Department of Biological Sciences, Tokyo Institute of Technology, 4259-B-19 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan
* Author for correspondence (email: shirose{at}bio.titech.ac.jp)
Accepted 21 April 2005
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Summary |
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Key words: mitochondria, mitofusin, mitochondrial morphology, mitochondrial membrane potential
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Introduction |
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Mitofusins, Mfn1 and Mfn2, were identified as homologues to the Drosophila fuzzy onions protein (Fzo), which is required for mitochondrial fusion during spermatogenesis of the fruit fly (Hales and Fuller, 1997). They are mitochondrial outer membrane proteins with large N-terminal and relatively short C-terminal domains exposed towards the cytosol (Hermann et al., 1998
; Rapaport et al., 1998
; Fritz et al., 2001
; Rojo et al., 2002
). Their domain structure is schematically shown in Fig. 1B, and consists of a GTPase domain near the N terminus, a coiled-coil domain, two transmembrane spans and a coiled-coil domain in the C-terminal tail facing the cytoplasm. Loss of the mitofusin function causes the mitochondrial network to rapidly fragment and no fusion of mitochondria occurs when this protein is defective or absent. Site-directed mutagenesis revealed that mitochondrial fusion machinery of fzo/mitofusin is regulated in a GTPase-dependent manner in fruit fly, yeast and mammals (Hales and Fuller, 1997
; Hermann et al., 1998
; Santel and Fuller, 2001
). In yeast Fzo1p, the short loop facing the intermembrane space has been demonstrated to associate with the contact sites between inner and outer membranes and to be essential for mitochondrial four-bilayer fusion (Fritz et al., 2001
). Rojo et al. (Rojo et al., 2002
) showed that a construct covering the double transmembrane spans and C-terminal tail of Mfn2 is targeted to mitochondria and the N- and C-terminal tails of Mfn2 are able to interact through their coiled-coil domains. Mitofusin has been suggested to be a component of multiprotein fusion machinery (Rapaport et al., 1998
; Fritz et al., 2001
; Santel et al., 2003
). Mice deficient in either Mfn1 or Mfn2 die in midgestation and the embryonic fibroblasts established from the knockout mice displayed fragmented mitochondria (Chen et al., 2003
). In mammals, two isoforms, Mfn1 and Mfn2, have been reported to cooperate to fuse mitochondria (Chen et al., 2003
; Santel et al., 2003
; Eura et al., 2003
). Functional differences between Mfn1 and Mfn2 are being revealed. For example, it has been shown that embryonic fibroblasts established from knockout mice lacking either Mfn1 or Mfn2, display distinct types of fragmented mitochondria (Chen et al., 2003
), that OPA1 requires Mfn1 but not Mfn2 to induce mitochondrial fusion (Cipolat et al., 2004
), and that Mfn1 tethers mitochondrial membranes with a higher efficiency than Mfn2 (Ishihara et al., 2004
). However, the mechanisms of mitochondrial fusion and the role of Mfn/Fzo family remain essentially unknown.
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Materials and Methods |
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Antibodies
Anti-Mfn2C was produced as described previously (Honda and Hirose, 2003). Anti-Hsp60 (SPA-807) was purchased from StressGen; anti-FLAG M2 (F3165) was from Sigma; and anti-mouse IgG Alexa Fluor 350 and anti-mouse IgG Alexa Fluor 488 were from Molecular Probes.
Immunofluorescence staining of transfected COS7 cells
COS7 cells were transfected with expression constructs for Mfn2 deletion mutants. After 16 hours, living cells were incubated with 500 nM MitoTracker Red CMXRos (Molecular Probes) at 37°C for 30 minutes, washed twice with PBS, fixed with 4% formaldehyde/PBS for 15 minutes, washed three times with PBS. Cells were permeabilized for 15 minutes in PBS with 0.2% Triton X-100 and blocked for 30 minutes with PBS containing 5% FBS. For double and triple staining, cells were first incubated with mouse anti-FLAG antibody for 3 hours at room temperature, washed three times with PBS, subsequently incubated with anti-mouse IgG Alexa Fluor 488 for 1 hour at room temperature, and washed three times with PBS. Cells were then incubated with rabbit anti-Mfn2C antibody overnight at room temperature, washed three times with PBS, subsequently incubated with anti-rabbit IgG Alexa Fluor 350 for 3 hours at room temperature, and washed three times with PBS. Coverslips were mounted in Fluoromount-G (Southern Biotechnology Associates, Inc., Birmingham, AL) on glass slides. Cells were examined with an Olympus fluorescence microscope, model IX70, equipped with a 100 x oil-immersion objective and filters optimized for triple-label experiments. Pictures were taken using a Princeton Instruments cooled CCD camera (MicroMAX5 MHz; Rooper Scientific) and analyzed using the MetaMorph software (Universal Imaging).
Immunoprecipitation
HEK293T cells were transfected with expression constructs for Mfn2-FLAG deletion mutants and Myc-Mfn2 deletion mutants. After 16 hours, the cells were washed three times with PBS and were lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 0.1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 10 mM MgCl2, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM PMSF). Aliquots of the lysates were incubated with 10 µl of anti-FLAG affinity agarose gel (Sigma) at 4°C for 3 hours. Immune complexes were washed three times with lysis buffer, eluted with 100 mM glycine (pH 3.5) and neutralized immediately with 1 M Tris-HCl (pH 8.0). Samples were analyzed by western blot analysis using alkaline phosphatase-conjugated goat anti-rabbit IgG or anti-mouse IgG as secondary antibody and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium chloride as the chromogenic substrate.
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Results |
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Deletion mutant lacking transmembrane spans acts as a dominant-negative mutant
Overexpression of Mfn in cultured cells by transient transfection is reported to induce perinuclear aggregation of mitochondria, independent of the mitochondrial fusion activity of Mfn (Santel and Fuller, 2001), as also observed in transient overexpression of the mitochondrial outer membrane protein OMP25 (Nemoto and De Camilli, 1999
). To avoid this artifactual effect, we made a deletion mutant of mouse Mfn2 that lacks double transmembrane spans (Mfn2
TM; Fig. 2A). When expressed in COS7 cells, Mfn2
TM protein caused mitochondria to fragment into small pieces (Fig. 2B,b). This result indicates that the balance between mitochondrial fusion and fission was shifted toward fission by the mutant protein. Such a shift is consistent with a block of the fusion by a dominant-negative mutant of a fusion factor such as Mfn2. Noteworthy is the fact that the dominant-negative effect was exerted by a mutant, Mfn2
TM, that became soluble because of deletion of the transmembrane spans (Fig. 2B,a), suggesting depletion by Mfn2
TM of soluble factors necessary for mitochondrial fusion.
Next, to determine the relationship between the dominant-negative effect and GTPase activity, we introduced a mutation in the active site of the GTPase domain (Mfn2T130A, TM; Fig. 2A), which corresponds to a loss-of-function mutation in the G2 motif of the GTPase domain of yeast Fzo1p (Fzo1T221A), a mutation that gives a similar phenotype as in G1 motif mutants such as Fzo1K200A (Hermann et al., 1998
), Mfn2K109T (Santel and Fuller, 2001
) and Mfn2K109A (Santel et al., 2003
). In the case of human Mfn1, overexpression of the G2-T109A mutant has been reported to result in fragmentation of mitochondria while introduction of point mutation in the G1 motif (G1-K88T mutant) only reduces mitochondrial networks (Santel et al., 2003
), suggesting that the G2 motif mutant exerts stronger inhibitory effects on mitochondrial fusion. We therefore decided to use the G2 mutant of mouse Mfn2
TM (i.e. Mfn2T130A,
TM). Mfn2T130A,
TM caused a loss of the dominant-negative activity, yielding normal mitochondrial morphology with an elongated tubular network (Fig. 2A; micrographs not shown). This normal mitochondrial morphology in the presence of Mfn2T130A,
TM is noteworthy since it has been shown, as mentioned above, that even a loss-of-function mutant of Mfn2 causes perinuclear clustering of mitochondria if it has the transmembrane spans (Santel and Fuller, 2001
). We next tried to determine which part of Mfn2
TM is responsible for the inhibitory effect, by expressing a series of deletion mutants of Mfn2
TM (Mfn2
R1/TM, Mfn2
R6/TM, Mfn2
R7/TM, Mfn2
R4/R5/TM, Mfn2
R3R5/TM; Fig. 2A) in COS-7 cells. Deletion mutants of Mfn2
TM lacking either the R1, R6 or R7 highly conserved region resulted in loss of the dominant-negative phenotype (Mfn2
R1/TM, Mfn2
R6/TM and Mfn2
R7/TM; Fig. 2A). Furthermore, Mfn2
TM lacking R3, R4 and R5 regions (Mfn2
R3R5/TM; Fig. 2A) also resulted in loss of the dominant-negative phenotype but this did not occur with Mfn2
TM lacking the R4 and R5 regions (Mfn2
R4/R5/TM; Fig. 2A,B,d). These results indicated that four highly conserved regions (R1, R3, R6 and R7) are necessary for the dominant-negative activity.
The N- and C-terminal tails of Mfn2 appear to interact through their highly conserved regions including coiled-coil domains in a GTPase-dependent manner
We next determined the effects of the N-terminal and C-terminal tails of Mfn2 on mitochondrial morphology when they were expressed as separate molecules (Fig. 3A). As expected, neither the N-terminal tail alone (Mfn2N-FLAG; Fig. 3B,f; asterisk) nor the C-terminal tail alone (Myc-Mfn2C; Fig. 3B,f; arrowhead) affected the mitochondrial morphology. Coexpression of Mfn2N-FLAG and Myc-Mfn2C (Fig. 3A), however, resulted in dominant-negative phenotypes (Fig. 3B,c; arrow) in a GTPase-dependent manner (Mfn2NT130A; Fig. 3A,B,i).
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To determine the regions involved in the interaction, we prepared deletion mutants of the large N-terminal tail as depicted in Fig. 3A, and examined their effects on mitochondrial morphology by coexpressing them with the C-terminal fragment Mfn2C. As summarized in Fig. 3A, deletion of either the R2-R4 regions (Mfn2NR2R4) or the GTPase domain including its N-terminal flanking region (Mfn2N
R1 and Mfn2N
R1/GTPase) resulted in complete loss of the dominant-negative activity that leads to fission of the mitochondrial reticulum (Fig. 3A). This dominant-negative activity correlated well with their ability to interact with the C-terminal fragment Mfn2C, as demonstrated by immunoprecipitation (Fig. 3C). An interesting finding was that interaction between Mfn2N
R1 and Mfn2C is relatively weak despite the fact that both constructs contain intact coiled-coil domains (i.e. R4/N-CC and R7/C-CC), indicating that the coiled-coil domains alone are not sufficient for tight association and the R1 region is also involved in the interaction between the N- and C-terminal tails (Fig. 3C). Furthermore, as mentioned above, deletion of R4 and R5, had no effect on the dominant-negative phenotype (Mfn2
R4/R5/TM; Fig. 2A,B,d). These results strongly suggest that the N- and C-terminal interaction leading to the dominant-negative effect occurs mainly through the R1-GTPase-R2-R3 regions. The fact that the conserved R regions alone (R1, R2, R3 and R4) are not sufficient for the interaction indicates that the GTPase contributes to maintain the conformation of the N-terminal R1-R4 region so as to fit the complementary structure of the C-terminal R6-R7.
GTPase domain- and transmembrane span-lacking soluble mutants of Mfn2 disrupt mitochondrial membrane potential
When a deletion mutant lacking the GTPase and transmembrane domains (Mfn2GTPase/TM; Fig. 4A) was expressed in COS7 cells, it caused loss of mitochondrial membrane potential and mitochondria became insensitive to MitoTracker, a membrane potential-sensitive dye (Fig. 4B,a,b). Further deletion of the two coiled-coil regions (Mfn2R2/R6; Fig. 4A) did not affect the membrane potential-disrupting activity (Fig. 4B,c,d), leaving two short regions as the sites responsible for the activity. Deletion of either of them (Mfn2
R2/GTPase/TM, Mfn2
GTPase/TM/R6; Fig. 4A) resulted in loss of the disrupting activity, yielding normal potential and morphology (data not shown). Interestingly, these regions (R2 and R6) are highly conserved among Mfn1 and Mfn2 of various species (Fig. 4A).
Loss of mitochondrial membrane potential is known to lead to fragmentation of mitochondria (Legros et al., 2002; Ishihara et al., 2003
), we therefore determined whether Mfn2
GTPase/TM induces mitochondrial fragmentation by staining mitochondria with anti-Hsp60, a mitochondrial protein present in the intermembrane space. Mfn2
GTPase/TM caused fragmentation of mitochondria (Fig. 4C) without causing loss of mitochondrial DNA (Fig. 4D). Concerning the mechanisms of Mfn2
GTPase/TM-induced loss of mitochondrial inner membrane potential, direct activation of the uncoupling protein (UCP) appears unlikely since UCP is an inner membrane protein and Mfn2
GTPase/TM is present in the cytosol and their direct interaction is physically impossible. Mfn2
GTPase/TM may interact with certain component(s) of the apoptotic signaling system in the cytosol and trigger the signal that leads to loss of mitochondrial membrane potential and fragmentation. However, the possibility remains that a minor part of the overexpressed molecules goes to mitochondria and exerts such a deteriorating effect.
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Discussion |
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Among the highly conserved regions of Mfn2, the N-terminal R1-GTPase-R2-R3-R4/CC segment was shown, by immunoprecipitation analysis, to interact with the C-terminal tail through not only the coiled-coil domains but also the R1 region in a GTPase-dependent fashion. Interestingly, the N-terminal flanking region of the Fzo/Mfn family members from yeast, fly and nematode, but not from mammals, has an additional third coiled-coil domain (Hermann et al., 1998; Mozdy and Shaw, 2003
), which might also be involved in the interaction between the N- and C-terminal tails, suggesting the functional importance of the N-terminal region throughout eukaryotic species. The N- and C-terminal interaction may create new binding site(s) for other fusion factor(s) and initiate the fusion reaction. In yeast, the following three proteins have been shown to interact directly in the mitochondrial outer membrane (Sesaki et al., 2003
; Wong et al., 2003
): (i) Fzo1p, a GTPase identified as a homologue of Drosophila fuzzy onions, which is required for mitochondrial fusion during fly spermatogenesis (Sesaki and Jensen, 1999
) and corresponds to mammalian mitofusins; (ii) Ugo1p, an integral membrane protein anchored in the mitochondrial outer membrane (Sesaki and Jensen, 2001
); (iii) Mgm1p, a dynamin-related GTPase located, as a peripheral membrane protein, in the intermembrane space (Sesaki et al., 2003
; Wong et al., 2003
). Gel filtration studies of detergent-solubilized mitochondria showed that Fzo1p is found in an
800-kDa complex (Rapaport et al., 1998
; Fritz et al., 2001
) and human Mfn1 in an
350-kDa complex (Santel et al., 2003
). Therefore similar multicomponent fusion machinery such as Fzo1p, Mgm1p and Ugo1p may also be functioning in the mammalian system. The inhibitory effect caused by such a soluble Mfn2N-Mfn2C complex(es) may be due to depletion of protein components essential for making an active fusion machinery around mitofusins on the mitochondrial surface, and the multiple regions of Mfn2 identified here as indispensable segments (R1, R2, R3, R6 and R7) may provide binding sites for the assembly.
Recently, Koshiba et al. (Koshiba et al., 2004) have reported that a heptad repeat region of the Mfn1 C-terminal tail (residues 660-735) forms a dimeric, antiparallel coiled coil and proposed that this interaction mediates tethering between adjacent mitochondria before fusion. The dominant-negative effect observed here is, however, not due to the direct interaction of Mfn2C with the heptad repeat region of endogenous mitofusins expressed on the surface of mitochondria, since addition of Mfn2C alone had no effect on mitochondrial morphology.
In addition, we unexpectedly found deletion mutants of Mfn2 (Mfn2GTPase/TM and Mfn2R2/R6; Fig. 4A) that can induce loss of mitochondrial membrane potential. Two groups have reported that mitochondrial fusion is completely inhibited by treatment with protonophores that dissipate mitochondrial membrane potential (Legros et al., 2002
; Ishihara et al., 2003
). Furthermore, loss or reduction of mitochondrial membrane potential has been observed in a significant population of embryonic fibroblasts established from Mfn1- or Mfn2-knockout mice (Chen et al., 2003
), and in cultured cells in which Mfn2 was depleted using an adenovirus vector expressing Mfn2-specific antisense cDNA (Bach et al., 2003
). These data indicate that the activity of Mfn is regulated directly or indirectly by mitochondrial membrane potential, and lowering of the membrane potential triggers the fusion reaction, leading to rescue of abnormal mitochondrial phenotypes by complementation with components of the fusion counterparts (normal mitochondria). R2 and R6 segments of Mfn2 identified here may be a key regulator of mitochondrial membrane potential or bind such regulator molecules; the soluble constructs Mfn2
GTPase/TM and Mfn2R2/R6 may be useful for identifying such factors. More analysis of the segments may shed new light on the relationship between mitochondrial fusion and mitochondrial membrane potential.
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Acknowledgments |
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