Unconventional PINK1 localization to the outer membrane of depolarized mitochondria drives ...

Unconventional PINK1 localization to the outer membrane of depolarized mitochondria drives ...

15 Pages · 2015 · 9.77 MB · English

for OMM localization, whereas mitochondrial translocase Tom40 (also known as TOMM40) and an (Deas et al., 2011; Greene et al., 2012; Jin et al., 2010; Meissner et al., 2011). Graham, R. L., Hess, S. and Chan, D. C. (2011).

Unconventional PINK1 localization to the outer membrane of depolarized mitochondria drives ... free download

depend on DY m (Fig 6), but instead is contingent on the TOM machinery (Fig 5) This feature is reminiscent of the Nterminal signalanchored proteins that localize to the OMM in a DY m independent and Tom40dependent manner, and which have the bulk of the polypeptide exposed to the cytosol (Ahting et al, 2005) PINK1 thus might be considered to be a unique Nterminal signalanchored protein containing an extra MTS and an IMM arrivaldependent degradation signal (TMD) MATERIALS AND METHODSPlasmids, siRNA and antibodiesThe plasmids used in this study are summarized in supplementarymaterial Table S1 The plasmid for weak PINK1 expression,pCMVTNT(d1), was generated by deleting the upstream 620 bp of theCMV promoter in pCMVTNT (Promega), as described previously(Okatsu et al, 2012b) For siRNA analysis, siGENOME siRNASMART pool (M012732000005, Thermo Fisher Scientific) andsiGENOME nontargeting siRNA pool (D0012061320, ThermoFisher Scientific) were used for the knockdown of Tom40 and as acontrol, respectively The following primary antibodies were used: antiParkin (clone PRK8, Sigma, 1:2000), antiPINK1 (BC100494, Novus,1:1000), antiGFP (ab6556, Abcam, 1:2000), antiTom20 (FL145, SantaCruz Biotechnology, 1:250 for immunoblotting or 1:2000 forimmunostaining), antiTom22 (clone 1C92, Sigma, 1:1000), antiOPA1(clone 18, BD Biosciences, 1:500), antiAIF (clone E1, Santa Cruz,1:500), antiHsp60 (clone N20, Santa Cruz Biotechnology, 1:500), anticytochrome c (6H2B4, BD Biosciences, 1:150), antiFoF1 ATPase(provided by Dr Takashi Ueno, Juntendo University, Tokyo, Japan;1:300) and antiTom40 (provided by Dr Katsuyoshi Mihara, KyushuUniversity, Fukuoka, Japan; 1:1000) We also used the followingsecondary antibodies: antimouseIgG, antirabbitIgG or antigoatIgGantibody conjugated to alkaline phosphatase (Santa Cruz, 1:5000) forimmunoblotting, and antimouseIgG or antirabbitIgG antibodyconjugated to Alexa Fluor 488, 568 or 647 (Life Technologies, 1:2000)for immunostaining Cells and DNA or siRNA transfectionHeLa cells were cultured at 37 C with 5% CO 2in Dulbecco’s modified Eagle medium (DMEM, Sigma) containing 10% fetal bovine serum(EquitechBIO Inc), 1 6 penicillinstreptomycinglutamine (Life Technologies), 1 6nonessential amino acids (Life Technologies) and 1 6 sodium pyruvate (Life Technologies) HeLa cells were transfected with theplasmids used in this study by using Fugene 6 (Roche or Promega) Forexpression of wildtype or mutant PINK1 in Tom40knockdown cells,Tom40 and control siRNAs were introduced into HeLa cells usingRNAiMAX reagent (Life Technologies), cells were incubated for 72 h andthen PINK1 plasmids were introduced into these cells using Fugene 6,followed by incubation for a further 24 h and then data collection CCCP treatmentFor mitochondrial depolarization, cells were treated with 10 mM CCCP for 1 h unless otherwise specified To examine the DY mdependency of mitochondrial import of Su9–GFP or PINK1N34–GFP, cells were pretreated with CCCP for 2 h prior to transfection with Su9–GFP or PINK1N34–GFP and further incubated in the presence of CCCP Following an18h incubation, the subcellular localizations of these GFPfused proteinswere observed Immunofluorescence, TMRE staining and MitoTracker stainingFor immunofluorescence experiments, cells were fixed with 4%paraformaldehyde, permeabilized with 50 mg/ml digitonin or 1% Triton X100, and then stained with the primary and secondary antibodiesdescribed above Cells were imaged using a confocal laserscanningmicroscope (LSM510, LSM710 and LSM780; Carl Zeiss, Inc) Tomonitor the DY m, cells were treated with 150 nM MitoTracker Orange CMH2TMRos (Life Technologies) or 50 nM TMRE (Sigma) for30 min Cells were washed two to three times and fixed as above for MitoTracker staining or subjected to livecell imaging for TMREstaining Image contrast and brightness were adjusted in Photoshop(Adobe) Mitochondrialenriched fractionation and proteinase Kprotection assayHeLa cells transfected with PINK1N34–GFP were suspended infractionation buffer [250 mM sucrose, 20 mM HEPESNaOH pH 81,protease and phosphataseinhibitor cocktail (Roche)] and disrupted by30 passages through a 25gauge needle using a 1ml syringe Debris wasremoved by centrifugation at 1000 gfor 7 min, and the supernatant was subjected to centrifugation at 10,000 gfor 10 min at 4 C to precipitate the mitochondriarich fraction The mitochondriarich fractions wereincubated with 200 mg/ml Pro K (Wako chemicals) on ice for 30 min The reaction was stopped with 1 mM phenylmethylsulfonyl fluoride(PMSF) before boiling for electrophoresis Phostag SDSPAGE and immunoblottingTo detect phosphorylated proteins, SDSPAGE with or without 50 mM Phostag acrylamide (Wako chemicals) and 100 mM MnCl 2were

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