INSERM U 523 Institut de Myologie, Groupe Hospitalier
Pitié-Salpêtrière, 47 boulevard de l'Hôpital, 75651
Paris Cedex 13, France
* Present address: INSERM U 505, Centre de Recherches Biomédicales des
Cordeliers, 75006 Paris, France
Author for correspondence (e-mail:
m.rojo{at}myologie.chups.jussieu.fr
)
Accepted 24 January 2002
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Summary |
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Key words: Transmembrane protein, Protein targeting, GTPase, Mitochondria, Membrane fusion
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Introduction |
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In contrast to other intracellular membranes, little is known about the
factors governing mitochondrial membrane dynamics. In the budding yeast,
mitochondrial fission is mediated by a dynamin-related protein (Dnm1p)
together with other recently discovered proteins (reviewed by
Yoon and McNiven, 2001).
Another dynamin-related protein, Mgm1p, is required for the maintenance of
fusion competent mitochondria (Wong et
al., 2000
). The fission activity of Dnm1p is antagonized by that
of a mitochondrial transmembrane protein, the predicted GTPase Fzo
(Bleazard et al., 1999
;
Sesaki and Jensen, 1999
).
Yeast Fzo is constitutively expressed, localizes to the mitochondrial outer
membrane and is essential for mitochondrial fusion and biogenesis
(Hermann et al., 1998
;
Rapaport et al., 1998
). The
mammalian homologs of the dynamin-related proteins Dnm1p (Drp1) and Mgm1p
(OPA1) are ubiquitously expressed and contribute to mitochondrial distribution
and function (Delettre et al.,
2000
; Smirnova et al.,
1998
). The expression of the Drosophila homolog of Fzo is
restricted to spermatids during the short time period where mitochondrial
fusion occurs and its absence leads to male sterility
(Hales and Fuller, 1997
). Two
human Fzo homologs, called mitofusins Mfn1 and Mfn2, have been recently
identified. Their expression pattern remains unknown. They appear to be
involved in fusion, as they can antagonize the fission activity of human Drp1,
like Fzo does in yeast (Santel and Fuller,
2001
).
Sequence analysis of the relatively large Fzo/Mfn proteins (90-100 kDa)
reveals a multidomain structure containing a GTP-binding and a transmembrane
domain, as well as two predicted coiled-coil regions upstream and downstream
of the transmembrane domain. The transmembrane domain of human Mfn2 is
necessary for mitochondrial targeting
(Santel and Fuller, 2001) and
yeast Fzo is imported into the outer membrane in a receptor-dependent manner
(Rapaport et al., 1998
).
However, the mechanisms and motifs responsible for mitochondrial targeting and
membrane insertion of Fzo/Mfn remain unknown. Mutations of conserved
GTP-binding motifs abolish the capacity of yeast Fzo to mediate mitochondrial
fusion (Hermann et al., 1998
)
and alteration of a small domain exposed to the intermembrane space interfere
with its capacity to mediate fusion (Fritz
et al., 2001
). However, the precise function of Fzo in the fusion
process remains unknown.
In this study we show that mitofusins Mfn1 and Mfn2 are ubiquitous mitochondrial proteins. We determine the membrane topology of Mfn2 and the role of its C-terminal domain in membrane targeting. Furthermore, we show that the coiled-coil domains of Mfn2 interact with each other, and that these interactions are important for mitochondrial targeting. Finally, we show that Mfn2 can modify mitochondrial morphology and bring mitochondrial membranes into close contact.
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Materials and Methods |
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Cloning and mutagenesis
Total RNAs were reverse transcribed with Superscript II (Gibco/BRL) using
oligo dT as a primer, and the chosen cDNA-fragments were amplified by PCR. For
semi-quantitative analysis of RT-PCR, gels were stained with ethidium bromide,
photographed with a CCD camera and stain-intensity of the bands was quantified
with NIH-Image. The open reading frame of human Mfn1 (deduced from overlapping
cDNA sequences AK000700 and U95822) was amplified by RT-PCR from total RNA of
human skin fibroblasts using Expand High Fidelity polymerase (Roche Molecular
Biochemicals). The cDNA encoding human Mfn2 [KIAA protein 0214
(Nagase et al., 1996)] was
kindly provided by Kazusa DNA Research Institute. Cloning was performed
according to standard procedures and all PCR products were verified by
sequencing. The cDNAs encoding two fragments of Mfn2, NG (residues 1-405) and
CT (residues 648-757), were amplified with primers appended with restriction
sites and inserted between the XhoI and BamHI sites of the
pET15b vector (Novagen). A mammalian expression vector coding for a
myc-epitope downstream of a Kozak sequence (pCB6-MYC) was generated by
ligation of annealed primers encoding a Kozak sequence and a mycepitope. Mfn1,
Mfn2 and truncated Mfn2 molecules (MF2-NT, MF2-TMCT, MF2-
C2, MF2-IYFFT)
were amplified with primers appended with restriction sites and the PCR
products were inserted in pCB6 (Rojo et
al., 2000
) or pCB6-MYC. Individual residues of the GTP binding
motifs of Mfn2 were mutagenized (K109A, S110N and R259L) with Quikchange
(Stratagene). Mfn2 molecules devoid of the GTP-binding domain (MF2-
GB:
residues 97-264 deleted) or of the first coiled-coil domain (MF2
C1:
residues 390-439 deleted) were generated by separate amplification of upstream
and downstream regions with primer couples containing a sequence overlap.
These PCR products were linked by fusion PCR
(Ho et al., 1989
), and the
final PCR products were cloned into PCB6-MYC. A GFP molecule targeted to the
mitochondrial matrix (mtGFP) was constructed as described
(Rizzuto et al., 1998
) and
cloned into pCB6.
Cell culture and microscopy
HeLa cells were maintained and transfected as described
(Rojo et al., 2000). A
permissive clone of the mouse myogenic cell line C2 [C27C4
(Pinset et al., 1988
)] was
maintained in DMEM (4.5 g Glc/l) supplemented with 20% fetal calf serum, 50
IU/ml penicillin and 50 µg/ml streptomycin. Cells were processed for
fluorescence microscopy as described (Rojo
et al., 1997
). Cells were viewed using a Zeiss Axiophot microscope
and images were recorded with a CCD camera or on Ilford HP5 Plus film.
Micrographs were processed with Metaview and Adobe Photoshop. Confocal laser
scanning microscopy and electron microscopy were performed as described
(Bakker et al., 2000
).
Reagents, proteins and antibodies
JC-1 was obtained from Molecular Probes, nocodazole from Calbiochem, and
FITC-Phalloidin, cytochalasin B and carbonyl cyanide m-chlorophenyl hydrazone
(cccp) were from Sigma. The stock solution of JC-1 (5 mg/ml in water) was kept
at 4°C; all other stock solutions (10 mM nocodazole in DMSO, 0.2 mg/ml
FITC-phalloidin in DMSO, 1 mg/ml cytochalasin B in ethanol, 20 mM cccp in
DMSO) were kept at -20°C. His-tagged Mfn2-fragments NG or CT were
expressed in E. Coli strain C41 (DE3)
(Miroux and Walker, 1996).
Inclusion bodies containing NG and CT were solubilized in buffers containing 6
M urea and purified with Ni-NTA agarose (Qiagen). Antisera against a mixture
of NG and CT (1:1 by mass) were generated in rabbits. For affinity
purification of antibodies, NG and CT were refolded on Ni-NTA columns with a
gradient of decreasing urea concentration. Refolded NG and CT were coupled
separately to Affi-Gel 10 (Bio-Rad) and antibodies were purified as described
(Rojo et al., 1997
). Protein
analysis (protein determination, SDS-PAGE and western blot) was performed as
described (Rojo et al., 1997
).
For western blot analysis, equal amounts of protein (20 µg) were loaded in
each lane, and equal loading was confirmed by Ponceau protein staining of
membranes. Antibodies specific for the following antigens and/or compartments
were used: ATP/ADP carrier (Rojo and
Wallimann, 1994
), subunits of cytochrome c oxidase [COX2 and COX4
(Bakker et al., 2000
)],
calnexin (StressGen Biotechnologies Corporation), unknown Golgi-specific
antigen [CTR433 (Jasmin et al.,
1989
)],
-tubulin (Amersham Pharmacia Biotech), Hsp60
(Sigma), cytochrome c (BD PharMingen) and mycepitope [9E10
(Evan et al., 1985
)].
Subcellular fractionation and protease digestion
Mitochondria from rat tissues were enriched as described
(Rojo and Wallimann, 1994)
except that the Percoll gradient step was omitted. Adherent culture cells were
homogenized in Hepes-Sucrose-medium (HS) as described
(Rojo et al., 1997
). For
sedimentation of nuclei, homogenates were centrifuged twice for 5 minutes at
200 g (Sigma tabletop centrifuge). To sediment mitochondria,
the post-nuclear supernatant was centrifuged for 10 minutes at 5000
g (Sigma tabletop centrifuge). For separation of microsomes
and cytosol, the postmitochondrial supernatant was centrifuged for 1 hour at
100,000 g (Beckman TLA-45 rotor). Freshly isolated
mitochondria (2 mg/ml in HS) were incubated with Proteinase K (10 µg/ml) at
0°C in the presence or absence of 0.5% Triton X-100. Digestions were
stopped by the addition of an excess of HS containing 2 mM PMSF.
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Results |
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The ubiquitous co-expression of the two mammalian mitofusins differs from
the restricted expression of Drosophila Fzo
(Hales and Fuller, 1997). This
discrepancy prompted us to search for other Fzo-homologs in DNA databases. We
identified Fzo-homologs in the genomes of C. elegans and S.
pombe (Fig. 1C, Ce-Fzo,
Sp-Fzo) and a new Fzo-homolog of in the genome of D. melanogaster
(Fig. 1C, Dm-FzoU). In
addition, we found expressed sequence tags (ESTs) originating from C.
elegans and D. melanogaster. In C. elegans, all ESTs
(n=18) were identical to the predicted protein Ce-Fzo. In
Drosophila, all ESTs (n=10) were identical to the new Fzo
homolog, Dm-FzoU, and originated from embryo, head and ovary libraries. This
predicts the existence of two Fzo homologs in Drosophila: Dm-FzoS,
whose expression is restricted to developing spermatids
(Hales and Fuller, 1997
) and
Dm-FzoU, whose ESTs are found at different developmental stages and in various
tissues. Interestingly, mammalian mitofusins are more similar to the
putatively ubiquitous Dm-FzoU than to the spermatid-specific Dm-FzoS
(Fig. 1C).
Mitofusins display, like all Fzo-homologs, a multi-domain structure that includes a GTP-binding domain, two coiled-coil regions and a predicted transmembrane domain (Fig. 2A, Mfn2). To investigate the localization of Mfn2, we generated a rabbit antiserum against Mfn2-fragments encoding N-terminal and C-terminal fragments of the protein (Fig. 2A, NG, CT). In western blots, this antiserum decorated a band with the expected apparent molecular mass of Mfn2 (86.4 kDa) that was highly enriched in mitochondrial fractions of mouse C2 and human HeLa cells (Fig. 2B, arrowhead). Affinity purified antibodies against each fragment identified a polypeptide of the same apparent molecular mass in mitochondrial fractions of human HeLa cells, mouse C2 cells, and different rat tissues (Fig. 2C), demonstrating the presence of Mfn2 in mitochondria of several tissues. Immunohistochemical analysis of human skin fibroblasts (Fig. 2D), human HeLa cells and mouse C2 cells (not shown) with affinity purified NG-antibodies confirmed the mitochondrial localization of Mfn2. It is important to note that endogenous Mfn2 distributes throughout the entire mitochondrial network (Fig. 2D). Affinity purified CT-antibodies were too weak to reveal endogenous levels of Mfn2 by immunofluorescence, but recognized the overexpressed protein in transfected cells (not shown).
|
The N-terminal and C-terminal domains of the transmembrane protein
Mfn2 are oriented towards the cytoplasm
The membrane topology of Mfn2 was investigated by protease treatment of
isolated mitochondria followed by western-blot analysis with domain-specific
antibodies. To confirm the specificity of affinity purified antibodies, we
analyzed extracts of cells expressing myc-tagged versions of Mfn1, Mfn2 and
Mfn2-mutants devoid of the C-terminal of N-terminal domain
(Fig. 3A). The blots shown in
Fig. 3B visualize the highly
abundant overexpressed proteins under conditions where endogenous Mfn2 is
hardly detected. Myc-specific antibodies
(Fig. 3B, myc) decorated Mfn1,
Mfn2 and the different Mfn2-fragments (arrows). Affinity purified antibodies
against N-terminal and C-terminal Mfn2-fragments (NG and CT,
Fig. 2A) were isoform and
domain-specific: (1) neither antibody decorated Mfn1; (2) NG decorated
MF2-IYFFT and MF2-NT but hardly recognized MF2-TMCT; and (3) CT revealed
MF2-TMCT, but did not decorate MF2-IYFFT and MF2-NT
(Fig. 3B). The weak recognition
of TMCT by NG-antibodies suggests that, despite affinity purification,
NG-antibodies were still contaminated with a minor amount of
CT-antibodies.
|
Next we treated freshly isolated mitochondria with proteinase K in the absence or presence of detergent. Proteins of the intermembrane space (Cyt.c) and of the matrix space (Hsp60) were protected in intact mitochondria (Fig. 3C) and were digested after membrane solubilization with detergent (Fig. 3D). By contrast, various Mfn2-fragments were generated during protease treatment of intact mitochondria (Fig. 3C, asterisks). Some of these fragments, which were labelled differentially by domain-specific antibodies (Fig. 3C, NG, CT), were smaller than MF2-NT (Fig. 3C, upper arrow) or MF2-TMCT (Fig. 3C, lower arrow), respectively. These results reveal the cleavage of the N-terminal and the C-terminal domains and demonstrate their exposure towards the protease-containing outside medium. The membrane topology revealed by these experiments is shown in Fig. 3E: Mfn2 is anchored in the mitochondrial outer membrane via its transmembrane domain and exposes N-terminal and C-terminal domains to the cytosol.
The C-terminal domain of Mfn2 specifies mitochondrial targeting
The transmembrane domain of Mfn2 has been shown to be required for
mitochondrial targeting (Santel and
Fuller, 2001). To better understand the targeting mechanism of
Mfn2, we analyzed the subcellular localization of Mfn2-mutants truncated
before the transmembrane domain (Fig.
4A, MF2-NT) and of Mfn2-molecules devoid of their N-terminal
domain (Fig. 4A, MF2-TMCT). The
cytoplasmic distribution of MF2-NT (not shown) confirmed that the
transmembrane domain of Mfn2 is necessary for mitochondrial targeting. The
colocalization of the MF2-TMCT molecule with mitochondrial GFP (mtGFP,
Fig. 4B) demonstrated that the
transmembrane and C-terminal domains of Mfn2 are sufficient for mitochondrial
targeting. The targeting properties of Mfn2 and Mfn2-fragments are similar to
those of tail-anchored proteins that expose their N-terminal domain towards
the cytosol (Borgese et al.,
2001
). Although their targeting mechanism is still poorly
understood, the available data indicate that their subcellular localization is
specified by the flanking region downstream of the transmembrane domain:
proteins are inserted in the mitochondrial outer membrane when this flanking
region contains basic residues and in the endoplasmic reticulum (ER) when it
contains neutral or hydrophobic amino acids
(Borgese et al., 2001
;
Isenmann et al., 1998
;
Kuroda et al., 1998
). Similar
motifs also function in proteins that display the opposite membrane
orientation (Kanaji et al.,
2000
).
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We noticed that all Fzo-homologs contain numerous basic amino acids
scattered along their C-terminal domains. To investigate whether they specify
insertion of Mfn2 in the mitochondrial outer membrane, we replaced the
C-terminal domain of Mfn2 by the stretch of hydrophobic amino acids (IYFFT)
that targets an isoform of the SNARE protein VAMP-1 (VAMP-1A) to the ER
(Isenmann et al., 1998).
MF2-IYFFT (Fig. 4A) was
targeted to the ER as demonstrated by its colocalization with the ER-marker
calnexin (Fig. 4C). The
C-terminal domain of Mfn2 was then replaced by the three polar residues (RRD)
that target the other VAMP-1 isoform (VAMP-1B) to mitochondria
(Isenmann et al., 1998
).
MF2-RRD (Fig. 4A) was targeted
mainly to mitochondria, as revealed by colocalization with mtGFP
(Fig. 4D). A minor fraction of
MF2-RRD was targeted to the ER-membranes, especially at high expression levels
(not shown). Together, these results show that mitochondrial targeting of Mfn2
is specified by the C-terminal domain via motifs and mechanisms resembling, at
least partially, those of tail-anchored proteins. The behaviour of MF2-RRD
suggests the presence of further targeting determinants in the C-terminal
domain of Mfn2.
Coiled-coil interactions are required for efficient mitochondrial
targeting
Even though coiled-coil domains (Fig.
2A, CC1, CC2) are conserved in all members of the Fzo-family
(Hales and Fuller, 1997;
Santel and Fuller, 2001
),
their function remains unknown. To investigate their function, we analyzed the
localization of Mfn2-molecules devoid of either coiled-coil domain
(Fig. 4A, MF2-
C2,
MF2-
C1). Although both molecules were targeted to mitochondria,
significant protein amounts remained cytosolic
(Fig. 4E,F).
The reduced targeting efficiency of MF2-C2 may be due to the removal
of C-terminal targeting determinants. In contrast, it is more difficult to
understand the reduced targeting efficiency of MF2-
C1, which has the
same C-terminal domain as full length Mfn2 and MF2-TMCT. The fact that both
deletion mutants display similar targeting properties suggest that, beside the
motifs of the C-terminal domain, coiled-coil interactions are required for
efficient mitochondrial targeting of full-length Mfn2. These interactions
could be established between the coiled-coil domains of Mfn2 or with the
coiled-coil domains of other proteins. To investigate these possibilities,
MF2-NT and a version of Mfn2-TMCT devoid of a myc-tag (F2-TMCT) were expressed
simultaneously by cotransfection. Co-expressed molecules were visualized with
antibodies against the myctag (MF2-NT) and with CT-antibodies (F2-TMCT).
Affinity purified CT-antibodies are too weak to reveal endogenous levels of
Mfn2, but recognized the CT epitope of F2-TMCT
(Fig. 5). We observed that
MF2-NT colocalized with F2-TMCT in mitochondria
(Fig. 5A). In contrast, MF2-NT
molecules devoid of their coiled-coil domain (MF2-NT
C1) remained
cytosolic despite the mitochondrial localization of co-expressed F2-TMCT
(Fig. 5B). Similarly, MF2-NT
remained cytosolic when co-expressed with a version of F2-TMCT devoid of its
coiled-coil domain (F2-TMCT
C2, not shown). Interestingly, MF2-IYFFT was
also targeted to mitochondria when cotransfected with F2-TMCT
(Fig. 5C,D). MF2-IYFFT
localized to the ER when the coiled-coil domain was deleted in any of the
co-transfected molecules (not shown). It is interesting to note that the
properties of cotransfected Mfn2-fragments resemble those of full length Mfn2:
they can cluster mitochondria in the perinuclear region at high expression
levels (Fig. 5D). Quantitative
analysis of these experiments revealed that mitochondrial localization of
MF2-NT or MF2-IYFFT by F2-TMCT was highly efficient and was abolished upon
deletion of the coiled-coil domain in any of the co-transfected molecules
(Fig. 5E). These results, which
demonstrate specific and strong interactions between both Mfn2-fragments,
further imply that the coiled-coil domains of truncated F2-TMCT and MF2-IYFFT
are oriented towards the cytoplasma. Although it is probable that the
coiled-coil domains of Mfn2-fragments interact directly with each other, it
cannot be excluded that unknown bridging molecules mediate these interactions.
The interactions between domains upstream and downstream of the transmembrane
domain confirm the membrane topology established previously by protease
protection experiments.
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Microtubules and actin filaments are dispensable for Mfn2-mediated
mitochondrial redistribution
Overexpression of human mitofusins leads to massive mitochondrial
clustering (Santel and Fuller,
2001). Also in this work, the overexpression of Mfn1 (not shown)
and Mfn2 (Fig. 6C,D) led to
perinuclear clustering of mitochondria in cells expressing high protein levels
(routinely in 35-45% of transfected cells). Immunofluorescence microscopy with
organelle-specific antibodies showed that Mfn2 overexpression did not affect
morphology or intracellular distribution of the endoplasmic reticulum and the
Golgi apparatus (not shown), demonstrating that Mfn2 acts specifically on
mitochondria. Mutations in key residues of their predicted G1 (K109A, S110N)
and G4 motifs (R249L) did not modify the capacity of excess Mfn to cluster
mitochondria (not shown), confirming that a functional GTP-binding domain is
not required for mitochondrial clustering
(Santel and Fuller, 2001
). A
mutant devoid of the entire GTP-binding domain
(Fig. 4A, MF2-
GB), which
was efficiently targeted to mitochondria, clustered mitochondria similarly to
wild-type Mfn2 (not shown). This definitely excludes the involvement of the
GTP-binding domain in the protein-protein interactions leading to
mitochondrial redistribution and clustering. Mfn2-molecules devoid of the
entire C-terminal domain (MF2-RRD) or of either coiled-coil domain
(MF2-
C1, MF2-
C2) retained the capacity to redistribute and
cluster mitochondria (Fig.
4D-F), albeit in a lower proportion of transfected cells (15-25%).
It is important to note that expression of Mfn2-fragments containing the
GTP-binding domain, but mistargeted to the cytosol (MF2-NT) or to the ER
(MF2-IYFFT) did not appear to influence mitochondrial morphology or
distribution (see below; Fig.
7C).
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Mitochondrial clustering by excess Mfn2 was paralleled by the
redistribution of mitochondria to the perinuclear region
(Fig. 6C,D). Since microtubules
and actin filaments modulate the intracellular distribution of mitochondria
(Bereiter-Hahn and Voth, 1994;
Krendel et al., 1998
;
Morris and Hollenbeck, 1995
),
we investigated whether excess Mfn2 required a functional cytoskeleton for
mitochondrial redistribution. To this end, we used drugs (nocodazole,
cytochalasin B) that severely interfere with the dynamics of the tubulin or
actin cytoskeleton. In untransfected cells, the distribution of elongated
mitochondria (Fig. 6A, control)
was mildly perturbed upon depolymerization of microtubules
(Fig. 6A, nocodazole). In
contrast, the depolymerization of actin filaments led to the appearance of
punctate mitochondria distributing throughout the cell
(Fig. 6B). In cells transfected
with plasmids encoding Mfn2 alone, or Mfn2 and mtGFP, the drug treatment
started 9 hours after transfection. At this early time point, only a minority
of cells (
2%) expresses any transgene. Fifteen hours later (24 hours after
transfection), when more than 30% of the cells expressed visible amounts of
mtGFP, cells were processed for immunofluorescence microscopy. The formation
of mitochondrial clusters and their redistribution to the perinuclear region
also occurred in the absence of a functional tubulin
(Fig. 6C) or actin cytoskeleton
(Fig. 6D). When drugs were
added after cluster formation (24 hours after transfection), they did not
affect the size or intracellular distribution of pre-existing mitochondrial
clusters (not shown). These results exclude the involvement of the tubulin and
actin cytoskeleton in Mfn2-mediated mitochondrial redistribution. They further
suggest that Mfn2 mediates intermitochondrial adhesion and that this leads to
the retraction of mitochondria from the cell periphery and to their
accumulation in the perinuclear region.
Excess Mfn2 modifies mitochondrial morphology and brings
mitochondrial membranes into close contact without damaging membrane
integrity
Next we analyzed the structure of clustered mitochondria by confocal
microscopy. In control cells expressing mtGFP alone, most mitochondria
appeared as tubules of several microns in length, and 400 to 450 nm diameter.
The inner membrane marker COX2 and the matrix marker mtGFP colocalized
extensively, and their intramitochondrial distributions displayed only minimal
differences (Fig. 7A, control).
In cells co-expressing Mfn2 and mtGFP, clustered mitochondria appear as
discrete spherical or ovoid structures whose diameters (860±160 nm,
n=34) were significantly larger than that of mitochondrial tubules in
control cells (Fig. 7A, +Mfn2).
Interestingly, the inner membrane appeared to surround the matrix space in
enlarged spherical mitochondria (Fig.
7A, +Mfn2). This emphasizes the significant increase in size and
suggests partial segregation of the inner membrane and the matrix. In cells
expressing high levels of MF2-IYFFT, MF2-IYFFT molecules segregated within the
ER and became enriched in discrete membrane domains
(Fig. 7B). The spherical
structure of these ER-subdomains is reminiscent of that of clustered
mitochondria.
We then investigated the integrity of mitochondrial membranes in clusters
induced by Mfn2 overexpression. Cytochrome c, a protein that is released from
the mitochondrial intermembrane space when the outer membrane is damaged
and/or permeabilized (for a review, see
Desagher and Martinou, 2000),
remained in mitochondrial clusters induced by Mfn2 expression (not shown). The
vital dye JC-1, which accumulates in mitochondria and forms red-fluorescent
aggregates in the presence of a high transmembrane potential
(Smiley et al., 1991
),
labelled normal and clustered mitochondria in a similar fashion (not shown).
Together, these experiments show that mitochondrial clustering by excess Mfn2
does not affect the integrity of mitochondrial inner and outer membranes.
The ultrastructure of HeLa cells transfected with an empty plasmid or with a plasmid encoding Mfn2 was analysed by electron microscopy. In control cells, mitochondria displayed a standard morphology: spherical or tubular mitochondrial profiles (minimum diameter 250-350 nm) delimited by a double membrane with an electron dense matrix space and closely apposed cristae membranes (Fig. 8A). Cells with mitochondrial clusters in the perinuclear region were found only in dishes transfected with Mfn2 (Fig. 8B,C). The size and localization of these mitochondrial clusters was in accordance with those observed by confocal microscopy. All mitochondrial profiles were spherical/ovoid, and their diameters were significantly increased, reaching up to 1 µm (Fig. 8B). The internal structure of clustered mitochondria was also affected: whereas the cristae membranes appeared normal in smaller mitochondria, they were swollen in mitochondria of intermediate size and were difficult to identify in the largest mitochondria (Fig. 8B,C). The increase in size and the change from tubular to globular morphology may lead to the relocalization of the inner cristae membranes (which are probably continuous with the inner membrane) to the periphery of enlarged mitochondria. This would explain why, in confocal microscopy, the inner membrane of enlarged mitochondria appears to surround the matrix (Fig. 7A). The higher magnification in Fig. 8C shows that the structure of the limiting inner and outer mitochondrial membranes was well preserved, and that the double membranes of apposed mitochondria could be in close contact without merging (Fig. 8C, arrowheads), confirming the capacity of excess Mfn2 to mediate intermitochondrial adhesion.
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Discussion |
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Targeting and membrane topology
The membrane topology of Mfn2, with N-terminal and C-terminal domains
exposed towards the cytosol, implies that the hydrophobic domain spans the
outer membrane twice, as seen in yeast Fzo
(Fritz et al., 2001). This
topology was confirmed by the interactions between N-terminal and C-terminal
domains of Mfn2. Given the fact that the domain organization is conserved
between all Fzo-homologs (Hales and
Fuller, 1997
; Santel and
Fuller, 2001
), it is probable that Fzo-proteins also share their
membrane topology.
It was found that the transmembrane and C-terminal domains of Mfn2 specify
its targeting via motifs resembling those of other transmembrane proteins of
the outer mitochondrial membrane (Borgese
et al., 2001; Isenmann et al.,
1998
; Kanaji et al.,
2000
; Kuroda et al.,
1998
). These targeting motifs are sufficient for localization of
Mfn2-fragments to the ER (MF2-IYFFT) or to mitochondria (MF2-TMCT), but not
for mitochondrial targeting of full-length Mfn2. Co-transfection experiments
revealed that the coiledcoil domains of Mfn2 mediate direct or indirect
interactions between different Mfn2-fragments and that these interactions are
necessary for efficient mitochondrial targeting of full-length Mfn2. These
experiments further showed that the mitochondrial targeting of interacting
Mfn2-fragments is dominant over the ER-targeting of MF2-IYFFT. Although
protein transport to the mitochondrial outer membrane is still poorly
understood, it has been recently shown that mitochondria and ER recruit
tail-anchored proteins directly from the cytosol and can compete for the same
polypeptide (Borgese et al.,
2001
). Co-expressed Mfn2-fragments could interact with each other
in the cytosol, mask the ER-targeting signal of MF2-IYFFT and the resulting
protein complex would then be translocated to mitochondria. Alternatively,
F2-TMCT could be first targeted to mitochondria, from where it would recruit
mistargeted MF2-NT and MF2-IYFFT. It is reasonable to assume that MF2-IYFFT
would be recruited by F2-TMCT before its insertion in the ER-membrane. The
fact that Mfn2-molecules devoid of either coiled-coil domain remain partially
cytosolic suggests that the mitochondrial transport machinery can discriminate
between proteins with and without assembled coiled-coils. This would argue for
the first hypothesis, that is, for the assembly of coiled-coil domains in the
cytosol, prior to mitochondrial import.
Modulation of mitochondrial distribution and morphology by Mfn2
Overexpressed mitofusins induced perinuclear clustering of mitochondria
without affecting membrane integrity. To our knowledge, the capacity to
cluster mitochondria upon overexpression has been reported only for the
mitochondrial outer membrane protein OMP25, and requires its PDZ domain
(Nemoto and De Camilli, 1999).
Numerous outer membrane proteins, including porin/VDAC isoforms
(Yu et al., 1995
), a SNARE
protein (Isenmann et al.,
1998
), a cytochrome b isoform
(Kuroda et al., 1998
) and
Tom20 (Kanaji et al., 2000
),
do not induce mitochondrial clustering when overexpressed in mammalian cells.
Although we cannot completely exclude that mitochondrial clustering is
provoked non-specifically, the ability of Mfn2 to redistribute mitochondria in
the absence of a functional cytoskeleton suggests that Mfn2 has the capacity
to mediate intermitochondrial adhesion. It is possible that the coiled-coil
domains of Mfn2-molecules mediate intermitochondrial adhesion/docking by the
formation of a four helix bundle, as seen in SNARE proteins
(Sutton et al., 1998
).
However, it is important to note that molecules devoid of a coiled-coil domain
also mediated mitochondrial clustering, albeit with lower efficiency. The
diminished clustering capacity of the coiled-coil mutants could reflect a
direct involvement of coiled-coil domains in adhesion, or result from the
lower efficiency of mitochondrial targeting. Therefore, it is possible that
Mfn2 mediates intermitochondrial adhesion by a mechanism other than the
formation of a (SNARE-like) four helix bundle and/or that it participates in
other membrane rearrangements leading to membrane merge and mitochondrial
fusion.
Since a functional GTP-binding domain is necessary for Mfn2-function
(Hales and Fuller, 1997;
Hermann et al., 1998
;
Santel and Fuller, 2001
), it
is tempting to speculate that this domain either modulates the activity of
Mfn2 or catalyzes further downstream reactions. Thus, mitochondrial clustering
by excess Mfn2 could be either provoked by Mfn2 molecules that overrun
modulation or result from a combination of increased intermitochondrial
adhesion and lack of downstream fusion events. Like other GTP-binding proteins
Mfn/Fzo could require modulator and/or effector molecules for proper function.
The fact that expression of mistargeted Mfn2-constructs (MF2-NT, MF2-IYFFT)
did not affect mitochondrial distribution and morphology, showed that
hypothetical factors were not titrated out. This may indicate that they are
highly abundant, membrane-bound (and cannot interact with mistargeted
molecules), or that they do not exist. Further characterization of Mfn/Fzo and
its GTP-binding domain, as well as identification and characterization of
further proteins (including hypothetical modulators of Mfn/Fzo) will improve
our understanding of Fzo-function and will help to unravel the mechanisms
governing mitochondrial fusion.
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Acknowledgments |
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References |
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Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J.,
Zhang, Z., Miller, W. and Lipman, D. J. (1997). Gapped BLAST
and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res. 25,3389
-3402.
Bakker, A., Barthelemy, C., Frachon, P., Chateau, D., Sternberg,
D., Mazat, J. P. and Lombes, A. (2000). Functional
mitochondrial heterogeneity in heteroplasmic cells carrying the mitochondrial
DNA mutation associated with the MELAS syndrome (mitochondrial encephalopathy,
lactic acidosis and strokelike episodes). Pediatr.
Res. 48,143
-150.
Bereiter-Hahn, J. and Voth, M. (1994). Dynamics of mitochondria in living cells: shape changes, dislocations, fusion, and fission of mitochondria. Microsc. Res. Tech. 27,198 -219.[Medline]
Bleazard, W., McCaffery, J. M., King, E. J., Bale, S., Mozdy, A., Tieu, Q., Nunnari, J. and Shaw, J. M. (1999). The dynamin-related GTPase Dnm1 regulates mitochondrial fission in yeast. Nat. Cell Biol. 1,298 -304.[Medline]
Borgese, N., Gazzoni, I., Barberi, M., Colombo, S. and
Pedrazzini, E. (2001). Targeting of a tail-anchored protein
to endoplasmic reticulum and mitochondrial outer membrane by independent but
competing pathways. Mol. Biol. Cell
12,2482
-2496.
Claros, M. G. and Vincens, P. (1996). Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur. J. Biochem. 241,779 -786.[Abstract]
Corpet, F. (1988). Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res. 16,10881 -10890.[Abstract]
Delettre, C., Lenaers, G., Griffoin, J. M., Gigarel, N., Lorenzo, C., Belenguer, P., Pelloquin, L., Grosgeorge, J., Turc-Carel, C., Perret, E. et al. (2000). Nuclear gene OPA1, encoding a mitochondrial dynamin-related protein, is mutated in dominant optic atrophy. Nat. Genet. 26,207 -210.[Medline]
Desagher, S. and Martinou, J. C. (2000). Mitochondria as the central control point of apoptosis. Trends Cell Biol. 10,369 -377.[Medline]
Enriquez, J. A., Cabezas-Herrera, J., Bayona-Bafaluy, M. P. and
Attardi, G. (2000). Very rare complementation between
mitochondria carrying different mitochondrial DNA mutations points to
intrinsic genetic autonomy of the organelles in cultured human cells.
J. Biol. Chem. 275,11207
-11215.
Evan, G. I., Lewis, G. K., Ramsay, G. and Bishop, J. M. (1985). Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol. Cell. Biol. 5,3610 -3616.[Medline]
Fritz, S., Rapaport, D., Klanner, E., Neupert, W. and
Westermann, B. (2001). connection of the mitochondrial outer
and inner membranes by Fzo1 is critical for organellar fusion. J.
Cell Biol. 152,683
-692.
Hales, K. G. and Fuller, M. T. (1997). Developmentally regulated mitochondrial fusion mediated by a conserved, novel, predicted GTPase. Cell 90,121 -129.[Medline]
Hermann, G. J., Thatcher, J. W., Mills, J. P., Hales, K. G.,
Fuller, M. T., Nunnari, J. and Shaw, J. M. (1998).
Mitochondrial fusion in yeast requires the transmembrane GTPase fzo1p.
J. Cell Biol. 143,359
-373.
Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. and Pease, L. R. (1989). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51-59.[Medline]
Hofmann, K. and Stoffel, W. (1993). TMbase a database of membrane spanning proteins segments. Biol. Chem. Hoppe-Seyler 347,166 -168.
Isenmann, S., Khew-Goodall, Y., Gamble, J., Vadas, M. and
Wattenberg, B. W. (1998). A splice-isoform of
vesicle-associated membrane protein-1 (VAMP-1) contains a mitochondrial
targeting signal. Mol. Biol. Cell
9,1649
-1660.
Jasmin, B. J., Cartaud, J., Bornens, M. and Changeux, J. P. (1989). Golgi apparatus in chick skeletal muscle: changes in its distribution during end plate development and after denervation. Proc. Natl. Acad. Sci. USA 86,7218 -7222.[Abstract]
Kanaji, S., Iwahashi, J., Kida, Y., Sakaguchi, M. and Mihara,
K. (2000). Characterization of the signal that directs tom20
to the mitochondrial outer membrane. J. Cell Biol.
151,277
-288.
Krendel, M., Sgourdas, G. and Bonder, E. M. (1998). Disassembly of actin filaments leads to increased rate and frequency of mitochondrial movement along microtubules. Cell Motil. Cytoskeleton 40,368 -378.[Medline]
Kuroda, R., Ikenoue, T., Honsho, M., Tsujimoto, S., Mitoma, J.
Y. and Ito, A. (1998). Charged amino acids at the
carboxyl-terminal portions determine the intracellular locations of two
isoforms of cytochrome b5. J. Biol. Chem.
273,31097
-31102.
Lupas, A., Van Dyke, M. and Stock, J. (1991). Predicting coiled coils from protein sequences. Science 252,1162 -1164.[Medline]
Miroux, B. and Walker, J. E. (1996). Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260,289 -298.[Medline]
Morris, R. L. and Hollenbeck, P. J. (1995). Axonal transport of mitochondria along microtubules and F-actin in living vertebrate neurons. J. Cell Biol. 131,1315 -1326.[Abstract]
Nagase, T., Seki, N., Ishikawa, K., Ohira, M., Kawarabayasi, Y., Ohara, O., Tanaka, A., Kotani, H., Miyajima, N. and Nomura, N. (1996). Prediction of the coding sequences of unidentified human genes. VI. The coding sequences of 80 new genes (KIAA0201-KIAA0280) deduced by analysis of cDNA clones from cell line KG-1 and brain. DNA Res. 3,321 -329.[Medline]
Nakada, K., Inoue, K., Ono, T., Isobe, K., Ogura, A., Goto, Y. I., Nonaka, I. and Hayashi, J. I. (2001). Inter-mitochondrial complementation: Mitochondria-specific system preventing mice from expression of disease phenotypes by mutant mtDNA. Nat. Med. 7, 934-940.[Medline]
Nemoto, Y. and De Camilli, P. (1999).
Recruitment of an alternatively spliced form of synaptojanin 2 to mitochondria
by the interaction with the PDZ domain of a mitochondrial outer membrane
protein. EMBO J. 18,2991
-3006.
Nunnari, J., Marshall, W. F., Straight, A., Murray, A., Sedat, J. W. and Walter, P. (1997). Mitochondrial transmission during mating in Saccharomyces cerevisiae is determined by mitochondrial fusion and fission and the intramitochondrial segregation of mitochondrial DNA. Mol. Biol. Cell 8,1233 -1242.[Abstract]
Ono, T., Isobe, K., Nakada, K. and Hayashi, J. I. (2001). Human cells are protected from mitochondrial dysfunction by complementation of DNA products in fused mitochondria. Nat. Genet. 28,272 -275.[Medline]
Parisi, M. A. and Clayton, D. A. (1991). Similarity of human mitochondrial transcription factor 1 to high mobility group proteins. Science 252,965 -969.[Medline]
Pinset, C., Montarras, D., Chenevert, J., Minty, A., Barton, P., Laurent, C. and Gros, F. (1988). Control of myogenesis in the mouse myogenic C2 cell line by medium composition and by insulin: characterization of permissive and inducible C2 myoblasts. Differentiation 38,28 -34.[Medline]
Rapaport, D., Brunner, M., Neupert, W. and Westermann, B.
(1998). Fzo1p is a mitochondrial outer membrane protein essential
for the biogenesis of functional mitochondria in saccharomyces cerevisiae.
J. Biol. Chem. 273,20150
-20155.
Rizzuto, R., Pinton, P., Carrington, W., Fay, F. S., Fogarty, K.
E., Lifshitz, L. M., Tuft, R. A. and Pozzan, T. (1998). Close
contacts with the endoplasmic reticulum as determinants of mitochondrial
Ca2+ responses. Science
280,1763
-1766.
Rojo, M. and Wallimann, T. (1994). The mitochondrial ATP/ADP carrier: interaction with detergents and purification by a novel procedure. Biochim. Biophys. Acta 1187,360 -367.[Medline]
Rojo, M., Pepperkok, R., Emery, G., Kellner, R., Stang, E.,
Parton, R. G. and Gruenberg, J. (1997). Involvement of the
transmembrane protein p23 in biosynthetic protein transport. J.
Cell Biol. 139,1119
-1135.
Rojo, M., Emery, G., Marjomaki, V., McDowall, A. W., Parton, R.
G. and Gruenberg, J. (2000). The transmembrane protein p23
contributes to the organization of the Golgi apparatus. J. Cell
Sci. 113,1043
-1057.
Santel, A. and Fuller, M. T. (2001). Control of
mitochondrial morphology by a human mitofusin. J. Cell
Sci. 114,867
-874.
Sesaki, H. and Jensen, R. E. (1999). Division
versus Fusion: Dnm1p and Fzo1p Antagonistically Regulate Mitochondrial Shape.
J. Cell Biol. 147,699
-706.
Smiley, S. T., Reers, M., Mottola-Hartshorn, C., Lin, M., Chen, A., Smith, T. W., Steele, G. D. and Bo Chen, L. (1991). Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc. Natl. Acad. Sci. USA 88,3671 -3675.[Abstract]
Smirnova, E., Shurland, D. L., Ryazantsev, S. N. and van der
Bliek, A. M. (1998). A human dynamin-related protein controls
the distribution of mitochondria. J. Cell Biol.
143,351
-358.
Sutton, R. B., Fasshauer, D., Jahn, R. and Brunger, A. T. (1998). Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution. Nature 395,347 -353.[Medline]
Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22,4673 -4680.[Abstract]
Tzagoloff, A. (1982).Mitochondria . New York: Plenum Press.
Wong, E. D., Wagner, J. A., Gorsich, S. W., McCaffery, J. M.,
Shaw, J. M. and Nunnari, J. (2000). The dynamin-related
GTPase, mgm1p, is an intermembrane space protein required for maintenance of
fusion competent mitochondria. J. Cell Biol.
151,341
-352.
Yoon, Y. and McNiven, M. A. (2001). Mitochondrial division: New partners in membrane pinching. Curr. Biol. 11,R67 -R70.[Medline]
Yu, W. H., Wolfgang, W. and Forte, M. (1995).
Subcellular localization of human voltage-dependent anion channel isoforms.
J. Biol. Chem. 270,13998
-14006.