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Structural Basis of Mitochondrial Tethering by Mitofusin Complexes was involved in suppression of virus pro-
duction during the HR. On the other hand,
induction of pathenogenesis-related (PR)
proteins, PR-1 and PR-2, continued after
the temperature shift in the VPE-silenced
leaves as in the non-silenced plants (Fig.
4B). The VPE deficiency did not affect the
production of the PR proteins, although it
affected TMV-induced cell death. These
results suggest that PCD and defense-
protein induction are not coupled during
the HR and that the HR is composed of two
independent processes, PCD and defense-
protein induction. VPE regulates PCD but
not defense-protein induction. There has been
a lot of discussion about whether PCD during
the HR is really critical for resistance (25).
Our results suggest that PCD contributes to
resistance to a virus infection.
Although the Arabidopsis genome does
not have a caspase family, it has a meta-
caspase family, which is distantly related to
the caspase family. A metacaspase was re-
ported to be involved in the cell death of
yeast (26 ). The gene expression of meta-
caspase was found in pathogen-infected to-
mato leaves (27), and a proteolytic activity
toward
Ac-VEID-MCA
(a
metacaspase/
caspase-6 substrate) was detected in dying
suspensor cells of Norway spruce (28). Meta-
caspases might function in the cytosol during
cell death as animal caspases. This is in con-
trast to the VPE functions in vacuole-mediated
cell death. VPE is structurally unrelated to the
caspase family, although it has caspase-1 activity
and an ability to bind caspase-1 inhibitor.
In animals, dying cells are engulfed by
phagocytes. However, in plants, which do not
have phagocytes, cells surrounded by rigid cell
walls must degrade their materials by them-
selves. Vacuolar collapse has been shown to
trigger degradation of the cytoplasmic struc-
tures and lead to cell death (21), although its
molecular mechanism is not known. Our find-
ings suggest that VPE functions as a key player
in vacuolar collapsetriggered cell death. VPEs
are distributed in mono- and dicotyledonous
plants. Arabidopsis VPE genes are up-regulated
in dying cells during development and senes-
cence of tissues (5, 13). Thus, VPE might reg-
ulate various types of PCD in higher plants.
Identification of the VPE-target proteins, which
are possibly associated with the vacuolar mem-
branes, would help to unravel the molecular
mechanism of VPE-mediated vacuolar collapse
underlying plant PCD. Because VPE acts as a
processing enzyme to activate various vacuolar
proteins, it might also convert the inactive hy-
drolytic enzymes to the active forms, which are
involved in the disintegration of vacuoles, to
initiate the proteolytic cascade in plant PCD.
Understanding the VPE-regulated mechanism,
which operates in the early process of the HR,
may also lead to practical applications for
strengthening disease resistance in crops.
References and Notes
1. G. M. Cohen, Biochem. J. 326, 1 (1997).
2. E. Lam, O. del Pozo, Plant Mol. Biol. 44, 417 (2000).
3. E. Lam, N. Kato, M. Lawton, Nature 411, 848 (2001).
4. E. J. Woltering, A. van der Bent, F. A. Hoeberichts,
Plant Physiol. 130, 1764 (2002).
5. I. Hara-Nishimura, M. Maeshima, in Vacuolar Com-
partments in Plants, A. D. G. Robinson, J. C. Rogers,
Eds. (Shefeld, London, 2000), pp. 2042.
6. J. T. Greenberg, Annu. Rev. Plant Physiol. Plant Mol.
Biol. 48, 525 (1997).
7. I. Hara-Nishimura, K. Inoue, M. Nishimura, FEBS Lett.
294, 89 (1991).
8. I. Hara-Nishimura, Y. Takeuchi, M. Nishimura, Plant
Cell 5, 1651 (1993).
9. K. Yamada, T. Shimada, M. Kondo, M. Nishimura, I.
Hara-Nishimura, J. Biol. Chem. 274, 2563 (1999).
10. N. Mitsuhashi et al., Plant Cell 13, 2361 (2001).
11. T. Shimada et al., J. Biol. Chem. 278, 32292 (2003).
12. K. Shirahama-Noda et al., J. Biol. Chem. 278, 33194
(2003).
13. T. Kinoshita, K. Yamada, N. Hiraiwa, M. Nishimura, I.
Hara-Nishimura, Plant J. 19, 43 (1999).
14. R. N. Goodman, A. J. Novacky, The Hypersensitive
Response Reaction in Plants to Pathogens: A Resis-
tance Phenomenon (American Phytopathological So-
ciety Press, St. Paul, MN, 1994).
15. N. Hiraiwa, M. Nishimura, I. Hara-Nishimura, FEBS
Lett. 447, 213 (1999).
16. M. Kuroyanagi, M. Nishimura, I. Hara-Nishimura,
Plant Cell Physiol. 43, 143 (2002).
17. S. M. Angell, D. C. Baulcombe, Plant J. 20, 357 (1999).
18. I. Hara-Nishimura, in Handbook of Proteolytic En-
zymes, A. J. Barrett, N. D. Rawlings, J. F. Woessner,
Eds. (Academic Press, London, 1998), pp. 746749.
19. I. Hara-Nishimura, T. Kinoshita, N. Hiraiwa, M. Nish-
imura, J. Plant Physiol. 152, 668(1998).
20. C. Becker et al., Eur. J. Biochem. 228, 456 (1995).
21. A. M. Jones, Plant Physiol. 125, 94 (2001).
22. N. Hatsugai et al., unpublished data.
23. R. Mittler, L. Simon, E. Lam, J. Cell Sci. 110, 1333
(1997).
24. A. Levine, R. I. Pennell, M. E. Alvarez, R. Palmer, C.
Lamb, Curr. Biol. 6, 427 (1996).
25. J. T. Greenberg, N. Yao, Cell. Microbiol. 6, 201 (2004).
26. F. Madeo et al., Mol. Cell 9, 911 (2002).
27. F. A. Hoeberichts, A. ten Have, E. J. Woltering, Planta
217, 517 (2003).
28. P. V. Bozhkov et al., Cell Death Differ. 11, 175 (2004).
29. We thank B. Baker (University of California and
USDAAgriculture Research Service) for donating a
binary vector with the TMV resistance gene N, D. C.
Baulcombe (John Innes Centre, UK) for donating a
Potato virus X vector (pgR107), and T. Kobayashi
(National Institute for Basic Biology, Japan) for his
help on pulsed-eld gel electrophoresis. Supported by
Core Research for Evolutional Science and Technolo-
gy (CREST) of the Japan Science and Technology
Corporation, and by Grants-in-Aid for Scientic Re-
search (no. 12138205) and for 21st Century Center
of Excellence Research, Kyoto University (A14), from
the Ministry of Education, Culture, Sports, Science
and Technology of Japan.
Supporting Online Material
www.sciencemag.org/cgi/content/full/305/5685/855/
DC1
Materials and Methods
SOM Text
Figs. S1 and S2
References and Notes
3 May 2004; accepted 9 July 2004
Structural Basis of Mitochondrial
Tethering by Mitofusin Complexes
Takumi Koshiba,
1
Scott A. Detmer,
1
Jens T. Kaiser,
2,3
Hsiuchen Chen,
1
J. Michael McCaffery,
4
David C. Chan
1*
Vesicle fusion involves vesicle tethering, docking, and membrane merger. We show
that mitofusin, an integral mitochondrial membrane protein, is required on adja-
cent mitochondria to mediate fusion, which indicates that mitofusin complexes act
in trans (that is, between adjacent mitochondria). Aheptad repeat region (HR2)
mediates mitofusin oligomerization by assembling a dimeric, antiparallel coiled coil.
The transmembrane segments are located at opposite ends of the 95 angstrom
coiled coil and provide a mechanism for organelle tethering. Consistent with this
proposal, truncated mitofusin, in an HR2-dependent manner, causes mitochondria
to become apposed with a uniform gap. Our results suggest that HR2 functions as
a mitochondrial tether before fusion.
Diverse membrane trafficking systems
including endoplasmic reticulumtoGolgi
transport, endosome fusion, Golgi-to-Golgi
fusion, and synaptic vesicle fusion use a
common set of steps to mediate the targeting
and fusion of intracellular vesicles to accep-
tor target membranes (1, 2). First, a vesicle
becomes tethered to its target membrane,
although the two membranes remain separat-
ed by a considerable gap. This step is often
mediated by the binding of activated Rab
guanosine triphosphatases (GTPases) on the
vesicle surface to effectors on the target
membrane (3). Second, a SNARE (soluble
N-ethylmaleimidesensitive factor attach-
ment protein receptor) protein on the teth-
ered vesicle surface forms a complex with
SNAREs on the target membrane; this
leads to closer apposition of membranes, a
state termed docking. Finally, the two
bilayers fuse, probably because of the close
proximity produced by SNARE complex
1
Division of Biology,
2
Division of Chemistry, California
Institute of Technology, 1200 East California Boulevard,
MC114-96, Pasadena, CA 91125, USA.
3
Howard Hughes
Medical Institute.
4
Integrated Imaging Center, Depart-
ment of Biology, Johns Hopkins University, 3400 North
Charles Street, Baltimore, MD 21218, USA.
*To whom correspondence should be addressed. E-
mail: dchan@caltech.edu
R
E P O R T S
6 AUGUST 2004 VOL 305 SCIENCE www.sciencemag.org
858 formation. Although much progress has been
made in understanding the structural basis of
docking and fusion by SNARE complexes (4,
5), less is understood about tethering, because
of difficulty in obtaining structures of the large
heterotypic protein complexes involved.
Mitochondria are dynamic organelles that
undergo continual cycles of fusion and fis-
sion, opposing processes that control the
overall morphology of the mitochondrial
population (68). Reduced mitochondrial fu-
sion causes greater autonomy for individual
organelles in the mitochondrial population, a
state that increases heterogeneity among or-
ganelles and results in dysfunction (9). Mito-
chondrial fusion is controlled by members of
the fuzzy onions (Fzo)/mitofusin (Mfn) fam-
ily of large GTPases that are localized to the
mitochondrial outer membrane (10). Mam-
mals h