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Supporting experimental procedures
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Organelle-Specific Fluorescent Dye Staining
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For observation of root cells, GFP-DRP1E transgenic plants (GFP-DRP1Eox) were grown on
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half-strength MS medium with 1% (w/v) sucrose and 1% (w/v) agar for 10 days. To visualize the
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plasma membrane, roots were stained with 8 μM FM4-64 (Invitrogen). To stain mitochondria,
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roots were stained with 500 nM MitoTracker Red CMXRos (Invitrogen) for 20 min at room
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temperature and then washed three times with distilled water. Root cells were observed at room
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temperature using a microscope (BX-61; Olympus) with the confocal laser scanning unit of a CSU
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10 (Yokogawa), a 488-nm laser (Furukawa) and a 561-nm laser (Cobolt AB, Stockholm, Sweden).
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Images were captured with the image analysis software SlideBook (Intelligent Imaging
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Innovations).
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Two Dimensional Differential Gel Electrophoresis (2D-DIGE)
2D-DIGE analysis was performed according to the method described previously
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(Minami et al., 2009). Briefly, plasma membrane fractions cleaned with a 2D-Clean-up kit (GE
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Healthcare) were resuspended in a lysis buffer [7 M urea, 2 M thiourea, 2% (w/v) ABS-14, 0.5%
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(w/v) Triton X-100 and 10 mM Tris-HCl (pH 8.5)]. 2D-DIGE analyses were then carried out
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according to the manufacturer’s CyDye DIGE Fluors protocol (minimal dyes, GE Healthcare).
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Protein samples (20 µg proteins) from wild-type or drp1e mutant plants were minimally labeled
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with either Cy3 or Cy5 dye. The IPG strips were rehydrated at 20°C for 12 h at 100 V after
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application of the protein samples and then isoelectrofocused at 20°C in the IPGphor system with
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a successive increase in voltage [500 V (1 h), 1,000 V (1 h), 2,000 V (1 h), 4,000 V (1 h), 5,000 V
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(1 h), 6,000 V (2 h) and 8,000 V (7 h)]. After isoelectrofocusing was completed, gel strips were
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subjected to 10% SDS-PAGE as described above. Two biologically independent samples and two
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technical replications of each sample were performed. Quantitative analysis was performed
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according to the method described previously (Minami et al., 2009).
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Purification of GST-Fused DRP1E Protein
DRP1E cDNA cloned into pENTR/D-TOPO was transferred into the pDEST15 vector
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containing GST tags with Gateway technology. The pDEST15-DRP1E plasmid was transformed
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into the E. coli strain BL21 (DE3) and expression of the GST-fused DRP1E protein was induced by
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incubating the cells overnight with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 20°C.
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The bacterial cells were lysed with 50 µg/mL lysozyme and sonication. After centrifugation, the
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supernatant was used to purify recombinant GST-DRP1E protein using Poly-prep
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Chromatography columns (Bio-Rad Laboratories) containing glutathione-sepharose 4B (GE
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Healthcare) according to the manufacturer’s protocol. Because the recombinant GST-DRP1E was
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not eluted under normal conditions, we used 1.5 M NaCl buffer containing 50 mM Tris-HCl (pH
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10.0), 50 mM reduced glutathione, 1.5 M NaCl and EDTA-free protease inhibitor cocktail (Roche
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Applied Science, Basel, Switzerland) to elute the recombinant protein. Recombinant His-GST
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protein was used as a control and purified using a standard protocol. Purified protein was dialyzed
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against 20 mM Tris-HCl and 500 mM NaCl and used for the protein-lipid overlay analysis.
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Protein-Lipid Overlay Assay
The in vitro lipid-binding assay was performed as described by Kim et al. (2001). Lipids
were obtained as follows: PA, PC, PI and PS from Doosan Serdary Research Laboratories
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(Etobicoke, Canada); PE and PG from Avanti Polar Lipids (Alabaster, AL); cholesterol from Wako
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Pure Chemical Industries (Osaka, Japan); plant glucocerebroside (GlcCer) from Matreya
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(Pleasant Gap, PA); and PI(3)P, PI(4)P and PI(4,5)P from Echelon Biosciences (Salt Lake City,
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UT). In all cases, 4 µL lipids (equivalent to 0.8 ng) were dissolved in chloroform:methanol:water
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(1:2:0.8, v/v/v) and spotted onto nitrocellulose membranes (Hybond-C Extra; GE Healthcare UK
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Ltd, Little Chalfont, UK). The membranes were blocked with a solution consisting of 20 mM
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Tris-HCl (pH 7.5), 150 mM NaCl and 0.1% (w/v) Tween 20 (TBS-T) overnight at 4°C and then
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incubated with 0.5 µg/mL purified recombinant protein in TBS-T containing 3% (w/v) BSA for 1 h at
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room temperature. After washing with TBS-T, the membrane was incubated with an anti-GST
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(mouse IgG2aκ) monoclonal antibody (Nacalai Tesque) for 2 h at room temperature. After
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re-washing with TBS-T, the membrane was incubated with a 1/2000 dilution of horseradish
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peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse IgG (H+L) secondary antibodies
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(Thermo Fisher Scientific, Waltham, MA) and washed again. The signal was detected using
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Super-Signal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL) in a Light Capture
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system (ATTO, Tokyo, Japan).
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Identification of DRP family proteins in co-immunoprecipitate fractions of GFP-specific
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antibody using microsomal fractions of DRP1E-GFPox seedlings
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One mg of microsomal fractions from DRP1E-GFPox and wild-type (control) plants were
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homogenized in buffer (10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 2 mM EDTA, 0.5% Nonidet P-40
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(v/v) and protease inhibitors) and incubated with anti-GFP antibody-coated Dynabeads
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(Invitrogen) overnight at 4˚C. After the samples were washed five times with TBS buffer, the
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immunoprecipitations were eluted by SDS sample buffer. Nano-LC−MS/MS (LTQ Orbitrap XL，
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Thermo Fisher) analysis according to Li et al. (2012a) and Takahashi et al. (2012) was used for
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identification of proteins in IP fractions.