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First published online 14 November 2006
doi: 10.1242/jcs.03253

Journal of Cell Science 119, 4913-4925 (2006)
Published by The Company of Biologists 2006
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Identification of a novel protein that regulates mitochondrial fusion by modulating mitofusin (Mfn) protein function

Yuka Eura, Naotada Ishihara, Toshihiko Oka and Katsuyoshi Mihara*

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


Figure 1
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  		Fig. 1. Identification of the Mfn-binding protein (MIB). (A) Isolation of MIB using MBP-Mfn{Delta}C-conjugated resin from rat liver cytosol. MBP-Mfn1{Delta}C- or MBP-conjugated resin were treated with the indicated concentrations of NaCl. The eluted proteins were analyzed by SDS-PAGE and CBB-staining. (B) Schematic representation and sequence identity of medium-chain dehydrogenase/reductase protein superfamily. dADH, medium-chain alcohol dehydrogenase; QOR, E. coli quinone oxidoreductase. (C) Hydropathy profile of rat MIB deduced by Kyte and Doolittle algorithm with a 10-amino-acid-residue window. (D) Elution profile of MIB-FLAG from MBP-Mfn{Delta}C-conjugated resin. Cytosolic fraction prepared from MIB-FLAG-expressing HeLa cells was incubated with MBP-Mfn1{Delta}C beads, then eluted by buffer containing the indicated concentrations of NaCl as in (A). The eluted proteins were analyzed by SDS-PAGE and subsequent immunoblotting using anti-flag antibody.

 

Figure 2
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  		Fig. 2. Tissue expression and membrane-binding properties of MIB. (A) Rat tissues were analyzed by SDS-PAGE and subsequent immunoblotting using anti-MIB and anti-TOM40 antibodies. (B) Subcellular fractionation of rat liver MIB. Rat liver was fractionated under isotonic buffer containing 150 mM NaCl into mitochondrial (mt), microsomal (ms) and cytosolic (cyt) fractions (20 µg per well), which were analyzed by SDS-PAGE and subsequent immunoblotting using anti-MIB, TOM40 (mitochondrial marker), calnexin (ER marker), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; cytosolic marker). (C) Sucrose-density-gradient-centrifugation profiles of rat liver post-nuclear membrane fraction (membrane) and mitochondrial fraction (mt). The post nuclear fraction or isolated mitochondria was subjected to sucrose-gradient centrifugation at 77,600 g, for 3 hours as described in Materials and Methods.

 

Figure 3
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  		Fig. 3. Effect of exogenously expressed MIB-FLAG on mitochondrial morphology in HeLa cells. (A) Rat MIB-FLAG was expressed in HeLa cells and the cells were counter-stained with MitoTracker and analyzed by immunofluorescence microscopy using anti-flag antibody. MIB-FLAG-stained fragmented mitochondria are indicated by arrowheads. Bars, 10 µm. Details of the boxed regions in upper panels are shown in the middle panels. Images of MIB-FLAG high expressed cells are shown in the lower panels. (B) Cells with filamentous network mitochondria, fragmented mitochondria and aggregated mitochondria were counted. More than 100 cells were counted for at least three different optical fields. (C) Effect of exogenously expressed MIB-FLAG on the morphology of the ER, Golgi and peroxisomes. The MIB-FLAG expressed HeLa cells were counterstained with antibodies against Sec61ß (ER marker), Golgin97 (Golgi marker), or Pex14 (peroxisome marker) as described in the Materials and Methods, and examined by fluorescence microscopy. Bars, 10 µm. (D) HeLa cells expressing the indicated constructs (FLAG-tagged) were fractionated and the postnuclear supernatant (PNS) and the mitochondrial fractions (20 µg per well) were analyzed by SDS-PAGE and subsequent immunoblotting using antibodies against flag and Tom40. (E) PNS from cells expressing the indicated constructs were subjected to sucrose-density-gradient centrifugation as described in Materials and Methods. The centrifuged solutions were fractionated and analyzed by SDS-PAGE and subsequent immunoblotting using antibodies against flag, Tom40 (mitochondria marker), calnexin (ER marker) and GAPDH (cytosolic marker).

 

Figure 4
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  		Fig. 4. Effect of MIB-FLAG constructs carrying mutation in the GGVG-motif on mitochondrial morphology. (A) MIB-FLAG proteins with the indicated mutations in the GGVG-motif were expressed in HeLa cells and examined by fluorescence microscopy as in Fig. 3. The constructs V13A and V190A carrying a mutation in the arbitrarily selected tetrapeptide region (containing GV) outside the GGVG-motif were examined as the negative controls. Bars, 10 µm. (B) Cells with the indicated mitochondrial morphology shown in A were quantified. More than 100 cells were counted in at least three distinct optical fields. Control: empty vector. (C) Expression levels of the indicated constructs were analyzed by SDS-PAGE and subsequent immunoblotting using anti-flag IgG. (D) Wild-type MIB-FLAG or the GGAG-mutant were exogenously expressed in HeLa cells, and the cytosolic fractions incubated with MBP- or MBP-Mfn1{Delta}C-conjugated beads. Proteins bound to the beads were eluted by buffer containing the indicated concentrations of NaCl, and eluted proteins were analyzed by SDS-PAGE and subsequent immunoblotting with anti-flag antibodies.

 

Figure 5
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  		Fig. 5. Exogenously expressed {zeta}-crystallin does not affect mitochondrial morphology, although it is targeted to the mitochondria. (A) Comparison of amino acid sequences of the GGVG-motif-containing regions of MIB and {zeta}-crystallin. (B) Mitochondrial morphology of HeLa cells expressing {zeta}-crystallin-FLAG. Intracellular localization of {zeta}-crystallin was analyzed by immunofluorescence microscopy. Mitochondria were counterstained with MitoTracker. Details of the boxed regions in top panels are shown in the bottom panels. Bars, 10 µm. (C) Expression levels of MIB-FLAG and {zeta}-crystallin-FLAG in HeLa cells were examined by SDS-PAGE and subsequent immunoblotting with anti-FLAG IgG. (D) Cells with the indicated mitochondrial morphology shown in B were quantified. At least 100 cells were counted in at least three distinct optical fields. Control: empty vector. (E) Binding of {zeta}-crystallin-FLAG to Mfn1 as examined by MBP-Mfn1{Delta}C-beads. HeLa cell extracts expressing {zeta}-crystallin-FLAG or MIB-FLAG were incubated with MBP- or MBP-Mfn1{Delta}C-conjugated beads. Other conditions were as described in Fig. 1D.

 

Figure 6
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  		Fig. 6. Mitochondrial fragmentation-stimulating activity of MIB is offset by the coexpression of Mfn proteins. (A) Mfn1-HA, MIB-FLAG, or both were transfected to HeLa cells and the mitochondrial structures of the cells were examined by immunofluorescence microscopy as described in Materials and Methods. Bars, 10 µm. (B) The cells containing fragmented mitochondria were quantified. At least 100 cells were counted in three distinct optical fields. (C) Mfn1-FLAG or Mfn2-FLAG (Eura et al., 2003Go) was coexpressed with MIB-HA in HeLa cells. The mitochondria were isolated from the cells and solubilized by 1% digitonin. The solubilized fractions were subjected to immunoprecipitation using anti-HA IgG under the indicated conditions. The immunoprecipitates were analyzed by SDS-PAGE and subsequent immunoblotting with anti-HA or anti-FLAG IgGs.

 

Figure 7
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  		Fig. 7. Knockdown of MIB induced extensive growth of mitochondrial network structures. (A) HeLa cells were transfected with MIB siRNA or, as a control, with GFP siRNA as described in Materials and Methods. The cell extracts (20 µg per well) were subjected to SDS-PAGE and subsequent immunoblotting using anti-flag or anti-Tom40 IgGs. (B) The MIB-RNAi cells and GFP-RNAi (control) cells were stained with MitoTracker and examined for mitochondrial morphology by fluorescence microscopy. Bars, 10 µm. (C) Quantification of cells with the indicated mitochondrial morphology. At least 100 cells were counted in three distinct optical fields. (D) RNAi cells for the indicated proteins were analyzed by immunofluorescence microscopy using the indicated antibodies or MitoTracker staining. Bars, 10 µm. (E) MIB knockdown did not affect morphology of peroxisomes, ER or Golgi complex. GFP-RNAi cells (control) or MIB-RNAi cells were analyzed by immunofluorescence microscopy using the indicated antibodies. Bars, 10 µm. (F) HeLa cells were split into eight dishes (6-cm dishes) and grown for 24 hours. Cells of one aliquot were counted after trypsin treatment using the Coulter counter (day 0). Cells in one aliquot were transfected with MIB RNAi or GFP RNAi (for a single transfection). The other aliquots were separately subjected to sequential siRNA-transfection (double or triple transfection) for MIB or GFP. Cells were all grown for 24 hours and then counted as described above. (G) HeLa cells were transfected with the indicated combinations of siRNAs according to the protocol as described in F, except that 3.5-cm dishes were used. (H) Polykaryonic cells were analyzed for cells transfected with the indicated plasmids. At least 100 cells were counted in three distinct optical fields. (I) HeLa cells exogenously expressing the indicated proteins were grown for 24 hours. The cells were cultured in the presence of 20 µM actinomycin D and 100 µM zVAD-fmk for 7 hours, which were then analyzed by immunofluorescence microscopy using anti-cytochrome c and anti-flag antibodies. (J) HeLa cells were subjected to the indicated treatments and the extract (20 µg per well) was analyzed by PARP-cleavage as a measure of apoptosis. *, full-length PARP; **, PARP fragment.

 

Figure 8
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  		Fig. 8. MIB affects the balance of mitochondrial fusion and fission by attenuating Mfn-dependent fusion. (A) Expression levels of the components that are involved in mitochondrial morphology or mitochondrial import in HeLa cells subjected to overexpression or knockdown of MIB, Mfn1 or Fis1. HeLa cells that were subjected to the indicated manipulations (20 µg per well) were analyzed by immunoblotting using the indicated antibodies. Left panel, siRNA-treated cells. Right panels, MIB-overexpressed or siRNA-treated cells; w/o, untreated cells. (B) HeLa cells subjected to the indicated manipulations were counterstained with MitoTracker and then the flag-tag signal was analyzed by immunofluorescence microscopy. Enlarged images are boxed. Bars, 10 µm. (C) HeLa cells with the indicated mitochondrial morphology shown in B were quantified. For each experiment, at least 100 cells were counted in three distinct fields. (D) The MIB-knockdown-induced mitochondrial morphology was attenuated by Mfn1-knockdown. HeLa cells were subjected to RNAi for MIB, Mfn1 or both proteins, and examined by fluorescence microscopy after MitoTracker staining. Bars, 10 µm. (E) HeLa cells with the indicated mitochondrial morphology shown in B were quantified. For each experiment, at least 100 cells were counted in three distinct fields.

 

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