1 Departments of Developmental Biology and Genetics, Stanford University School
of Medicine, Stanford, CA 94305, USA
2 Biochemistry Section, Surgical Neurology Branch, NINDS, National Institutes of
Health, Bethesda, MD 20892, USA
3 BD Biosciences CLONTECH, Palo Alto, CA 94303, USA
4 Department of Neuropathology, University of Bonn, 53105 Bonn, Germany
Author for correspondence (e-mail:
fuller{at}cmgm.stanford.edu
Accepted 14 March 2003
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Summary |
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Key words: Mitochondria, Fusion, Fission, Fzo, GTPase, Organelle, Human, mtDNA
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Introduction |
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Mitochondria take on different characteristic morphologies in different
cell types and under different conditions in the same cell type
(Bereiter-Hahn and Voth, 1994;
Griparic and van der Bliek,
2001
). Although many examples of mitochondrial morphogenesis have
been reported (reviewed in (Griparic and
van der Bliek, 2001
; Hermann
et al., 1998
; Shaw and
Nunnari, 2002
; van der Bliek,
2000
; Westermann,
2002
), the physiological roles of regulated changes in
mitochondrial morphology are not yet understood. Formation of extensive
mitochondrial networks has been proposed to be important for efficient
intracellular energy transfer into different cell compartments
(Skulachev, 2001
).
Mitochondrial morphology also changes during the process of apoptosis and
remodeling of the mitochondrial cristae has been proposed to mobilize
cytochrome c release during apoptosis
(Scorrano et al., 2002
). In
addition, the mitochondrial fission protein Drp1 participates in regulating
apoptosis by controlling mitochondrial fragmentation
(Frank et al., 2001
).
In Drosophila and yeast, mitochondrial fusion is mediated by the
nuclear encoded mitochondrial GTPase fuzzy onions (fzo)
(Hales and Fuller, 1997;
Hermann et al., 1998
). The
Drosophila Fzo protein was identified through its role in a
developmentally regulated massive mitochondrial fusion event leading to the
formation of a specialized mitochondrial structure (the Nebenkern) during
spermatogenesis. Its homologue in Sacharomyces cerevisiae, yFzo1, is
required for mitochondrial fusion after mating and also for the continuously
ongoing mitochondrial fusion events that maintain the dynamic network of
mitochondrial filaments in vegetatively growing yeast
(Bleazard et al., 1999
;
Shaw and Nunnari, 2002
;
van der Bliek, 2000
). The
Drosophila and yeast fzo proteins are the founding members
of a family of conserved, large, transmembrane GTPases that constitute the
only known protein mediators of mitochondrial fusion. We previously described
the identification of two human Fzo homologues, Mitofusin1 (Mfn1) and
Mitofusin2 (Mfn2) (Santel and Fuller,
2001
) and reported that overexpression of Mfn2 influences
mitochondrial morphology in cultured mammalian cells.
Here we provide evidence that human Mfn1 controls mitochondrial morphology by regulating fusion of mitochondria. The human Mfn1 gene encodes a ubiquitously expressed protein associated with mitochondria. Overexpression of Mfn1 by transient transfection of cultured cells resulted in formation of characteristic networks of interconnected mitochondria. A point mutation in the conserved G1 motif of the GTPase domain (Mfn1K88T) dramatically decreased formation of mitochondrial networks, while a point mutation in the G2 motif of the GTPase domain (Mfn1T109A) resulted in mitochondrial fission upon overexpression in cultured cells, suggesting that the predicted GTPase activity of Mfn1 plays an important mechanistic role in the effects of Mfn1 on mitochondrial morphology. Expression of different ratios of wild-type Mfn1 protein versus the apparently dominant negative, fission promoting, Mfn1T109A GTPase-mutant variant in the same cell appeared to titrate the effects of Mfn1 overexpression on mitochondrial morphology, resembling the dynamic balance of fusion and fission in vegetatively growing yeast. Such ongoing fusion and fission of mitochondria in mammalian cells may provide a mechanism for functional complementation of mutants in mtDNA by ensuring mixing of protein and membrane components among mitochondria throughout the cell.
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Materials and Methods |
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Immunofluorescence and microscopy
About 15-20 hours after transfection, cells were fixed for 15 minutes at
room temperature in 4% formaldehyde/PBS. All subsequent steps for indirect
immunofluorecence staining were carried out as described previously
(Santel and Fuller, 2001).
MitoTracker Red (CMXRos; Molecular Probes) was added to cell medium at a
concentration of 0.1 µM for 15 minutes. Electron microscopy and live
imaging of transfected cells by confocal microscopy was carried out as
described (Frank et al.,
2001
).
Antibodies
To generate anti-Mfn-1 antibodies, an EcoRI-PstI-fragment
from pBSfzoH1 (human EST HFBDS57) was subcloned in frame into pGEX-KG. The 35
kDa GST-fusion protein was expressed in E. coli XL-1 Blue cells with
IPTG-induction under standard conditions. The soluble GST-fusion protein (2
mg) was purified under native conditions according to the manufacturer's
protocol and used for a standard immunization of two rabbits (BabCo/Covance,
CA). Polyclonal antisera from both rabbits were affinity-purified against the
GST-Mfn fusion protein coupled to AminoLink column (Pierce) and subsequently
tested in western blot experiments. Both purified antibodies gave identical
results when used in western blot experiments.
Polyclonal antibodies specific to Mfn2 protein were raised against an internal peptide sequence [(C)-KNSRRALMGYNDQVQ-RPIPLTPAN] by Zymed Laboratories (South San Francisco, CA) and affinity-purified against the immunogenic peptide. The affinity purified Mfn1- and Mfn2-antibodies were used at a concentration at 1:500-1000 in western blot experiments and 1:50 for immunofluorescence staining
Polyclonal rabbit anti-rTOM40 antibodies
(Suzuki et al., 2000) were
kindly provided by Hiroyuki Suzuki and Katsuyoshi Mihara (Kyushu University
Fukuoka, Japan) and used at 1:1000. Monoclonal anti-cytochrome c (clone
6H2.B4) purchased from Pharmingen (San Diego, CA) was used at 1:50. Monoclonal
anti-actin antibody obtained from Chemicon International (Temecula, CA) and
mouse anti-porin monoclonal antibody (Calbiochem) were used according to the
manufacturer's recommendation. The monoclonal anti-myc antibodies (clone 9E10;
kindly provided by Alan J. Zhu, Stanford University, CA) was used 1:1000 and
monoclonal anti-HA (BabCo/Covance) at a concentration of 1:500.
Expression constructs
For expression constructs carrying the Mfn1 coding region, the Mfn1 ORF was
amplified by PCR using primers A (5'-ATGGCAG-AACCGTTTCTCCAC-3')
and B (5'-CATGGTCACCAAAGC-AATC-3'), A-tailed by Taq polymerase and
subcloned in pcDNA3.1V5/HISTOPO resulting in pcDNA3.1::Mfn1. The GFP-Mfn1
expression construct (pEGFPC2::Mfn1)
(Santel and Fuller, 2001) was
altered by site-directed mutagenesis to generate mutant expression constructs
pEGFPC2::Mfn1(K88T) and pEGFPC2::Mfn1(T109A) using the Quickchange Mutagenesis
kit (Stratagene) with appropriate PCR primers. Generation of introduced point
mutations was confirmed by sequencing. For N-terminal in frame fusion of an
V5/HIS-tag to the Mfn1 ORF, B-primer was replaced by primer C lacking the stop
codon (5'-GGATTCTTCATTGCTTGAAGG-3') for PCR amplification
resulting in pcDNA3.1::Mfn1V5-HIS. The expression construct
pEGFPC2::Mfn1(1-418) was generated by excising a SalI fragment from
the original pEGFPC2::Mfn1 construct. GFP-Mfn1
GTPase was generated by
PCR and subsequent in frame subcloning into pcDNA-NT-GFPTOPO (Invitrogen). PCR
reactions were performed using proof-reading polymerase pfu (Perkin
Elmer) or the ThermalAce DNA amplification kit (Invitrogen). The HA-tagged
Drp1(K38A) expression construct was kindly provided by A. van der Bliek (UCLA)
(Smirnova et al., 1998
).
Protein extracts and western blot analysis
Proteins were extracted from mammalian tissue-culture cells by treating
cells with NP-40 buffer (150 mM NaCl, 1% NP-40, 50 mM Tris-HCl pH 8.0) for 15
minutes at 4°C and subsequent centrifugation at 3000 g for
an additional 10 minutes. Human protein extracts were purchased from Clontech
(Protein Medley Kidney and Heart). For mitochondrial protein extracts and
subcellular fractionation, mitochondria were isolated and fractionated
following a previously described protocol
(Spector et al., 1998).
Protein extracts were separated in an 8% SDS-PAGE and then blotted onto Hybond
ECL membrane. Membranes were blocked in 5% nonfat dried milk (NFDM) in PBT
(PBS with 0.1% Tween-20) for 2 hours at room temperature and probed with the
primary antibody in PBT+NFDM overnight at 4°C. After washing with PBT for
30 minutes at room temperature, samples were incubated with
HRP-peroxidase-coupled secondary antibodies (anti-mouse from Roche,
anti-rabbit from Amersham) for 2 hours at room temperature, followed by a 30
minute wash step at room temperature. The reaction products were visualized by
enhanced chemiluminescence using ECLplus (Amersham) and BioMax films
(Kodak).
Gel filtration
Cells were spun down, washed once in Iscove's medium and resuspended at a
cell density of 1x108/ml in Lysis buffer (20 mM HEPES, pH
7.4, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 250
mM sucrose and 0.1 mM phenylmethylsulfonyl fluoride). After a 30 minute
incubation on ice, the lysate was Dounce-homogenized and spun at 900
g to pellet the nuclei. The postnuclear supernatant was then
spun at 10,000 g to pellet the mitochondria. Isolated
mitochondria (2 mg protein) were lysed in 200 µl digitonin buffer [1% (w/v)
digitonin, 150 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 1 mM
phenylmethylsulfonyl fluoride] for 30 minutes on ice. Following a clearing
spin (30 minutes, 226,000 g, TLA45 rotor, Beckman TL-100
ultracentrifuge), the supernatant was applied to a Superose 6 gel-filtration
column (25 ml column volume, Pharmacia) equilibrated with the same digitonin
buffer. Fractions (0.5 ml) were collected, precipitated by adding
trichloroacetic acid to a final concentration of 12.5% (w/v) and analyzed by
SDS-PAGE and western blotting.
Northern blot analysis and RNA-chip analysis
Human Multiple-Tissue-Northern-/MTN-Northern blots were purchased from
Clontech and hybridized with radiolabelled probes (32PC-labeling
kit from Amersham) specific for Mfn1 and Mfn2. The RNA Chip contained 128
different human poly-A+ RNAs derived from a wide range of human tissues and
cell lines, normalized to six house-keeping genes, and printed in duplicate
spots onto chemically treated microscope slides (BD Biosciences-Clontech).
Several negative control spots were added to each RNA Chip: Yeast total RNA,
Yeast tRNA, E. coli rRNA, E. coli DNA, Poly r(A), Human
Cot-1 DNA, Human genomic DNA. 32P-labeled DNA-probes (specific activity of the
probe was >108 cpm/µg DNA) were used for hybridization
according to the manufacturer's protocol. The RNA Chip was analyzed by
phosphorimaging and autoradiography. The following probes were applied as
positive control to assess equal RNA quantity and specificity: actin,
liver-specific TAT (data not shown).
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Results |
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The other two human potential Mfn genes appeared to be pseudogenes. PsiMfn3 and psiMfn4 were found on chromosome X (Xq23-24) and chromosome 9, respectively. Although psiMfn3 and psiMfn4 were highly homologous to the Mfn1 gene both showed characteristic features of pseudogenes, including deletions and multiple stop codons throughout the predicted coding region, suggesting that they no longer encode proteins.
Mfns are widely expressed but show tissue-specific differences in
expression level
Human Mfn1 and Mfn2 mRNAs were expressed at low levels in all tissues
tested (Fig. 2). However, the
levels of Mfn1 and Mfn2 mRNA expression varied among different tissues, with
Mfn1 mRNA levels being increased in more tissue and cell types than Mfn2 mRNA.
An Mfn1-cDNA probe detected transcripts of 6.4kb and 3.6kb in a variety of
adult human tissues, including heart, pancreas, skeletal muscle, brain, liver,
placenta, lung, and kidney. An Mfn2-specific probe detected a single 5.5kb
mRNA in the same range of tissues. (Fig.
2A). Both Mfn1 and Mfn2 mRNA levels were high in heart compared to
other tissues (Fig. 2A). In
addition, Mfn2 mRNA was also high in skeletal muscle
(Fig. 2A). In contrast, Mfn1
mRNA levels appeared slightly elevated in pancreas and liver, but not in
skeletal muscle (Fig. 2A). As
expression of the actin mRNA loading control was also elevated in heart and
skeletal muscle in Northern blots, we analyzed relative levels of Mfn mRNA
expression using an RNA-chip with mRNA from 128 tissues (including fetal
tissues) normalized to six different housekeeping genes
(Fig. 2B). The RNA-chip results
confirmed that Mfn1 and Mfn2 transcripts are widely expressed at low level in
most tissues. Relatively elevated levels of Mfn2 mRNA expression were detected
in heart and skeletal muscle, as well as in tongue
(Fig. 2B). Expression of both
Mfn1 and Mfn2 was detected in fetal tissues, with Mfn1 mRNA more abundant than
Mfn2 (Fig. 2B, box 4). The chip
results and Northern blot analysis (data not shown) revealed that both Mfn1
and Mfn2 mRNAs were present in several cultured cell lines, indicating that
both genes can be co-expressed in the same cell type. Strikingly, the level of
Mfn1 mRNA was elevated in certain carcinoma derived cancer cell lines while
the level of Mfn2 mRNA was not (Fig.
2B; for example, compare Mfn1 expression to Mfn2 mRNA in human
HL-60 carcinoma cells). In addition, Mfn1 and Mfn2 mRNAs appeared to be
differentially expressed in two Burkitt's lymphoma cell lines
(Fig. 2B).
|
Differential expression of mitofusin proteins
Mfn1 protein was widely expressed, based on immunoblotting experiments
using antibodies that recognized Mfn1 but not Mfn2
(Fig. 3; see Materials and
Methods). The affinity-purified anti-Mfn1 antibodies recognized a single
endogenous protein with an apparent size of 86 kDa in mammalian cell
extracts (Fig. 3A), matching
the size of 84 kDa predicted from the Mfn1 primary sequence. The anti-Mfn1
antibodies also recognized recombinant GFP-tagged Mfn1 fusion protein
transiently expressed in mammalian cells, but did not crossreact with Mfn2
protein similarly expressed from an epitope-tagged construct (Mfn2-myc) in
COS-7 cells (Fig. 3A, lower
panel) or expressed in yeast under control of an inducible GAL1 promoter
(Fig. 3C). In addition, the
anti-Mfn1 antibodies recognized overexpressed Mfn1 but not Mfn2 in
immunofluorescence analysis of cells transiently transfected with various Mfn1
and Mfn2 expression constructs (data not shown). Immunoblotting experiments
with anti-Mfn1 revealed expression of endogenous Mfn1 protein in HeLa cells,
human kidney and heart (Fig.
3B), as well as in mouse heart, liver, kidney, NIH3T3 fibroblasts,
mouse C2C12 myoblasts, and differentiated myotubes, and rat clone9 cells (data
not shown). Strikingly, the level of Mfn1 protein appeared to be only slightly
elevated in human heart protein extracts compared to kidney, using the level
of the mitochondrial protein TOM40 as a loading control. Thus, the high level
of Mfn1 mRNA detected in heart tissues may not be reflected in correspondingly
elevated levels of Mfn1 protein.
|
Expression of Mfn2 protein appeared more tissue-specific than Mfn1 protein, as suggested from the mRNA expression studies. An Mfn2-specific antibody was raised against a peptide from an internal region of Mfn2 that had relatively low homology to Mfn1 (Fig. 1, bracket). The anti-Mfn2 antibody detected recombinant Mfn2 protein expressed in yeast and a doublet of proteins migrating at an apparent size of approximately 86 kDa in extracts from human tissues (Fig. 3C). Mfn2 protein appeared to be present in significantly higher levels in human heart than human kidney, in contrast to Mfn1, which was expressed at more similar levels in these two tissues. As Mfn1 mRNA and protein appeared to be more generally expressed than Mfn2, we investigated the subcellular localization and possible role of the Mfn1 protein in controlling mitochondrial morphology in mammalian cells.
Mitofusin 1 is a mitochondrial protein
Mfn1 protein from both HeLa cells and mouse heart co-fractionated with
mitochondrial markers during differential centrifugation and was either not
detected or was greatly reduced in the cytosolic postmitochondrial supernatant
(Fig. 4A). Mfn1 protein
released from mitochondria by detergent extraction migrated in an apparent
high molecular mass complex through a size exclusion gel filtration column. A
mitochondrial preparation derived from human HL-60 cells was treated with 1%
digitonin to solubilize membrane proteins, fractionated by gel filtration, and
fractions were probed for Mfn1 by western blotting. Mfn1 immunoreactivity
peaked in the fraction corresponding to a molecular mass of 350 kDa
(Fig. 4B). Mfn1 was not
detected in the monomeric fraction corresponding to 86 kDa.
|
Both GFP-tagged and untagged Mfn1 protein localized to mitochondria when expressed transiently in cultured cells, as demonstrated by counterstaining with Mitotracker or antibodies against the mitochondrial intermembrane space protein cytochrome c (Fig. 5A,B). In contrast, a C-terminal truncated GFP-Mfn11-418 fusion protein lacking the conserved transmembrane domain and predicted coiled-coil tail did not localize to mitochondria (data not shown), suggesting that Mfn1, like Mfn2, carries the mitochondrial targeting signal in the C-terminal half.
|
Overexpression of Mfn1 in mammalian tissue culture cells induced
formation of mitochondrial networks
Overexpression of either untagged or GFP-tagged Mfn1-fusion protein
resulted in formation of perinuclear clusters of mitochondria in COS-7 cells
(Fig. 5), as well as in HeLa
cells, rat clone9 cells, C2C12 myoblasts, and NIH3T3 fibroblasts (data not
shown). This dramatic change in mitochondrial morphology appeared to correlate
with substantially elevated levels of Mfn1 protein: anti-Mfn1 antibodies
strongly stained the protein in transfected cells with perinuclear networks of
mitochondria, while under the same conditions endogenous Mfn1 protein was not
detected by standard immunofluorescence microscopy in presumably untransfected
cells, which had well spread mitochondria
(Fig. 5B). Staining with the
dye Mitotracker Red (CMXRos) indicated that the clustered mitochondria in Mfn1
transfected cells still had an active membrane potential and therefore most
likely unaffected respiration activity
(Fig. 5A).
Detailed analysis of transfected cells by high magnification light microscopy (Fig. 5C-E) and ultrastructural analysis by electron microscopy (Fig. 5F) revealed a peculiar and characteristic structural arrangement in the perinuclear networks of mitochondria. Simultaneous visualization of GFP-Mfn1 and cytochrome c, a marker of the mitochondrial intermembrane space, revealed that GFP-Mfn1 was adjacent to, but did not colocalize with, cytochrome c in the structures. Instead, anti-cytochrome c stained grape-like clusters of spherical structures, while the GFP-Mfn1 both surrounded the cytochrome c stained spheres in a thin layer and appeared concentrated in the interstices between the tightly packed spheres (Fig. 5C-E). Similarly, GFP-Mfn1 appeared to circumscribe the mitochondrial matrix as indicated by the Mitotracker marker in transfected cells (data not shown). Strikingly, the cytochrome c positive spheres were smaller in size toward the center of the array.
Ultrastructural analysis by electron microscopy confirmed the peculiar structure of the mitochondrial networks (Fig. 5F). Overexpression of GFP-Mfn1 after transient transfection induced formation of tight clusters of mitochondria, with smaller mitochondria near the center surrounded by larger mitochondria with typically scant cristae. Immunogold-labeling using antibodies against GFP indicated that the GFP-Mfn1 was largely associated with the mitochondrial outer membrane (Fig. 5F), consistent with the appearance of GFP-Mfn1 in a thin layer surrounding the cytochrome c containing spheres observed by light microscopy (Fig. 5C). GFP-Mfn1 protein appeared most abundant at the interfaces between the tightly packed small mitochondria inside the clusters. The enlarged mitochondria around the periphery of the cluster were less densely stained by immunogold particles. We propose that overexpression of Mfn1 by transient transfection triggers aggregation of mitochondria into tight networks through close interactions between outer membranes, followed by outer membrane fusion events that result in formation of the enlarged mitochondria characteristically observed around the periphery of the grape-like clusters.
Formation of mitochondrial networks required a wild type Mfn1
GTPase-domain
To test whether formation of the grape-like mitochondrial networks
(Fig. 5, Fig. 6B) was dependent on the
predicted GTPase activity of the overexpressed Mfn1 protein, we expressed a
mutant form of GFP-Mfn1 carrying a K to T substitution at amino acid residue
88 in the G1 motif of the signature GTPase domain (GFP-Mfn1K88T;
Fig. 6A). Substitutions in the
analogous residue in the Drosophila Fzo and yeast Fzo1p proteins
blocked mitochondrial fusion activity
(Hales and Fuller, 1997;
Hermann et al., 1998
). The
Mfn1K88T GTPase mutant greatly reduced the formation of
mitochondrial networks. The majority of Mfn1K88T expressing cells
exhibited mitochondria with apparently normal morphology and distribution,
compared with mitochondria in adjacent non-transfected cells
(Fig. 6C). Whereas 93% of the
cells expressing detectable levels of wild type Mfn1 after transient
transfection exhibited perinuclear clusters of mitochondria
(Fig. 6B), only 30-40% of the
cells expressing Mfn1K88T showed collapsed mitochondrial
aggregates. In addition, most of these aggregates that were observed appeared
structurally different from those mitochondrial clusters induced by wild-type
Mfn1 expression. In the perinuclear mitochondrial aggregates observed in cells
overexpressing GFP-Mfn1K88T, the GFP-Mfn1 and the cytochrome c
intermembrane space marker appeared well aligned at the light microscope level
(Fig. 6D), in contrast to the
cluster-of-grapes appearance characteristic of mitochondrial networks observed
in cells transfected with wild type Mfn1
(Fig. 5). We suggest, that in
the case of Mfn1K88T induced mitochondrial aggregates, mitochondria
might be tightly packed rather than `hyperfused' as observed for Mfn1 induced
mitochondrial clusters. The difference in structure of the mitochondrial
clusters/aggregates for wild type versus mutant Mfn1 could be accounted for if
individual mitochondria in cells overexpressing GFP-Mfn1K88T did
not fuse into the large mitochondria observed at the periphery of the
grape-like clusters in cells transfected with wild type Mfn1. Expression of a
GFP-Mfn1 fusion construct lacking the entire GTPase domain
(Mfn1
aa1-248) caused formation of mitochondrial
aggregates resembling those observed in cells expressing the
Mfn1K88T mutant variant (Fig.
6E) in almost all transfected cells (>80%).
|
The GTPase mutant Mfn1T109A blocked endogenous fusion
activity
Mutation of the predicted G2 motif of the GTPase domain by a T to A
substitution at amino acid residue 109 (Mfn1T109A) produced a form
of the Mfn1 protein that appeared to act as a dominant negative and block
endogenous mitochondrial fusion when expressed at high levels in COS-7 cells
(Fig. 7).
GFP-Mfn1T109A was still targeted specifically to mitochondria
(Fig. 7A). Counterstaining with
MitoTracker in live cells expressing GFP-Mfn1T109A showed an active
membrane potential (Fig. 7A).
However, in more than 80% of the transfected cells, mitochondria were
distributed as small punctate structures throughout the cytoplasm, in contrast
to the normal filamentous morphology observed in non-transfected cells
(Fig. 7A, lower cell) or cells
exhibiting only very low expression of the GFP-Mfn1T109A construct
(Fig. 7B, upper cell).
|
To test whether mitochondria might be fragmented due to interference with
the endogenous activity of Mfn1, we shifted the balance of mutant versus wild
type Mfn1 in the cells by co-transfecting with GFP-Mfn1T109A and
wild type Mfn1. Cells doubly transfected with GFP-Mfn1T109A and
wild type Mfn1 showed a range of mitochondrial morphologies. However,
mitochondria in these cells often exhibited a more filamentous morphology
(Fig. 6C) than mitochondria in
cells transfected with GFP-Mfn1T109A alone. To test whether the
different mitochondrial morphologies observed correlated with relative levels
of wild type and Mfn1T109A mutant protein expressed in individual
cells, we co-transfected COS-7 cells with GFP-Mfn1T109A and a
construct driving expression of a HIS-tagged wild type Mfn1 (Mfn1-V5/HIS)
fusion protein and compared relative levels of GFP-Mfn1T109A and
His-tagged Mfn1 in doubly transfected cells. Cells expressing
GFP-Mfn1T109A and relatively low levels of Mfn1-V5/HIS tended to
have fragmented and/or lightly fused mitochondria
(Fig. 7D, upper panels, double
arrow: cell with low Mfn1-V5/HIS; lower panels, cell with no detectable
Mfn1-V5/HIS). Cells showing relatively similar levels of
GFP-Mfn1T109A and Mfn1-V5/HIS tended to have more normal,
filamentous mitochondria. High levels of expression of Mfn1-V5/HIS appeared to
override and block the negative interfering activity of
GFP-Mfn1T109A, resulting in formation of mitochondrial networks
resembling those observed in cells overexpressing wild-type Mfn1 alone
(Fig. 7D, arrow). We propose
that Mfn1T109A encodes a dominant negative form of the protein able
to block mitochondrial fusion activity of endogenous Mfn1. If so, then
overexpression of Mfn1T109A may cause fragmentation of mitochondria
due to unopposed mitochondrial fission, much as loss of function mutations in
the Saccharomyces cerevisiae mitofusin gene yfzo1 causes
fragmentation of mitochondria in vegetatively growing yeast. To test, whether
the observed mitochondrial fragmentation in Mfn1T109A expressing
cells can be prevented by blocking opposed mitochondrial fission, we
co-expressed GFP-Mfn1T109A with the dominant-negative version of
the fission protein Drp1 (HA-tagged Drp1K38A)
(Smirnova et al., 1998)
transiently in COS-7 cells. In doubly-transfected cells, mitochondrial fusion
rather than fission occurred (Fig.
8). Fragmented mitochondria were no longer observed indicating
that mitochondrial fission was blocked. The expression of Drp1K38A
interfered with the expression of GFP-Mfn1T109A resulting in
altered mitochondrial morphology (Fig.
8). Clustered and aggregated mitochondria as well as filamentous
and interconnected mitochondria were observed. In a few cases, mitochondria
displayed an interconnected network of tubular GFP-positive structures.
(Fig. 8, right panel).
|
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Discussion |
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Our results indicate that at both the mRNA and the protein levels human
Mfn1 may be more widely and abundantly expressed than the previously described
Mfn2 isotype (Santel and Fuller,
2001), which was expressed mainly in heart and skeletal muscle.
The differential expression we observed by Northern and dot blot disagrees
with the conclusions drawn by Rojo et al., who observed similar levels of Mfn1
and Mfn2 mRNA expression by an RT-PCR assay
(Rojo et al., 2002
). Northern
blot analysis revealed two different transcripts detected by an Mfn1-specific
probe. Database searches uncovered cDNAs derived from the Mfn1 locus that may
represent alternative splice variants encoding truncated forms of the Mfn1
protein (A.S. and M.T.F., unpublished). Interestingly, it has been reported
that expression of a corresponding Mfn1-splice variant was specifically
up-regulated in tumor tissues (Chung et
al., 2001
). Our investigation did not independently either confirm
or rule out the existence of such mRNA or protein variants, especially in
diseased tissues or cancer cells.
The Fzo-GTPase family members Drosophila Fzo and yeast Fzo1p have
been shown to be key mediators of mitochondrial fusion
(Hales and Fuller, 1997;
Hermann and Shaw, 1998
). We
propose that the human Mitofusin-1 protein (Mfn1) represents the main mediator
of mitochondrial fusion in many human cell types. This idea is in agreement
with two recent reports on the characterization of Mitofusin function
(Chen et al., 2003
)
(Legros et al., 2002
).
Loss-of-function analysis of the mouse orthologs clearly demonstrated a
contribution of Mfns in mitochondrial fusion
(Chen et al., 2003
). Consistent
with our model, high level overexpression of Mfn1 in cultured cells led to
formation of characteristic grape-like perinuclear clusters of mitochondria
containing many large mitochondria around the outer edge. Strikingly,
immunofluorescence staining for cytochrome c showed that the large peripheral
mitochondria in the clusters had highly enlarged regions of intermembrane
space (Fig. 5E,F). The large
mitochondria with abnormal internal structure in Mfn1 overexpressing cells
could arise if Mfn1 protein mediates fusion of outer membranes but not the
inner membranes of adjacent and closely apposed mitochondria. Action at the
outer mitochondrial membrane is consistent with a number of biochemical
studies indicating that Mfn family proteins are associated with the
mitochondrial outer membrane (Fritz et al.,
2001
; Rapaport et al.,
1998
; Rojo et al.,
2002
). In addition, several lines of evidence support the
prediction that Mfn family proteins have a characteristic bipartite
transmembrane domain that passes through the outer membrane twice, leaving
both the N-terminal GTPase domain and the C-terminal coiled-coil motif facing
the cytoplasm (Fritz et al.,
2001
), where they would be available to interact with Mitofusins
or other proteins displayed on the surface of adjacent mitochondria. Fusion of
inner mitochondrial membranes to allow mixing of matrix components could
either be mediated by different proteins or require additional components not
overexpressed in our studies.
The ability of overexpressed human Mfn1 to cause mitochondrial fusion in
mammalian tissue culture cells was dependent on signature GTPase motifs, as
demonstrated for Drosophila Fzo and yeast Fzo1p. Mutation of the
conserved residue K88 to T in the G1 GTPase motif of Mfn1 abolished ability of
the overexpressed Mfn1K88T protein to induce formation of the
characteristic mitochondrial networks and grape-like clusters containing
enlarged mitochondria. The G1 motif (GxxxxGKS/T) is the conserved core of the
P-loop, which in Ras (Bourne et al.,
1991) and several other members of the GTPase superfamily forms a
critical part of the nucleotide binding pocket interacting with the
and ß phosphates of GTP or GDP. The three dimensional structure of the G1
region of Ras was found to be similar in the GTP versus the GDP bound forms
(Krengel et al., 1990
;
Pai et al., 1989
). The K
residue is conserved throughout the GTPase superfamily, as well as in ATPase
motor proteins such as myosins and kinesins
(Vale, 1996
). In Ras, the
-amino group of the corresponding lysine residue (K16) with its positive
charges stabilizes GTP phosphates by forming ionic interactions with the
ß- and
-phosphates of the nucleotide
(Maegley et al., 1996
;
Prive et al., 1992
). The loss
of the corresponding K residue in the Mfn1K88T mutant form might
affect affinity of guanine nucleotide binding, and therefore result in reduced
hydrolysis rates for GTPase activity. Our finding that the K88 to T mutation
in human Mfn1 blocked the ability of overexpressed Mfn1 to induce formation of
the characteristic enlarged mitochondria suggests that mitochondrial fusion by
Mfn1 protein depends on the GTPase cycle and raises the possibility that
mitochondrial fusion in the cell may be regulated by additional proteins, much
as GTPase activating proteins (GAPs) and guanine nucleotide exchange factors
(GEFs) regulate the action cycle of many GTPases.
In contrast, overexpression of a version of the Mfn1 protein mutated in a
different GTPase subdomain (Mfn1T109A) had a strikingly different
effect on mitochondrial morphology. Amino acid residue T109 of Mfn1 is located
in the predicted G2 motif, a region that in Ras is part of a large loop that
forms a roof over the GTP binding pocket. A threonine is conserved in an
analogous position throughout the GTPase superfamily
(Bourne et al., 1991). In the
crystal structure of Ras complexed with the GTP analogue Gpp(NH)p, this
conserved threonine contacts the gamma phosphate of the nucleotide and also
binds the critical Mg2+ ion that coordinates the
and ß
phosphates of GTP (Bourne et al.,
1991
). Comparison of the structures of RAS in the GTP vs.
GDP bound forms revealed that the G2 loop undergoes a conformational change
upon GTP binding, most likely due to a dramatic reorientation of the conserved
threonine (Bourne et al.,
1991
).
We found that a form of human Mfn1 in which the critical G2 motif threonine
was mutated (Mfn1T109A) acted as a dominant negative when
overexpressed by transient transfection in tissue culture cells. Cells
expressing Mfn1T109A displayed many small mitochondria, resembling
the punctate mitochondria that rapidly accumulate after loss of function of
the yeast Mfn, yeast Fzo1p (Bleazard et
al., 1999; Hermann et al.,
1998
). Consideration of the activity cycle of GTPase family
members suggests a model for why Mfn1T109A may interfere with the
function of endogenous Mfn protein in the transfected cells. Many GTPases rely
on interaction with a guanine exchange factor (GEF) protein to induce release
of bound GDP, which must occur to allow GTP to enter the nucleotide binding
pocket after each activity cycle. Analysis of the GTPase family member EF-Tu
suggested that the protein passes through a transient empty state, lacking
nucleotide, while still bound to its GEF partner EF-Ts
(Romero et al., 1985
). Binding
of GTP then allows release of EF-Ts, possibly due in part to the dramatic
change in conformation of the GTPase upon GTP binding. If a similar GTPase
cycle involving a partner GEF operates as a critical part of Mfn function in
mitochondrial fusion, then high levels of the mutant Mfn1T109A
protein may act as a dominant negative by binding up and titrating out the
GEF. Without the critical G2 motif threonine residue, the Mfn1T109A
protein may not undergo the dramatic conformational change upon GTP binding
needed to release the GEF for use by endogenous Mfn proteins in the cell. As a
consequence, loss of mitochondrial fusion activity could allow unbalanced
mitochondrial fission, leading to accumulation of small, punctiform
mitochondria, as observed in yeast lacking function of yeast Fzo1p.
Our results suggest that normal mitochondrial morphology in mammalian cells
is maintained by a dynamic balance of Mfn-mediated mitochondrial fusion and
Drp1-mediated mitochondrial fission. Consistent with this model, co-expression
of Drp1K38A, a dominant interfering form of the dynamin GTPase Drp1
implicated in mitochondrial fission
(Smirnova et al., 2001;
Smirnova et al., 1998
) with
human Mfn1 resulted in formation of interconnected filamtentous mitochondrial
tubules of mitochondrial outer membrane (S. Frank, R. J. Youle, A. Santel and
M. T. Fuller, unpublished) much as observed for Mfn2 in similar experiments
(Santel and Fuller, 2001
). In
addition, co-expression of dominant negative Drp1 (Drp1K38A) with
the Mfn1 mutant form Mfn1T109A resulted in an inhibition of
mitochondrial fission, but leading to mitochondria with altered morphology
similar to those observed in cells expressing Drp1K38A alone or
co-expressed with wild-type Mfn1/Mfn2, respectively
(Santel and Fuller, 2001
).
This observation resembles the situation in yeast
dnm1
/fzo1-1 double mutants, where mitochondrial
morphology was similar to those observed in
dnm1
-cells
(Bleazard et al., 1999
).
Interestingly, Chan and colleagues also observed altered mitochondrial
morphology in Mfn-mutant mouse embryonic fibroblasts expressing
Drp1K38A compared to restored mitochondrial morphology in
Mfn-deficient cells rescued by epitope-tagged mitofusins
(Chen et al., 2003
).
Mfn1 migrated in a gel filtration column as part of a high molecular
protein complex, which may represent either a mitochondrial fusion machine or
Mfn1 protein complexed with regulatory GEFs or GAPs. In extracts from S.
cerevisiae, the Fzo1p behaved in biochemical experiments as part of an
800 kDa protein complex (Rapaport et al.,
1998) rather than the 350 kDa complex observed in human HL-60
cells. The nature of the protein complex and the other components are not yet
known for either yeast or human cells. Presumably due to the tissue-specific
expression of Mfn2 we could not detect Mfn2 as a component of the Mfn1-complex
by gel filtration fractionation in HL-60 cell extracts. In addition,
preliminary experiments with extracts from cultured cells transiently
transfected with Mfn1 and Mfn2 did not uncover biochemical interaction between
these two isoforms. It is possible that Mfn1 and Mfn2 reside in different
protein complexes, even in tissues were expression of both proteins
overlaps.
The human Mfn1 and Mfn2 proteins had subtly different behavior with respect
to their effects on mitochondrial morphology after transient overexpression in
cell culture. Overexpression of Mfn1 alone was sufficient to cause GTPase
dependent fusion of mitochondria, resulting in the formation of large
mitochondria around the periphery of the perinuclear mitochondrial clusters.
In contrast, GTPase dependent effects of overexpression of Mfn2 were best
revealed in cells also undergoing overexpression of the dominant interfering
Drp1K38A form of the mitochondrial fission mediator Drp1
(Santel and Fuller, 2001).
Conversely, overexpression of Mfn2 caused mitochondrial clustering that was
dependent on the C-terminal coiled-coil domain of Mfn2
(Rojo et al., 2002
;
Santel and Fuller, 2001
). It
may be that the more tissue specific Mfn2 regulates or mediates a specialized
form of mitochondrial fusion characteristic of only certain cell types.
Alternatively, the Mfn2 protein may play a completely different or more
specialized function related to mitochondrial morphology. Recently, it has
been proposed that Mfn2 may be in involved in regulating BAX-mediated
apoptosis (Karbowski et al.,
2002
).
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Acknowledgments |
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Footnotes |
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References |
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