Figure S1, related to Figure 1. Mitochondrial localization of Mfn2 ActA and ER localization of Mfn2 IYFFT and their functional analyses.

Transcription

1 Molecular Cell, Volume 51 Supplemental Information MITOL Regulates Endoplasmic Reticulum-Mitochondria Contacts via Mitofusin2 Ayumu Sugiura, Shun Nagashima, Takeshi Tokuyama, Taku Amo, Yohei Matsuki, Satoshi Ishido, Yoshihisa Kudo, Heidi M. McBride, Toshifumi Fukuda, Nobuko Matsushita, Ryoko Inatome, and Shigeru Yanagi Inventory of Supplemental Information Figure S1, related to Figure 1. Mitochondrial localization of Mfn2 ActA and ER localization of Mfn2 IYFFT and their functional analyses. Figure S2, related to Figure 2. MITOL is not involved in the degradation of Mfn2. Figure S3, related to Figure 3. Effect of MITOL knockdown on Mfn2 localization and ER morphology. Figure S4, related to Figure 4 and Figure 7. MITOL and ubiquitination of K192 of Mfn2 are critical for intracellular Ca 2+ homeostasis. Figure S5, related to Figure 5. MITOL knockdown inhibited the formation of high-molecular-weight Mfn1 complexes. Figure S6, related to Figure 7. Ubiquitination at K192 of Mfn2 by MITOL is critical for Mfn2 activation. Supplemental Experimental Procedures cdna cloning, expression vectors, and mutagenesis, Antibodies and reagents, shrna construct, shrna-resistant MITOL, shmitol stable cell lines, Morphological analysis by immunofluorescence microscopy, Electron microscopy, Subcellular fractionation, Immunoprecipitation, GST pull-down assay, Ca 2+ imaging, Blue Native PAGE, GTP-binding assay, GTP hydrolysis assay Supplemental References

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3 Supplemental Figure 1, related to Figure 1. Mitochondrial localization of Mfn2 ActA and ER localization of Mfn2 IYFFT and their functional analyses. (A) Structures of the 2 Mfn2 mutants used in this study. TM, transmembrane domain; RBD, Ras-binding domain; HR, heptad repeat; ActA tail, mitochondria-insertion signal; IYFFT, ER membrane-targeting signal. (B) Mitochondrial localization of Mfn2 ActA and ER localization of Mfn2 IYFFT. Immunofluorescence analysis with Mito-GFP and ER-DsRed was performed on HeLa cells transfected with Mfn2 ActA -FLAG or Mfn2 IYFFT -FLAG. Merged images indicate the mitochondrial localization of Mfn2 ActA and ER localization of Mfn2 IYFFT. (C) Subcellular fractionation demonstrated mitochondrial localization of Mfn2 ActA and ER localization of Mfn2 IYFFT. IB analysis was performed using the indicated Abs in HEK293 cells transfected with wild-type Mfn2, Mfn2 ActA -FLAG, or Mfn2 IYFFT -FLAG. (D) Mitochondrial fusion activity of Mfn2 ActA. Mfn2-/- MEFs were co-transfected with Mfn2 ActA -3 FLAG and Mito-GFP. Mitochondrial morphological changes in Mfn2-/- MEFs co-transfected with Mfn2 ActA -3 FLAG and Mito-GFP were analyzed by confocal microscopy. Percentages of cells with elongated mitochondria obtained from 3 independent experiments (103 Mfn2-/- cells and 73 Mfn2-/- cells expressing Mfn2 ActA ) are shown on the right. Mfn2-/-, FLAG-negative cells. (E) Rescue of MAM formation in Mfn2-/- MEFs expressing Mfn2 ActA and Mfn2 IYFFT. Immunofluorescence analysis was performed on Mfn2-/- MEFs co-transfected with Mfn2 ActA and Mfn2 IYFFT using Mito-GFP and ER-DsRed. Three-dimensional images were reconstructed, and MAM formation as indicated in the merged image was quantified by Manders s coefficient. Error bars indicate ± SE (n = 50). Scale bars represent 10 µm. (F) Relationship between MITOL and Mfn1. The GST-HR1 fragment of Mfn1 does not pull down MITOL. A GST pull-down assay was performed on lysates from HEK293 cells expressing HA-tagged MITOL and GST, GST-Mfn2 HR1 ( aa), or GST-Mfn1 HR1 ( aa), followed by IB analysis with the indicated Abs.

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5 Supplemental Figure 2, related to Figure 2. MITOL is not involved in the degradation of Mfn2. (A) No obvious change in Mfn2 levels following MITOL knockdown. An IB assay was performed on shgfp- or shmitol-expressing HeLa cells with anti-mfn2 and anti-drp1 Abs, and each protein was quantified. Error bars indicate ± SD. *, p < 0.05 (Student s t-test, n = 3). (B) (C) The rate of Mfn2 degradation was not altered by MITOL knockdown or MITOL overexpression. A cycloheximide (CHX)-chase assay was performed on (B) shgfp- and shmitol-expressing cells or (C) HeLa cells co-transfected with Mfn2-HA and either empty vector (vec) or MITOL. Cells were treated with 10 µg/ml CHX for the indicated times. The level of Mfn2 was monitored by IB with the indicated Abs, and the statistical analysis was performed as described in (A). Error bars indicate ± SD, n = 3. (D) Mfn1 slightly accumulates in shmitol-expressing cells. IB analysis was performed on shgfp- or shmitol-expressing HeLa cells using the indicated Abs. (E) A CHX chase assay showed delayed degradation of Mfn1. Error bars indicate ± SD (n = 3). The reason for Mfn1 accumulation in shmitol-expressing cells is unknown at present. It is possible that increased Mfn1 expression compensates for Mfn2 dysfunction in shmitol-expressing cells. (F) MITOL knockdown induced mitochondrial elongation. Mitochondria of shgfp- and shmitol-expressing HeLa cells were labeled with DsRed-Mito. Mitochondrial morphology was analyzed by confocal microscopy. Graph indicates the mean ± SD of 3 independent experiments (50 cells in each experiment). Scale bar represents 10 µm.

6 Supplemental Figure 3, related to Figure 3. Effect of MITOL knockdown on Mfn2 localization and ER morphology. (A) MITOL knockdown induced Mfn2 mislocalization. An immunofluorescence assay was performed on shgfp- and shmitol-expressing cells transfected with Mfn2-FLAG and DsRed-Mito by staining with anti-flag Ab. Arrowheads indicate punctiform Mfn2 not co-localized to mitochondria. These were quantified by Manders s coefficient. The data in the graph represent the mean ± SE (40 cells in each experiment). Scale bar represents 10 µm.

7 (B) An electron microscopy analysis showed a decrease in the number of mitochondria interacting with ER in MITOL-knockdown cells. Contacted and dissociated positions between the ER and mitochondria are indicated by closed and open arrowheads, respectively. The data in the graph represent the percentages of mitochondria interacting with ER obtained from 70 mitochondria in 7 shgfp or shmitol cells. Error bars indicate ± SD of 3 independent experiments. Scale bar represents 200 nm. (C) MITOL knockdown induced abnormal ER morphology. Morphological analysis using confocal microscopy was performed on shgfp- and shmitol-expressing cells transfected with ER-DsRed. Changes in ER morphology were evaluated from 3 independent experiments (50 cells/experiment). Abnormal ER morphology was defined as disrupted reticular morphology with aggregation or fragmentation. Graph represents the mean ± SD. *, p < 0.05 (chi-square test data from 3 independent experiments). Scale bar represents 10 µm. (D) MITOL localizes to the MAM domain. Each subcellular fraction was isolated from HeLa cells, and IB was performed with the indicated Abs. IP 3 R3 and Calnexin, ER/MAM proteins; FACL4 (long chain fatty acid-coa ligase4), MAM protein; Mfn2, mitochondrial/mam/microsomal protein; VDAC, MAM and mitochondrial outer membrane protein; Tom20, mitochondrial outer membrane protein; α-tubulin, cytosolic protein.

8 Supplemental Figure 4, related to Figure 4 and Figure 7. MITOL and ubiquitination of K192 of Mfn2 are critical for intracellular Ca 2+ homeostasis. shgfp- and shmitol-expressing HeLa cells (A, B), or Mfn2-/- MEFs (C, D) were transfected with the indicated vectors. Mitochondrial (A, C) and cytosolic (B, D) Ca 2+ mobilization was monitored as described in Fig. 4 and Materials and Methods. The data in

9 the graphs represent the mean ± SE (50 cells). Supplemental Figure 5, related to Figure 5. MITOL knockdown inhibited the formation of high-molecular-weight Mfn1 complexes. (A) To detect Mfn1 complexes, a blue native (BN)-PAGE retardation assay was performed on MAM fractions derived from shgfp- or shmitol-expressing cells. IB after SDS-PAGE (lower panel) is shown as an Mfn1 loading control. (B) MITOL knockdown inhibited the interaction of ER-Mfn2 with Mfn1. An IP-IB assay was performed on shgfp- and shmitol-expressing cells transfected with indicated vectors. The amount of Mfn1 that co-immunoprecipitated with ER-Mfn2 was quantified by densitometry. Relative values are shown in the graph. Error bars indicate ± SD. *, p < 0.05 (Student s t-test, n = 3).

10 Supplemental Figure 6, related to Figure 7. Ubiquitination at K192 of Mfn2 by MITOL is critical for Mfn2 activation. (A) Comparison of the amino acid sequences in the GTPase domains of Mfn1 and Mfn2.

11 The GTPase domain sequences of Mfn1 and Mfn2 were aligned with ClustalW. The arrowhead indicates the lysine residue in Mfn2 that is not conserved in Mfn1. (B) MITOL ubiquitinates the GTPase-defective K109A Mfn2 mutant as well as wild-type Mfn2. Lysates of HeLa cells transfected with the indicated vectors were subjected to an IP-IB assay with the indicated Abs. (C) No changes in the degradation of Mfn2 K109A and K192R mutants. A cycloheximide (CHX)-chase assay was performed on HeLa cells transfected with the indicated vectors. Cells were treated with 10 µg/ml CHX for the indicated times. The level of Mfn2 was monitored over time by IB with an anti-flag Ab. Error bars indicate ± SD, n = 3. (D) (E) Lysine 192 is required for high efficiency binding to and hydrolysis of GTP. Mitochondrial lysates from HEK293 cells transfected with the indicated vectors were used in a GTP-binding assay. Eluted and GTP-bound Mfn2 was analyzed by IB with an anti-ha Ab (D). Immunopurified FLAG-tagged Mfn2 from Mfn2-/- cells was incubated with [ -32 P]GTP at 37 C for 1 h, and analyzed by thin layer chromatography followed by digital autoradiography. (F) (G) The Mfn2 K192R mutant rescue altered mitochondrial morphology, but not ER morphology in Mfn2-/- cells. Mfn2-/- MEFs were cotransfected with empty vector (Vec), FLAG-tagged Mfn2, or Mfn2 K192R and Mito-GFP. Mitochondrial and ER morphology was analyzed by confocal microscopy. The data shown in the graphs indicate the percentage of cells with elongated mitochondria (vec: 87, Mfn2: 62, Mfn2 K192R : 141 cells were observed respectively) or reticular ER (vec: 92, Mfn2: 41, Mfn2 K192R : 95 cells were observed respectively). Graph indicates the mean ± SD of 3 independent experiments (50 cells in each experiment). Scale bars represent 10 µm.

12 Supplemental Experimental Procedures cdna cloning, expression vectors, and mutagenesis To clone cdna encoding human Mfn1 and Mfn2, total RNA isolated from HeLa cells was reverse-transcribed using the Superscript RT kit (Invitrogen) according to the manufacturer s protocol. Full-length cdnas were obtained by PCR and subcloned into pflag CMV14 (Sigma). To generate localization-restricted Mfn2, mitochondrial Mfn2 (Mfn2 ActA ) and ER Mfn2 (Mfn2 IYFFT ) were constructed as previously described (Rojo et al., 2002; Zhu et al., 1996). cdna containing the Mfn2 point mutations K192R or K109A or ubiquitin mutants were generated with the site-directed mutagenesis kit (Stratagene) Antibodies and reagents The anti-mitol rabbit polyclonal Ab was previously described (Yonashiro et al., 2006). Anti-FLAG (M2) and mouse monoclonal anti- -tubulin Abs were purchased from Sigma. The mouse monoclonal anti-c-myc Ab was from Roche. The mouse monoclonal anti-ha Ab was from Babco. The rabbit polyclonal anti-v5 Ab was from MBL. The mouse monoclonal anti-gst Ab was from WAKO. The rabbit polyclonal anti-his Ab was from Cell Signaling Technology. The mouse monoclonal Abs against Drp1, Tom20, Tim23, PDI, and IP 3 R3 were from BD Biosciences. The mouse monoclonal anti-mitofusin2 Ab (XX-1), mouse monoclonal anti-ubiquitin Ab (P4D1), and goat polyclonal anti-calnexin Ab (C-20) were from Santa Cruz. The rabbit polyclonal anti-facl4 Ab was from ABGENT. The rabbit polyclonal anti-vdac Ab was from Calbiochem. The rabbit polyclonal anti-mfn1 Ab was from Abcam. The rabbit polyclonal anti-cytochrome P450 Ab was from Chemicon. The anti-ub K48- and K63-specific Abs were from Millipore. shrna construct For the shrna experiments, 2 complementary oligonucleotides (sense with an added BamHI linker, 5'-gat cgg TTT ACG TCT TGG ATC TTG Ccg aag CAA GAT CCA AGA CGT AAA CCt ttt tt-3'; antisense with an added HindIII linker, 5'-agc taa aaa agg TTT ACG TCT TGG ATC TTG Ctt cgg CAA GAT CCA AGA CGT AAA CC-3') were designed and inserted into the psilencer 3.1-H1 neo vector according to the manufacturer s

13 instructions (Ambion). shrna-resistant MITOL An shrna-resistant MITOL construct was generated by site-directed mutagenesis using following primers: 1st (sense, 5'-AAA ATT GGG TCC AGT TGT GTA CGT GTT GGA TCT TGC AGA T-3'; antisense, 5'-ATC TGC AAG ATC CAA CAC GTA CACA ACTG GAC CCA ATT TT-3'), 2nd (5'-AAA ATT GGG ACC GGT TGT GTA CGT GCT GGA TCT TGC AGA T-3'; antisense, 5'-ATC TGC AAG ATC CAG CAC GTA CAC AAC CGG TCC CAA TTTT-3'). shmitol stable cell lines To establish MITOL-knocked down cell lines, HeLa cells were transfected with shmitol or shgfp as a control. Cells were selected with 1 mg/ml G418 neomycin. Morphological analysis by immunofluorescence microscopy Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 1 h at room temperature, then washed twice with PBS, permeabilized with 0.2% Triton X-100 in PBS for 5 min, then washed 4 times with PBS, and blocked with 1% bovine serum albumin in PBS, all at room temperature. For double staining, the cells were incubated with appropriate primary Abs for 1 h at room temperature, washed 3 times with PBS, and then incubated with appropriate secondary Abs for 30 min. The samples were washed as described above, mounted using Fluorescent Mounting Medium (Dako), and analyzed using an Olympus IX81 confocal fluorescence microscope. To analyze the interaction between mitochondria and ER, a z-axis image series of cells expressing mitochondrial GFP and ER-DsRed were obtained and stacked. The background signals were removed using the BG subtraction function of ImageJ, and then Manders s colocalization coefficient was calculated. For ER morphological analysis, DsRed-labeled ER was observed in shrna-transfected stable cell lines with a confocal fluorescence microscope. ER morphology (reticular or not,

14 aggregation or fragmentation) was determined. Data are the mean ± SD of 3 independent experiments. Electron microscopy HeLa cells stably expressing shgfp or shmitol were fixed with 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (ph 7.4). They were postfixed with 2% OsO 4 in the same buffer. After dehydration with a graded series of ethanol, they were substituted by propylene oxide and embedded in Epoxy resin. Silver and gold thin sections were doubly stained with uranyl acetate and lead citrate, and examined with an H-7000 transmission electron microscope (HITACHI, Tokyo, Japan). Subcellular fractionation MAM fractions were isolated from HeLa cells or mouse brain as previously described (Szabadkai et al., 2006; Vance, 1990). HeLa cells or mouse brain were homogenized with homogenization buffer (HB) (5 mm HEPES ph 7.4, 0.5 mm EDTA, 250 mm mannitol) and the crude mitochondrial fraction (pellet after centrifugation at 8,000 g) was subjected to further separation by 30% Percoll gradient centrifugation at 9,500 g for 30 min at 4 C. A low-density band (denoted as the MAM fraction) was resuspended in HB and further purified by removing the pellet after centrifugation at 6,300 g for 10 min. The mitochondrial fraction in the high-density band was resuspended in HB and pelleted by centrifugation (6,300 g for 10 min). The MAM fraction was obtained by centrifugation at 100,000 g for 1 h at 4 C. Microsomes were pelleted from the mitochondrial supernatant by centrifugation at 100,000 g for 1 h. Cytosol was recovered as the supernatant from this centrifugation. SDS-PAGE sample buffer was added to each fraction and analyzed by IB. Immunoprecipitation To analyze the interaction between endogenous MITOL and Mfn2, crude mitochondria from mouse brain cells were solubilized with lysis buffer (0.5% NP-40, 50 mm Tris-HCl ph 7.4, 150 mm NaCl, 5 mm EDTA, 20% sucrose, protease inhibitors). Lysates were centrifuged at 15,000 rpm for 10 min at 4 C, and the supernatant was subjected to IP using

15 an anti-mitol Ab and normal rabbit IgG as a control. For the ubiquitination assay, HeLa cells transfected with vectors as indicated in the figures were lysed with 0.1% SDS RIPA buffer (0.1% SDS, 0.05% DOC, 1% TritonX-100, 10 mm Tris-HCl ph 7.4, 150 mm NaCl, 5 mm EDTA, protease inhibitors), and then incubated with the appropriate Ab. For the in vitro ubiquitination assay, FLAG-tagged Mfn2 and HA-tagged MITOL were purified from HEK293 cells by IP. Immunoprecipitates were incubated with reaction buffer containing 50 mm Tris-HCl, ph 7.4, 2 mm MgCl 2, 4 mm ATP, 100 ng of E1 (Biomol), 400 ng of UbcH5b (Biomol), and 2 g of His-Ub (Biomol) for 2 h at 30 C, and then terminated with 3 SDS sample buffer. GST pull-down assay To determine the specific sites required for MITOL and Mfn2 interaction, GST-fusion deletion mutants were prepared as described previously (Yonashiro et al., 2006). Lysates from mouse brain were incubated with the purified GST-fusion proteins. Precipitates were analyzed by IB. Ca 2+ imaging HeLa cells stably expressing shrnas were seeded on a glass-bottom dish 1 day before measurement. For transfection, HeLa cells or Mfn2-/- MEFs were seeded on a glass-bottom dish 2 days before measurement. After 1 day, cells were transfected with the expression vectors described in supplemental Fig. 5. Cells were washed with BSS buffer (20 mm HEPES, 130 mm NaCl, 5.4 mm KCl, 2 mm CaCl 2, 1 mm MgCl 2, 5.5 mm glucose), stained with 3 µm XRhod 1-AM (Molecular Probes) for 1 h or 3 µm Fura 4-AM (Molecular Probes) for 20 min at RT, washed with BSS, and then incubated in BSS for 30 min. Ca 2+ mobilization induced by treatment with 1 µm histamine (Sigma) for HeLa cells or 200 µm ATP (Sigma) for MEFs was detected by a quantitative fluorescence system using a CCD camera (Orca-ER; Hamamatsu Photonics K.K., Hamamatsu, Shizuoka) through the 40 objective lens of an inverted microscope. The images were processed using software specific for Ca 2+ imaging (Aquacosmos; Hamamatsu Photonics). The time course of the change in [Ca 2+ ]i at a region of interest (ROI) in each cell was calculated as the

16 average brightness of each ROI. For the preparation loaded with fura-4f, the ratio of the fluorescence (>500 nm) induced by alternative excitation at 340 nm and 380 nm (F340/F380) was calculated. In the X rhod-1-stained cells, the ratio of the fluorescence (>580 nm) induced by excitation at 540 nm was calculated using the initial image as a reference. In the present study, F340/F380 data for fura-4f, and F540/F540 data for X rhod-1 were normalized to the time point just before exposure to histamine, which was set to 1.0 (10 cells/experiment, n = 3) Blue Native PAGE Blue-native PAGE (BN-PAGE) was performed according to Schagger (Schagger, 2001). Mitochondria or MAM domains isolated from HeLa cells were solubilized with solubilization buffer (1% digitonin, 20 mm NaCl, 50 mm imidazole ph 7.0, 1 mm EDTA, 10% glycerol, 500 mm 6-aminocaproic acid, protease inhibitors). After centrifugation at 14,000 g for 10 min at 4 C Coomassie Brilliant blue G-250 (0.2% final concentration) was added, and the samples were electrophoresed through 3 12% polyacrylamide gradient gels. The gels were subjected to IB using the Abs described in the figures. GTP-binding assay Mitochondria were isolated from HeLa cells and solubilized in 1% digitonin buffer (50 mm Tris-HCl, 150 mm NaCl, protease inhibitors). After centrifugation at 14,000 g for 10 min at 4 C, the supernatant was incubated with reaction buffer (50 mm Tris-HCl ph 7.4, 150 mm NaCl, 1 mm MgOAc 2, 1 mm DTT, protease inhibitors) containing GTP-agarose beads on ice or at 30 C for 60 min. The bound proteins were eluted with elution buffer (1% digitonin, 50 mm Tris-HCl ph 7.4, 150 mm NaCl, 10 mm GTP, protease inhibitors) for 1 h at 4 C, and 3 SDS-PAGE sample buffer was added to the eluates including GTP-agarose. The amount of Mfn2 bound to GTP was determined by IB with an anti-mfn2 Ab. For the expressed Mfn2, mitochondria from HEK293 cells were lysed in reaction buffer containing 1% Triton X-100 (50 mm Tris-HCl, ph 7.4, 150 mm NaCl, 5 mm MgCl 2, 1 mm DTT, protease inhibitors) and incubated on ice for 20 min. After centrifugation at 20,000 g for 15 min at 4 C, supernatant was incubated with GTP-agarose for 1 h at 37 C.

17 The bound proteins were eluted with elution buffer (1% digitonin, 50 mm Tris-HCl ph 7.4, 150 mm NaCl, 20 mm EDTA, 10 mm GTP, protease inhibitors). GTP hydrolysis assay Endogenous or expressed Mfn2 was purified from mitochondria by IP using an anti-mfn2 or anti-flag Ab. The purified proteins were incubated with 20 L of 20 mm HEPES-KOH buffer (ph 7.4) containing 2 mm MgCl 2, 1 mm dithiothreitol, and 1% Triton X-100 with 1 Ci of [ - 32 P]GTP at 37 C for 1 h. One microliter of the reaction mixture was removed and spotted on a polyethyleneimine-cellulose filter (Merck). The reaction products were resolved by thin layer chromatography in 1 M LiCl 2 and 2 M formic acid. The products were analyzed and quantified using a Bioimage Analyzer BAS2000 (Fuji Film Co.).

18 Supplemental References Rojo, M., Legros, F., Chateau, D., and Lombes, A. (2002). Membrane topology and mitochondrial targeting of mitofusins, ubiquitous mammalian homologs of the transmembrane GTPase Fzo. J Cell Sci 115, Schagger, H. (2001). Blue-native gels to isolate protein complexes from mitochondria. Methods Cell Biol 65, Szabadkai, G., Bianchi, K., Varnai, P., De Stefani, D., Wieckowski, M.R., Cavagna, D., Nagy, A.I., Balla, T., and Rizzuto, R. (2006). Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels. J. Cell Biol. 175, Vance, J.E. (1990). Phospholipid synthesis in a membrane fraction associated with mitochondria. J. Biol. Chem. 265, Yonashiro, R., Ishido, S., Kyo, S., Fukuda, T., Goto, E., Matsuki, Y., Ohmura-Hoshino, M., Sada, K., Hotta, H., Yamamura, H., et al. (2006). A novel mitochondrial ubiquitin ligase plays a critical role in mitochondrial dynamics. EMBO J. 25, Zhu, W., Cowie, A., Wasfy, G.W., Penn, L.Z., Leber, B., and Andrews, D.W. (1996). Bcl-2 mutants with restricted subcellular location reveal spatially distinct pathways for apoptosis in different cell types. EMBO J. 15,