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First published online 30 November 2004
doi: 10.1242/jcs.01565

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Mitofusin 1 and 2 play distinct roles in mitochondrial fusion reactions via GTPase activity

Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan

View larger version (50K): [in a new window]   Fig. 1. GTP and Mfn1-dependent mitochondrial tethering. (A) Schematic drawing of the mitochondrial tethering reaction using isolated mitochondria. (B) Mitochondria (mit, a-e) or salt-washed mitochondria (wash mit, f and g) were prepared from HeLa cells coexpressing mit-GFP and Mfn1-FLAG, or mit-RFP and Mfn1-FLAG. Mitochondria labeled with GFP or RFP were mixed, and incubated with (b-e and g) or without (a and f) GTP at 30°C. The reaction mixtures were analyzed by confocal microscopy. Magnified images of bound mitochondria are shown (c-e). (C) GFP-labeled mitochondria and RFP-labeled mitochondria prepared from the cells expressing Mfn1-FLAG (filled bars), or without Mfn1-FLAG (open bars) were incubated as in B, and the number of bound mitochondria were counted as described in Materials and Methods. (D) Schematic drawing of pull-down assay for the binding reaction between sonicated mitochondrial vesicles. (E) The PNS fractions from HeLa cells expressing Mfn1-HA or Mfn1-FLAG were sonicated, mixed and incubated in the presence or absence of GTP. The reaction mixtures were then treated with or without Triton X-100 and subjected to immunoprecipitation using anti-HA antibody. The immunoprecipitates were analyzed by immunoblotting using anti-FLAG antibody. In a separate experiment, Mfn1K88T-FLAG-harboring mitochondria were used in lieu of Mfn1-FLAG-harboring mitochondria.

View larger version (39K): [in a new window]   Fig. 2. GTP-dependent formation of trans-mitochondrial complex with Mfn1 protein. (A) Assay for the formation of Mfn1-mediated trans-mitochondrial complex. (B) Post nuclear supernatant (PNS), mitochondria (mit) or salt-washed mitochondria (wash mit) fractions were prepared from HeLa cells expressing HA-tagged or FLAG-tagged Mfn1. Fractions with HA-tagged and FLAG-tagged Mfn1 were mixed, and incubated with or without GTP at 0°C or 30°C for 30 minutes. In a reaction with PNS, mitochondria (p) and the supernatant (s) were separated by centrifugation after the reaction. All these fractions were solubilized with 1% digitonin and subjected to immunoprecipitation using anti-HA antibodies. The immunoprecipitates were subjected to SDS-PAGE and subsequent immunoblotting using anti-FLAG and anti-HA antibodies. As controls, the reaction mixtures using mock-transfected cells instead of Mfn1-HA (w/o HA), or immunoprecipitation without antibodies (w/o IgG) were analyzed. The reaction mixture prior to immunoprecipitation was also analyzed by immunoblotting by anti-FLAG (input). (C) Time course of co-immunoprecipitation. PNS or mitochondria as prepared in B were incubated for the indicated time intervals at 0°C or 30°C. The recovery of Mfn1-FLAG from the reaction mixture is shown. (D) The PNS fractions with HA-tagged or FLAG-tagged Mfn1 were mixed and incubated with 1 mM ATP, GTP, GTPS or 1 mM ATP plus 1 mM GTP at 30°C for 30 minutes, then subjected to immunoprecipitation and immunoblotting as in B. (E) The PNS fractions obtained from HeLa cells expressing wild-type Mfn1 (wt) or its mutant on the GTPase (K88T) with HA- or FLAG-tag were incubated with GTP in the indicated combinations, then analyzed as in B. (F) The PNS fractions obtained from HA-tagged or FLAG-tagged Mfn1-expressed cells were mixed and incubated at 30°C for 1 hour with or without GTP, in the presence of 5 mM NADH plus 20 mM sodium succinate (NaSuc), or 20 µM CCCP to dissipate membrane potential. In a separate experiment, PNS fractions with HA-Mfn1 or FLAG-Mfn1 were sonicated, then mixed and incubated as above (sonic). These samples were solubilized by digitonin and subjected to immunoprecipitation and subsequent immunoblot analysis.

View larger version (40K): [in a new window]   Fig. 5. Comparison of activity of Mfn1 and Mfn2 for GTP-dependent complex formation. (A,B) HA-tagged or FLAG-tagged Mfn1 or Mfn2 was separately expressed in HeLa cells, and the intact mitochondria (A) or digitonin-solubilized mitochondria (B) were mixed in the indicated combinations and incubated with 0.4 mM GTP at 30°C for the indicated times. The complex formation was analyzed by immunoprecipitation using anti-HA antibodies and subsequent immunoblotting by anti-FLAG or anti-HA antibodies as in Fig. 2. The immunoprecipitated bands were quantified. (C) The solubilized (+dig) or intact (–dig) mitochondria from the HeLa cells expressing Mfn1-FLAG or Mfn2-FLAG were mixed, and incubated with or without GTP. The incubation mixtures with intact mitochondria were solubilized by digitonin. All the reaction mixtures were then analyzed by sucrose density gradient centrifugation in the presence of digitonin as in Fig. 4. (D) The solubilized (+dig) or untreated (–dig) rat liver mitochondria were incubated with or without GTP. The reaction mixtures were analyzed by sucrose density gradient centrifugation in the presence of digitonin as in Fig. 4. Recovery (%) of Mfn proteins in the peak fractions is shown.

View larger version (47K): [in a new window]   Fig. 3. Complex formation of Mfn1 protein in solubilized conditions. (A) Assay for GTP-dependent homotypic Mfn1 complex formation. (B) PNS, post mitochondrial supernatant (PMS) and mitochondria fractions (mit) were prepared from HeLa cells expressing HA-tagged or FLAG-tagged Mfn1, and solubilized by digitonin. The lysates with HA-tagged or FLAG-tagged Mfn1 were mixed, and incubated with or without GTP, at 0°C or 30°C for 1 hour. The reaction mixtures were subjected to immunoprecipitation using anti-HA antibodies, and analyzed by immunoblotting using anti-FLAG and anti-HA antibodies. The reaction mixtures were also analyzed by immunoblotting using anti-FLAG antibodies (input). (C) Time course of co-immunoprecipitation. The solubilized PNS or mitochondrial fractions with HA-tagged or FLAG-tagged Mfn1 were mixed and incubated with GTP on ice or at 30°C. Co-immunoprecipitated Mfn1-FLAG by anti-HA antibodies was quantified. (D) Solubilized mitochondrial fractions with HA-tagged or FLAG-tagged Mfn1 were mixed and incubated with 1 mM nucleotides as indicated. The reaction mixtures were analyzed as in B. (E) Wild-type Mfn1 (wt) or the mutant K88T with HA or FLAG tags were mixed in the indicated combinations and incubated with GTP. The reaction mixtures were analyzed as in B. (F) The mitochondria (mit), or salt washed mitochondria (wash mit) were solubilized by digitonin. The solubilized (dig+) or intact (dig–) mitochondria were incubated with or without GTP, and analyzed as in B. (G) The mitochondria harboring Mfn1-HA or Mfn1-FLAG were incubated in the digitonin-solubilized (dig+) or unsolubilized (dig–) conditions with 0.4 mM GTP at 30°C for 1 hour (1st GTP). After incubation, 2 mM GTPS or digitonin was added and further incubated at 30°C for the indicated time intervals (2nd GTPS), then subjected to immunoprecipitation and immunoblotting.

View larger version (62K): [in a new window]   Fig. 4. Mfn1-containing complexes as analyzed by sucrose density gradient centrifugation and BN-PAGE. (A) Mitochondria prepared from Mfn1-FLAG or Mfn1-HA-expressing HeLa cells were solubilized with digitonin (w/o), mixed, and incubated with 0.4 mM GTP (GTP) for 1 hour at 30°C. The lysates were subjected to sucrose density gradient centrifugation in digitonin as described in Materials and Methods, and the separated fractions were analyzed by immunoblotting with anti-FLAG and anti-HA antibodies. Recovery of Mfn1 protein in the peak fractions was quantified and shown as % of total signals after centrifugation. (B) Mitochondria prepared from Mfn1-FLAG or Mfn1K88T-FLAG-expressing HeLa cells were solubilized by digitonin (w/o), and incubated with 0.4 mM GTPS or GTP for 15 minutes or 1 hour at 30°C. In a separate experiment, after incubation with 0.4 mM GTP for 1 hour, 2 mM GTPS was added and incubated for an additional 1 hour at 30°C (GTP-GTPS). The reaction mixtures were subjected to sucrose density gradient centrifugation and analyzed as in A. (C) The mitochondria harboring Mfn1-FLAG were incubated with GTP at 30°C for the indicated time intervals. The reaction mixtures were then solubilized with digitonin and subjected to sucrose density gradient centrifugation, and analyzed as in A. (D) Mitochondria (mit) or salt-washed mitochondria (wash mit) were prepared from Mfn1-FLAG expressing HeLa cells, and incubated in the presence (+dig) or absence (–dig) of digitonin with (+GTP) or without (–GTP) GTP for 30 minutes at 30°C. The incubated mitochondria (for –dig) were solubilized by digitonin and subjected to sucrose density gradient centrifugation as in A. (E) Mitochondria (mit) or salt-washed mitochondria (wash mit) were prepared from HeLa cells expressing Mfn1-HA or Mfn1-FLAG, mixed, and incubated with GTP at 30°C for 1 hour (–dig). The membranes were solubilized by digitonin. Separately, mitochondria solubilized by digitonin (+dig) were also incubated with GTP. The reaction mixtures were subjected to sucrose density gradient centrifugation as above. Fractions 8 and 9 (peak 1), 11 and 12 (peak 2) or 14 and 15 (peak 3) were analyzed by immunoprecipitation using anti-HA antibodies, and subsequent immunoblotting using anti-FLAG antibodies as in Fig. 2. (F) BN-PAGE of mitochondria harboring Mfn1-FLAG or Mfn1-HA. Mfn1-harboring mitochondria were incubated with GTP as in D, and subjected to BN-PAGE. The separated protein complexes were analyzed by immunoblotting using anti-HA antibody. Marker proteins used were serum albumin (66 kDa), lactate dehydrogenase (140 kDa), catalase (232 kDa), apoferritin (440 kDa) and thyroglobulin (669 kDa).

View larger version (45K): [in a new window]   Fig. 6. GTP-binding and hydrolysis activity of two Mfn proteins. (A) FLAG-tagged Mfn2 or Mfn1 was expressed in HeLa cells and their PNS were solubilized by digitonin. The lysates were incubated with GTP-agarose beads on ice for 60 minutes, or at 30°C, for the indicated times. The bound proteins and PNS used (5%) were analyzed by immunoblotting using anti-FLAG antibodies. (B) FLAG-tagged Mfn2, Mfn2K109T, Mfn1, or Mfn1K88T was incubated with GTP-agarose at 30°C for 1 hour and bound proteins were analyzed by immunoblotting using anti-FLAG antibodies. (C) His6-tagged Mfn2 and Mfn1 proteins were expressed in E. coli and purified as described. The purified proteins were analyzed by SDS-PAGE followed by Coomassie Brilliant blue staining. (D,E) His-tagged Mfn2 or Mfn1 was incubated with [-32P]GTP at 37°C for the indicated times, and analyzed by thin layer chromatography followed by digital autoradiography as described, using bovine serum albumin as a control. The positions of GDP, GTP, and the origin are shown by an arrow, filled arrowhead, and open arrowhead, respectively. GTP and GDP radioactivity was quantified using an image analyzer (E).

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