits protein phosphatase activity toward Ser/Thr (Takeda et al, 2009) and His (Panda et al, 2016). Recent studies highlighted the importance of PGAM5 in a variety of cellular processes, including mitochondrial dynamics (Wilkins et al, 2014; Lavie et al, 2017; O'Mealey et al, 2017), mitophagy (Chen et al, 2014; Wu et al, 2014; Hawk et al, 2018), programmed cell death (Wang et al, 2012; Lin et al, 2013; Zhuang et al, 2013; Xu et al, 2015; He et al, 2017), the WNT/β-catenin pathway (Rauschenberger et al, 2017; Bernkopf et al, 2018), immune responses (Kang et al, 2015; Moriwaki et al, 2016), and longevity (Borch Jensen et al, 2017). Several lines of evidence suggest the close relationship of PGAM5 with Parkinson's disease. PGAM5 deficiency suppresses the loss-of-function mutation of the mitochondrial protein kinase PINK1 in Drosophila (Imai et al, 2010) and causes a Parkinson's-like movement disorder and resistance to metabolic stress in mammals (Lu et al, 2014, 2016; Sekine et al, 2016). Moreover, PINK1 and PGAM5 are alternative substrates for the mitochondrial rhomboid protease PARL: while PARL complexed with SLP2 (Wai et al, 2016) continuously cleaves the intramembrane domain of PINK1 in healthy cells (Jin et al, 2010), upon mitochondrial injury PARL cleaves PGAM5 instead of PINK1 (Sekine et al, 2012), leading to the expression of PINK1 on the mitochondrial surface (Jin et al, 2010). In Parkin-expressing cells, PINK1 appearance on the mitochondrial surface facilitates the recruitment of cytosolic Parkin to dysfunctional mitochondria, followed by ubiquitination and engulfment of the mitochondria for mitophagy (Pickrell & Youle, 2015; Hattori et al, 2017). Syntaxin 17 (Stx17) is a SNARE protein located in the MAM and mitochondria, and functions not only as a fusion protein but also as a scaffold at the ER–mitochondria interface (Arasaki et al, 2015; Tagaya & Arasaki, 2017). Our previous results suggest that Stx17 binds to microtubules through MAP1B-LC1 phosphorylated at Thr217 (Arasaki et al, 2018) and promotes mitochondrial fission in fed cells by preventing Drp1 from binding to Rab32 (Arasaki et al, 2015), a mitochondrial GTPase serving as an anchor for protein kinase A that inactivates Drp1 through phosphorylation at Ser637 (Bui et al, 2010). Starvation causes the dephosphorylation of MAP1B-LC1 at Thr217 and allows Stx17 to dissociate from Drp1 (Arasaki et al, 2018) and associate with Atg14L (Hamasaki et al, 2013), a subunit of the Vps34-containing class III phosphatidylinositol (PI) 3-kinase (Matsunaga et al, 2010). This binding induces the attachment of this kinase to the MAM, promoting the formation of phosphatidylinositol 3-phosphate (PI3P), followed by the expansion and formation of autophagosomes (Axe et al, 2008). The function of Stx17 as a scaffold requires not the SNARE motif, but the C-terminal hydrophobic domain (CHD) separated by Lys254 (Arasaki et al, 2015; Fig 1A). Stx17 and Atg14L function not only at the early stage of autophagy, but also at the late stage, that is, the fusion of autophagosomes with lysosomes (Itakura et al, 2012; Diao et al, 2015). 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. Source Data for Figure 1 [embj201798899-sup-0003-SDataFig1.zip] Download figure Download PowerPoint To explore the multiple functions of Stx17, we searched for its binding proteins by means of immunoprecipitation and identified PGAM5. In this study, we show that PGAM5-mediated Drp1 dephosphorylation is regulated by Stx17 in healthy cells and that the PGAM5-Stx17 interaction is essential for the function of the mitophagy receptor FUNDC1 (Wei et al, 2015), which is dephosphorylated by PGAM5 at the onset of mitophagy (Chen et al, 2014; Wu et al, 2014). Results Stx17 binds to PGAM5 Among the proteins co-immunoprecipitated with FLAG-Stx17, we focused on PGAM5, a protein phosphatase that was reported to catalyze Drp1 dephosphorylation under certain conditions such as cell death stimuli (Wang et al, 2012; Lin et al, 2013; Xu et al, 2015). We first determined which regions of Stx17 are responsible for the interaction with PGAM5. Deletion of the C-terminal cytoplasmic tail had no effect on the binding to PGAM5 (Fig 1B, lane 7), and the CHD plus the C-terminal cytoplasmic tail retained the ability to bind to PGAM5 (lane 8), suggesting that the CHD is responsible for the association with PGAM5. Notably, replacement of Lys254, which divides the CHD into two segments, markedly reduced the binding to PGAM5 (Fig 1B, lane 6). The requirement of Lys254 of Stx17 for PGAM5 association was corroborated by in situ proximity ligation assay (PLA) (Fig 1C). Using recombinant proteins, the direct binding between Stx17 and PGAM5 was verified (Fig 1D, lane 6). The results also confirmed that the CHD of Stx17 is required for binding to PGAM5 (lanes 7 and 8). Our previous study demonstrated that Stx17 also interacts with Drp1 (Arasaki et al, 2015). To define the Drp1-binding site on Stx17, we used a GTPase-deficient mutant of Drp1, i.e., Drp1 K38A, in which Lys38 was replaced by Ala (Smirnova et al, 1998). This mutant was found to co-immunoprecipitate with Stx17 (Arasaki et al, 2015). Immunoprecipitation and pull-down experiments demonstrated that although the CHD and the following C-terminal cytoplasmic tail of Stx17 can bind to Drp1 (Fig EV1A, lane 8), as in the case for binding to PGAM5, deletion of the C-terminal tail abolished the binding ability (Fig EV1A, lane 7 and B, lane 7). Thus, Drp1 and PGAM5 likely bind to overlapping but partially different sites on Stx17. PGAM5 has an N-terminal transmembrane domain (amino acids 7–29), followed by a KEAP1 domain and PGAM-like domain (Sadatomi et al, 2013; Fig 1A). Removal of the N-terminal transmembrane domain abolished the binding to Stx17 (Fig 1E, lane 5), whereas the transmembrane domain itself could bind to Stx17 (lane 6). The transmembrane domain of PGAM5 was found not to bind to Stx18 (Fig 1F), a syntaxin localized in the ER (Hatsuzawa et al, 2000). The requirement of the transmembrane domain of PGAM5 for binding to Stx17 was confirmed by pull-down experiments using recombinant proteins (Fig EV1C). Click here to expand this figure. 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. Download figure Download PowerPoint PGAM5 localizes to the ER–mitochondria interface Although immunoreactivity for both Stx17 (Arasaki et al, 2015) and PGAM5 (Fig 2A, upper row) was predominantly detected on mitochondria, PLA combined with the expression of the ER and mitochondria fluorescence markers revealed that Stx17 interacts with PGAM5 principally on and in the vicinity of the ER (Fig 2B). This finding prompted us to examine whether PGAM5 localizes to the ER–mitochondria interface in addition to mitochondria. Subcellular fractionation revealed this to be the case. PGAM5 was recovered not only in the mitochondrial fraction (Fig 2C, lane 5), but also in the MAM fraction (lane 4). The localization of PGAM5-GFP at the ER–mitochondria interface was confirmed by electron microscopy using an ascorbate peroxidase 2 (APEX2)-GFP-binding peptide (Fig 2D). 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. Source Data for Figure 2 [embj201798899-sup-0004-SDataFig2.zip] Download figure Download PowerPoint The MAM is rich in cholesterol and sphingolipids, thus resembling lipid rafts (Herrera-Cruz & Simmen, 2017). Because the cholesterol-rich structure is sensitive to low concentrations of digitonin (Oliferenko et al, 1999), we treated cells with 0.03 mg/ml digitonin, a concentration only effective to solubilize cholesterol-rich membranes such as the plasma membrane, but not intracellular membranes. Similar to the case of Stx17 (Arasaki et al, 2015), this treatment caused partial dissociation of PGAM5 from mitochondria (Fig 2A, lower low). Albeit less effective, treatment of cells with cholesterol depletion reagents, methyl-β-cyclodextrin (MβCD; Ohtani et al, 1989) and nystatin (Rothberg et al, 1992), also caused an increase in diffuse staining for PGAM5 (Fig EV2A). Click here to expand this figure. 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. Download figure Download PowerPoint To determine whether ER–mitochondria contact is required for the association of FLAG-Stx17 with PGAM5, we knocked down Mfn2. Although the role of Mfn2 in MAM–mitochondria tethering is under debate (Naon et al, 2016), it is, at least, widely accepted that Mfn2 depletion abolishes the MAM function (Hailey et al, 2010; Hamasaki et al, 2013; Arasaki et al, 2015). PLA revealed that the FLAG-Stx17-PGAM5 proximity was diminished upon depletion of Mfn2, but not the non-tethering protein Mfn1 (Fig 2E). Knockdown of PACS-2, a multifunctional sorting protein required for maintaining MAM integrity (Simmen et al, 2005), also reduced the PLA signal for the FLAG-Stx17-PGAM5 proximity (Fig 2E). PGAM5 promotes mitochondrial fission through interaction with Stx17 Previous studies demonstrated that PGAM5 dephosphorylates Drp1 in the context of cell death (Wang et al, 2012; Lin et al, 2013; Xu et al, 2015) and that its overexpression causes mitochondrial fragmentation (Wilkins et al, 2014). As calcineurin and PP2A were reported to be also responsible for the dephosphorylation of Drp1 (Cereghetti et al, 2008; Merrill et al, 2013), we first sought to confirm that PGAM5 regulates mitochondrial division in healthy cells. Knockdown of PGAM5 caused mitochondrial elongation (Fig EV2B, lower row), and the defect in mitochondrial fission was confirmed by incubation of PGAM5-depleted cells with the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP; Fig EV2C), which is known to induce robust mitochondrial fission in a Drp1-dependent manner (Ishihara et al, 2006). Immunoblotting (IB) demonstrated that PGAM5 depletion (Fig EV2D, lane 2), as well as Stx17 depletion (lane 4), increased the phosphorylation level of Drp1 at Ser637. Contrary to PGAM5 depletion, its overexpression caused mitochondrial fission (Fig EV2E, upper row), as reported previously (Wilkins et al, 2014), whereas the overexpression of a phosphatase-dead H105A mutant (Takeda et al, 2009), in which His105 was replaced by Ala, caused mitochondrial elongation (lower row). The proximity between endogenous PGAM5 and Drp1 was detected on PLA, and this proximity was also abrogated upon Stx17 depletion as well as depletion of Mfn2 or PACS-2 (Fig 2F). These results suggest that PGAM5 promotes mitochondrial fission by dephosphorylating Drp1 at Ser637 in a Stx17, MAM-dependent manner. To understand why Stx17 depletion abrogates the link between PGAM5 and Drp1, we examined the localization of PGAM5 in Stx17-depleted cells. Strikingly, Stx17 depletion caused aggregation of PGAM5 within mitochondria, and some Tom20-positive tubules were found to be devoid of PGAM5 (Fig 3A, lower row), suggesting that Stx17 regulates PGAM5 localization. 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. Source Data for Figure 3 [embj201798899-sup-0005-SDataFig3.zip] Download figure Download PowerPoint PGAM5 was often observed as two bands on IB: the upper and lower bands correspond to the full-length and PARL-cleaved forms, respectively (Fig 3B, lane 1; Sekine et al, 2012; Wai et al, 2016). Loss of mitochondrial membrane potential halts PINK1 cleavage and instead enhances PGAM5 cleavage by PARL (Sekine et al, 2012). When Stx17 was knocked down in 293T cells, the amount of the upper band decreased concomitant with an increase in the amount of the lower band (Fig 3B, lane 2 vs. lane 1), implying a link between PGAM5 and Stx17. As reported previously (Sekine et al, 2012), CC" @default.
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• W2890597757 title "Syntaxin 17 regulates the localization and function of PGAM5 in mitochondrial division and mitophagy" @default.
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• W2890597757 doi "https://doi.org/10.15252/embj.201798899" @default.
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