T H E J O U R N A L O F C E L L B I O L O G Y
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 184 No. 1 129–141
Correspondence to Toshiya Endo: email@example.com
Y. Tamura ’ s present address is Dept. of Cell Biology, The Johns Hopkins University
School of Medicine, Baltimore, MD 21205.
H. Yamamoto ’ s present address is Dept. of Cell Biology, National Institute for
Basic Biology, Myodaiji, Okazaki 444-8585, Japan.
Abbreviations used in this paper: BPA, benzoylphenylalanine; DHFR, mouse di-
hydrofolate reductase; IMS, intermembrane space; MMC, mtHsp70-associated
motor and chaperone; mtHsp70, mitochondrial Hsp70; PK, proteinase K.
Normal eukaryotic cell functions rely on dedicated systems of
protein traffi c control that ensure correct assembly of specifi c
sets of proteins in each membrane-bounded compartment or or-
ganelle ( Schatz and Dobberstein, 1996 ). Mitochondria are two-
membrane bounded organelles consisting of ? 1,000 different
proteins, most of which are synthesized in the cytosol as precur-
sor proteins, imported into mitochondria, and sorted to one of
the four subcompartments, the outer membrane, intermembrane
space (IMS), inner membrane, and matrix. Protein import and
sorting are mediated by membrane protein assemblies called
translocators, including the TOM40 and TOB/SAM complexes
in the outer membrane and the TIM23 and TIM22 complexes in
the inner membrane ( Koehler, 2004 ; Kutik et al., 2007 ; Neupert
and Herrmann, 2007 ). These translocators are not tightly linked
to each other, but instead cooperate dynamically to pass precur-
sor proteins onto each other, thereby achieving precise as well as
effi cient protein delivery to their destinations ( Endo et al., 2003 ;
Kutik et al., 2007 ). The TOM40 complex is the entry site for
most mitochondrial proteins, and after translocation through the
TOM40 complex, protein sorting pathways branch out for differ-
ent intramitochondrial locations. Mitochondrial precursor pro-
teins with an N-terminal cleavable presequence use the TIM23
complex to reach the matrix or inner membrane. Presequence-
less polytopic membrane proteins including carrier proteins use
the TIM22 complex to be inserted into the inner membrane.
Translocation of presequence-containing proteins requires
cooperation of the TOM40 complex, TIM23 complex, and im-
port motor proteins in the matrix. Presequences are fi rst recog-
nized by the general import receptor Tom20 of the TOM40
complex on the mitochondrial surface, and then move through
the Tom40 channel to reach the trans site for presequence bind-
ing, which consists of Tom40, Tom22, and Tom7 on the IMS
side of the outer membrane ( Endo and Kohda, 2002 ; Esaki et al.,
2004 ). After crossing the outer membrane, precursor proteins
are transferred to the TIM23 complex and sorted to the matrix
or inner membrane. Transfer of presequence-containing proteins
plex translocates and inserts proteins into the mitochon-
drial inner membrane. Here we analyze the intermembrane
space (IMS) domains of Tim23 and Tim50, which are es-
sential subunits of the TIM23 complex, in these functions.
We fi nd that interactions of Tim23 and Tim50 in the IMS
facilitate transfer of precursor proteins from the TOM40
itochondrial protein traffi c requires coordinated
operation of protein translocator complexes in
the mitochondrial membrane. The TIM23 com-
complex, a general protein translocator in the outer mem-
brane, to the TIM23 complex. Tim23 – Tim50 interactions
also facilitate a late step of protein translocation across
the inner membrane by promoting motor functions of mi-
tochondrial Hsp70 in the matrix. Therefore, the Tim23 –
Tim50 pair coordinates the actions of the TOM40 and
TIM23 complexes together with motor proteins for mito-
chondrial protein import.
Tim23 – Tim50 pair coordinates functions of
translocators and motor proteins in mitochondrial
Yasushi Tamura , 1 Yoshihiro Harada , 1 Takuya Shiota , 1 Koji Yamano , 1 Kazuaki Watanabe , 1 Mihoko Yokota , 1
Hayashi Yamamoto , 1 Hiromi Sesaki , 2 and Toshiya Endo 1
1 Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
2 Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205
© 2009 Tamura et al. This article is distributed under the terms of an Attribution–
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tion date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
JCB • VOLUME 184 • NUMBER 1 • 2009 130
each other likely through their predicted coiled-coil regions
( Bauer et al., 1996 ; Geissler et al., 2002 ; Yamamoto et al., 2002 ).
To assess the roles of Tim23 – Tim50 interactions in the IMS, we
systematically introduced mutations in the predicted coiled coil
and its fl anking regions of Tim23 to disrupt its possible interac-
tions with Tim50 and tested their effects on yeast cell growth.
Among them, we observed growth defects for the yeast strains
that have Leu → Ser mutations at positions 64, 71, and/or 78 of
Tim23, which are expected to form a hydrophobic side of the
coiled coil for protein – protein interactions ( Fig. 1 A ). tim23
mutant strains with the L71S mutation ( tim23-71 ), but not L64S
( tim23-64 ) or L78S ( tim23-78 ) alone, showed growth defects at
elevated temperature ( Fig. 1 B ). The L78S mutation showed
synthetic growth defects with L71S ( tim23-71,78 ), and the
L64S mutation showed those with L71,78S ( tim23-64,71,78 )
( Fig. 1 B ), whereas the tim23 mutant with fi ve mutations at resi-
dues 60 – 64 ( tim23-60-64 ) did not cause any growth defects.
The L68S and L73S mutations also showed synthetic growth
defects with the L71,78S mutation ( tim23-68,71,78 and tim23-
71,73,78 in Fig. 1 B ). In contrast, although negative charges in
the IMS domain of Tim23 were suggested to be important for its
receptor function for positively charged presequences ( Bauer
et al., 1996 ; Komiya et al., 1998 ), the tim23 mutant with re-
placement of four negatively charged residues at positions 72,
74, 75, and 76 with positively charged Lys ( tim23-4K ) did not
cause obvious growth defects ( Fig. 1 B ).
Mitochondria with mutations in the IMS
domain of Tim23 are defective in protein
We conducted in vitro protein import into mitochondria with
Tim23 containing mutations at positions 64, 71, and/or 78.
The mitochondria isolated from the tim23-71,78 and tim23-
64,71,78 mutants, which show strong growth defects, exhib-
ited the normal levels of translocator components and of ? ? ,
which is essential for protein import via the TIM23 complexes
( Fig. 1, C and D ). We fi rst analyzed import of radiolabeled
matrix-targeted precursor proteins with an N-terminal prese-
quence, i.e., pSu9 – mouse dihydrofolate reductase (DHFR), a
fusion protein between the presequence of F 0 -ATPase subunit
9 and DHFR, a precursor to mitochondrial Hsp60 (pHsp60),
and pb 2 (167) ? 19-DHFR and pb 2 (220) ? 19-DHFR, the fi rst
167 and 220 residues of the yeast cytochrome b 2 precursor
with deletion of the 19-residue inner membrane – sorting sig-
nal, respectively, fused to DHFR. Import of those proteins via
the TIM23 complex with the aid of the import motor machin-
ery comprising mtHsp70 and MMC proteins was retarded
signifi cantly for tim23-71 , tim23-71,78 , and tim23-64,71,78
mitochondria or moderately for tim23-64 and tim23-78 mito-
chondria as compared with wild-type mito chondria ( Fig. 2
and Fig. S1 A, available at http://www.jcb.org/cgi/content/full/
We next analyzed import of inner membrane – sorted
proteins. Import of the presequence-containing fusion proteins
pb 2 (167)-DHFR and pb 2 (220)-DHFR, the fi rst 167 and 220 resi-
dues of the yeast cytochrome b 2 precursor fused to DHFR,
respectively, via the TIM23 complex was retarded for tim23-71 ,
from the trans site of the TOM40 complex to the TIM23 com-
plex was proposed to be facilitated by (a) the receptor functions
of the TIM23 complex specifi c for presequences ( Bauer et al.,
1996 ; Komiya et al., 1998 ; Yamamoto et al., 2002 ; Mokranjac
et al., 2003 ), (b) recruitment of the TIM23 complex to the mito-
chondrial contact sites, where the outer and inner membranes
are closely apposed and protein translocation across the two
membranes takes place ( Donzeau et al. 2000 ; Vogel et al., 2006 ),
and (c) recruitment of the TIM23 complex to the TOM40 com-
plex by their possible transient interactions ( Chacinska et al.,
2005 ; Mokranjac et al., 2005 ). Tim50 of the TIM23 complex
is the fi rst component to receive the translocating precursor
protein from the TOM40 complex at the inner membrane
( Yamamoto et al., 2002 ; Mokranjac et al., 2003 ).
The presequence is then translocated across the inner mem-
brane through the Tim23(-Tim17) channel, depending on the
membrane potential across the inner membrane ( ? ? ; Truscott
et al., 2001 ; Meinecke et al., 2006 ; Alder et al., 2008b ). Subsequent
translocation and unfolding of the matrix-targeted precursor pro-
tein is driven by the import motor mitochondrial Hsp70 (mtHsp70)
or Ssc1p in yeast, which binds to and dissociates from the incom-
ing polypeptide segment at the outlet of the TIM23 channel upon
ATP hydrolysis ( Neupert and Brunner, 2002 ; Yamano et al.,
2008 ). mtHsp70 functions in cooperation with partner proteins,
mtHsp70-associated motor and chaperone (MMC) proteins, that
facilitate protein import into the matrix. MMC proteins include
Tim44, Tim14/Pam18, Tim16/Pam16, and Pam17 as subunits of
the TIM23 complex and Yge1p/Mge1p and Tim15/Zim17/Hep1
in the matrix. Tim14 is a J-protein that forms a complex with a
J-like protein Tim16 and stimulates ATPase activity of mtHsp70.
Tim44 directly interacts with the translocating polypeptide and
both Tim44 and Pam17 ( van der Laan et al., 2005 ) link Tim14 –
Tim16 to the TIM23 complex. Translocation of the inner mem-
brane sorted proteins with a hydrophobic sorting signal after the
matrix-targeting signal in the presequence is arrested at the inner
membrane by the interactions of the sorting signal part with the
TIM23 complex and is released laterally into the inner membrane
( Glick et al., 1992 ; Esaki et al., 1999 ).
Although the IMS domains of Tim23 and Tim50 interact
with each other ( Geissler et al., 2002 ; Yamamoto et al., 2002 ;
Alder et al., 2008a ), precise roles of those interactions in mito-
chondrial protein import remain unclear. We thus generated yeast
strains with Tim23 or Tim50 mutants that would deteriorate
Tim23 – Tim50 interactions. Systematic analyses of those mutants
revealed that Tim23 – Tim50 interactions in the IMS are essential
for both the initial and late steps of protein translocation through
the TIM23 complex. On the basis of the obtained results, we pro-
pose a model for protein translocation across and insertion into
the inner membrane via the TIM23 complex in which the Tim23 –
Tim50 pair in the IMS plays central and multiple roles.
Mutations in the IMS domain of Tim23
cause defective cell growth
The IMS domains of both Tim23 and Tim50 (residues 1 – 101
and 133 – 476, respectively) are indispensable and interact with
131TIM23 – TIM50 IN MITOCHONDRIAL PROTEIN IMPORT • Tamura et al.
Mitochondria with mutations in the IMS
domain of Tim50 are defective in protein
We asked if mutations in the IMS domain of Tim50 that disrupt
possible interactions with Tim23 in turn affect cell growth and
protein import. On the basis of the analyses of Tim50 mutants
with a deletion of predicted coiled-coil regions, we found that
the coiled-coil regions 274 – 290 and 349 – 365 are important for
Tim50 functions (unpublished data). We thus made tim50 strains
with mutations L279,282,286S ( tim50-279,282,286 ) or A352S/
F355S ( tim50-352,355 ) ( Fig. 3 A ), and found that tim50-
279,282,286 cells, but not tim50-352,355 cells, showed strong
growth defects ( Fig. 3 B ). Mitochondria isolated from tim50-
279,282,286 cells exhibited the normal levels of translocator
components and of ? ? ( Fig. 3, C and D ).
We then conducted in vitro protein import into mitochon-
dria with Tim50 containing the L279,282,286S or A352S/F355S
mutation and obtained the results essentially similar to those for
the Tim23 IMS domain mutant mitochondria. Import rates of
matrix-targeted fusion proteins, pSu9-DHFR, pHsp60, pb 2 (167) ?
19-DHFR, and pb 2 (220) ? 19-DHFR, decreased for tim50-
279,282,286 mitochondria, but less signifi cantly for tim50-
352,355 mitochondria ( Fig. 3 E and Fig. S1 B). In contrast,
although import of inner membrane – sorted fusion proteins,
pb 2 (167)-DHFR and pb 2 (220)-DHFR, into tim50-279,282,286
mitochondria was retarded, they were imported into tim50-
352,355 mitochondria nearly as effi ciently as wild-type mito-
chondria ( Fig. 3 E and Fig. S1 B). Import of Tim23 or ADP/ATP
carrier (AAC) was not impaired by the L279,282,386S or
A352S/F355S mutation ( Fig. 3 E and Fig. S1 B).
Mutations in the coiled coils of Tim23 and
Tim50 impair their interactions in the IMS
We analyzed the interactions between the IMS domains of
Tim23 and Tim50 in the tim23 and tim50 mutant strains that
are defective in cell growth and protein import in detail. First,
we detected interactions of mutant Tim23 with wild-type
Tim50 by coimmunoprecipitation of digitonin-solubilized
mitochondria. The L71S mutation signifi cantly reduced the
amounts of Tim23 bound to Tim50FLAG and of Tim50 bound
to Tim23 in tim23-71 , tim23-71,78 , and tim23-64,71,78 mito-
chondria, whereas Tim23 bound to Tim50 was not reduced in
tim23-78 mitochondria or reduced partially in tim23-64 mito-
chondria ( Fig. 4, A and B ). We obtained essentially the similar
results by measuring binding of solubilized tim23 mutant mito-
chondria to the recombinant IMS domain of Tim50 ( Fig. 4 C )
and by performing glycerol density-gradient centrifugation of
solubilized tim23 mutant mitochondria (Fig. S2 A, available at
reversed-charge mutant ( tim23-4K ) resulted in only moderate
reduction of the amount of Tim50 bound to Tim23 (Fig. S2 B).
We then analyzed interactions of mutant Tim50 with wild-
type Tim23 by coimmunoprecipitation and found that the
L279,282,386S mutation reduced the amounts of Tim50 bound
to Tim23FLAG ( Fig. 4 D ), although glycerol density-gradient
centrifugation was not sensitive enough to detect such reduced
interactions (Fig. S2 C).
tim23-71,78 , and tim23-64,71,78 mitochondria, whereas they
were imported into tim23-64 and tim23-78 mitochondria as ef-
fi ciently as wild-type mitochondria ( Fig. 2 and Fig. S1). In con-
trast, import of presequenceless polytopic inner membrane
proteins, Tim23 and ADP/ATP carrier (AAC), via the TIM22
complex was not affected by the mutations of L64S, L71S,
L78S, L71,78S, or L64,71,78S in the IMS domain of Tim23
( Fig. 2 and Fig. S1 A). Therefore, import of presequence-
containing proteins via the TIM23 complex are impaired by the
mutations in the Tim23 IMS domain, although import defects
tend to be more pronounced for the matrix-targeted proteins
than for the inner membrane – sorted proteins.
Figure 1. Tim23 mutants with mutations in the coiled-coil region in the
IMS. (A) Amino acid sequences of the IMS domains of Tim23 mutants in
tim23 mutant strains (top) and coiled-coil regions in Tim23 (bottom) pre-
dicted by COILS (http://www.ch.embnet.org/software/COILS_form.html).
Mutation points are shown in black. (B) Serial dilutions of tim23 mutant
cells were plated on SCD ( ? Trp) and YPGlycerol and grown at the indi-
cated temperature for 2 and 3 d, respectively. (C) Mitochondria were iso-
lated from wild-type control (WT) and tim23 mutant cells grown at 30 ° C.
Indicated amounts of mitochondrial proteins were analyzed for indicated
proteins by SDS-PAGE followed by immunoblotting. (D) ? ? of wild-type
control (WT) and tim23 mitochondria measured by DiSC 3 (5) ( Sims et al.,
1974 ). mito., mitochondria; val., valinomycin.
JCB • VOLUME 184 • NUMBER 1 • 2009 132
of BPA at, e.g., position 71 of Tim23 did not appear to disrupt
the interaction with Tim50 completely. Cross-linked partners
involving Tim23 were assigned to the subunits of the TIM23
complex or Tim14 (Fig. S3 B). BPA at positions 64, 71, and 78
was cross-linked to Tim50 and Tim17 ( Fig. 4 E ), confi rming the
proximity of these residues to Tim50. BPA at positions 71 and
78 was also cross-linked to Tim21 and BPA at residue 64 to
Tim14 ( Fig. 4 E ), although Tim23 is not stoicheometrically
saturated by these proteins (unpublished data). Therefore, the
IMS domain of Tim23 is apparently in contact with multiple sub-
units of the TIM23 complex, yet coimmunoprecipitation with
To directly probe proteins interacting with the IMS do-
main of Tim23, we took the approach of site-specifi c photo-
cross-linking. We incorporated a photoreactive unnatural amino
acid, benzoylphenylalanine (BPA), into residue 64, 71, or 78 in
Tim23 in vitro by the suppressor tRNA method ( Kanamori et al.,
1997 , 1999 ). Tim23 with incorporated BPA was imported into
isolated mitochondria and subjected to UV irradiation to trigger
cross-linking of BPA with nearby proteins. Imported Tim23 was
assembled into the TIM23 complex correctly as judged from
blue native – PAGE analyses (Fig. S3 A, available at http://www
.jcb.org/cgi/content/full/jcb.200808068/DC1) and introduction
Figure 2. Tim23 IMS domain mutants impair import into the matrix and sorting to the inner membrane differently. Mitochondria were isolated from wild-
type control (WT) and tim23 mutant cells after cultivation in lactate medium at 30 ° C. Radiolabeled mitochondrial precursor proteins were incubated with
those mitochondria at 30 ° C for indicated times. For quantifi cation of the imported, protease-protected proteins, amounts of protease-resistant proteins in
wild-type mitochondria after the longest incubation time were set to 100%. p, precursor form; i, processing-intermediate form; m, mature form.
133 TIM23 – TIM50 IN MITOCHONDRIAL PROTEIN IMPORT • Tamura et al.
complex in such a way that the presequence reaches the IMS
side of the TOM40 complex, i.e., the trans site, whereas the
DHFR domain is bound to the Tom40 channel in an unfolded
state ( Kanamori et al., 1999 ). The amount of the bound interme-
diate was not sensitive to salt concentrations and was not af-
fected by the L64,71,78S or L279,282,286S mutation in the
IMS domain of Tim23 or Tim50, respectively ( Fig. 5 A ,
Binding). However, when we relieved the translocation arrest
by replenishment of ? ? , effi ciency in the chase into the matrix
was reduced by both mutations ( Fig. 5 A , Chase). These results
show that Tim23 – Tim50 interactions in the IMS affect the step
after accumulation of the precursor protein at the trans site of
the TOM40 complex.
tim23-64,71,78 mitochondria shows that mutations on the hydro-
phobic side of the coiled coil in Tim23 reduced the amount of
bound Tim50, but not of bound Tim21, Tim14, or Tim17 ( Fig. 4 F ).
Tim23 – Tim50 interactions in the IMS are
important for presequence transfer from
the TOM40 complex to the TIM23 complex
What are the roles of the coiled-coil interactions between Tim23
and Tim50 in the IMS in protein import? To address this ques-
tion, we performed two-step import using tim23 or tim50 mutant
mitochondria. In brief, we made use of an in vitro translocation
intermediate of pSu9-DHFR generated in the absence of ? ? at
high temperature. This intermediate is arrested at the TOM40
Figure 3. Tim50 mutants with mutations in the coiled-coil region in the IMS. (A) Amino acid sequences of the IMS domains of wild-type control (WT) and
mutant Tim50 (top) and coiled-coil regions in Tim50 (bottom) predicted as in Fig. 1 A . Mutation points are shown in black. (B) Serial dilutions of wild-type and
tim50 mutant cells were plated on SCD ( ? Trp) and YPGlycerol and grown at the indicated temperature for 2 and 3 d, respectively. (C) Mitochondria were iso-
lated from wild-type and tim50 mutant cells grown at 23 ° C in lactate medium. Indicated amounts of mitochondrial proteins were analyzed for indicated proteins
by SDS-PAGE followed by immunoblotting. (D) ? ? of wild-type and tim50-279,282,286 mitochondria measured as in Fig. 1 D . (E) Mitochondria were isolated
from wild-type and tim50 mutant cells after cultivation in lactate medium at 23 ° C. Radiolabeled precursor proteins were incubated with those mitochondria at
25 ° C for indicated times, and import reactions were analyzed as in Fig. 2 . p, precursor form; i, processing-intermediate form; m, mature form.
JCB • VOLUME 184 • NUMBER 1 • 2009 134
the TIM23 complex to the membrane contact sites and/or di-
rectly to the TOM40 complex. The former was proposed to in-
volve penetration of the N-terminal 50 residues of Tim23 into
the outer membrane ( Donzeau et al. 2000 ; Vogel et al., 2006 ;
Popov- Č eleketi ć et al., 2008 ), although such a role of Tim23
was questioned by others ( Chacinska et al., 2003 ). The latter
may be facilitated by direct interactions between the compo-
nents of the TIM23 complex, including Tim21, and those of the
TOM40 complex, including Tom22 ( Chacinska et al., 2005 ;
Mokranjac et al., 2005 ). We assessed possible effects of tim23
or tim50 mutations on the N-terminal interactions of Tim23
with the outer membrane by analyzing accessibility of the N
terminus of Tim23 to the externally added protease ( Donzeau
et al., 2000 ; Yamamoto et al., 2002 ). The mutations L71S,
L71,78S, and L64,71,78S in Tim23 and L279,282,286S and
A352S/F355S in Tim50 indeed reduced the susceptibility of
Tim23 to proteinase K (PK) added outside the mitochondria
( Fig. 5 C ). When we attached a folded protein A to the N termi-
nus of Tim23, the N-terminal clipping of Tim23 by added PK
was prevented (Fig. S4 A, available at http://www.jcb.org/cgi/
content/full/jcb.200808068/DC1) as in the case of N-terminal
50-residue deletion of Tim23 ( Donzeau et al., 2000 ). We then
made tim23 mutant strains expressing Tim23 with mutations
that deteriorate its interactions with Tim50 as well as the dele-
tion of the N-terminal 50 residues (Tim23 ? 50) or N-terminal
attachment of protein A (protein A – Tim23). Cells expressing
We then analyzed the effects of deteriorated Tim23 – Tim50
interactions on the step of presequence transfer from the trans
site to the TIM23 complex. Because a translocation intermediate
lodged at the TOM40 complex in the absence of ? ? is cross-
linked to Tim50 ( Yamamoto et al., 2002 ; Mokranjac et al., 2003 ),
we asked by cross-linking experiments if Tim50 could receive
the presequence from the trans site in tim23 or tim50 mutant
mitochondria. pSu9-DHFR with BPA at position 21 in the prese-
quence was accumulated at the TOM40 complex in the absence
of ? ? . Subsequent UV irradiation yielded cross-linked products
involving BPA at residue 21 with Tim50 as well as with Tom40
and Tom22 in wild-type mitochondria ( Fig. 5 B ). We found that
the amounts of the cross-linked products between pSu9-DHFR
and Tim50 signifi cantly decreased in mitochondria with the mu-
tation, L71S in Tim23 or L279,282,286S in Tim50 ( Fig. 5 B ).
These results indicate that the Tim23 – Tim50 interactions are im-
portant for effi cient transfer of the presequence from the trans
site of the TOM40 complex to Tim50, which likely refl ects the
Tim23-aided receptor ability of Tim50 for presequences as the
import defects of tim23-71 mitochondria were not resumed after
rupturing the outer membrane (Fig. S3 C).
Tim23 and Tim50 physically link the TIM23
complex to the TOM40 complex
The effi cient presequence transfer from the TOM40 complex to
the TIM23 complex may be related to effi cient recruitment of
Figure 4. Tim23 and Tim50 IMS domain mutants are defective in Tim23 – Tim50 interactions. (A) Wild-type control (WT) and tim23 mutant mitochondria
with FLAG-tagged Tim50 were solubilized with 1% digitonin and subjected to immunoprecipitation with the anti-FLAG antibody. 5%, 5% of the loaded
proteins; F, 10% of unbound proteins; E, 100% of proteins eluted with 100 mM glycine-HCl, pH 2.5. (B) Immunoprecipitation was performed as in A
(Tim50 is not FLAG tagged) using anti-Tim23 antibodies. (C) wild-type, tim23-64 , tim23-71 , tim23-78 , tim23-71,78 , and tim23-64,71,78 mitochondria
were solubilized with 0.5% Triton X-100 and subjected to incubation with Ni-NTA agarose loaded with purifi ed recombinant Tim50 IMS. Bound proteins
were eluted with 500 mM imidazole and analyzed by SDS-PAGE and immunoblotting with anti-Tim23 antibodies. 5%, 5% of the loaded proteins; FT, 10%
of unbound proteins; W2-W3, 100% of washed fractions; E, 100% of eluted proteins. (D) Wild-type and tim50 mutant mitochondria with FLAG-tagged
Tim23 were solubilized with 1% digitonin and subjected to immunoprecipitation with the anti-FLAG antibody as in A. 15%, 15% of the loaded proteins;
F, 10% of unbound proteins; E, 100% of eluted proteins. (E) Radiolabeled Tim23 with BPA incorporated at position 64, 71, or 78 was imported into mito-
chondria (W303-1A) at 25 ° C for 1 h and subjected to UV irradiation. Identifi ed cross-linked partners are indicated. (F) Mitochondria with the FLAG-tagged
version of Tim23 (WT) or L64,71,78S Tim23 ( tim23-64,71,78 ) were solubilized with 1% digitonin and subjected to immunoprecipitation as in A. Error
bars represent SDs from three independent experiments.
135 TIM23 – TIM50 IN MITOCHONDRIAL PROTEIN IMPORT • Tamura et al.
Figure 5. Roles of Tim23 – Tim50 interactions in the IMS in the early stage of protein translocation across the inner membrane. (A, Binding) Radiolabeled
pSu9-DHFR was incubated with CCCP-treated mitochondria isolated from wild-type control (WT), tim23 mutant, and tim50 mutant cells for 15 min at 30 ° C.
Mitochondria were then washed with buffer containing indicated concentration of KCl, and bound proteins were analyzed by SDS-PAGE and radioimaging.
(Chase) Mitochondria after binding of pSu9-DHFR in the presence of 100 mM KCl were incubated in chase buffer for indicated times (without following
PK treatment). Proteins were analyzed by SDS-PAGE and radioimaging, and the chased, mature form was quantifi ed. C, bound pSu9-DHFR (100%); p,
precursor form; m, mature form. (B) Radiolabeled pSu9-DHFR containing BPA at position 21 was bound to wild-type control (WT), tim23 mutant, and tim50
mutant mitochondria in SM buffer with 10 mM KCl, 5 mM MgCl 2 , and 2 mM methionine with 10 mg/ml valinomycin at 30 ° C for 15 min. Mitochondria
were washed with SM buffer containing 150 mM KCl and kept on ice with (UV+) or without (UV ? ) UV irradiation in the same buffer for 5 min. The UV-
irradiated mitochondria were solubilized and subjected to immunoprecipitation with anti-Tim50 antibodies (+IP), and proteins were analyzed by SDS-PAGE
and radioimaging. Closed and open triangles indicate cross-linked products with Tim50 and Tom22, respectively. (C) Mitochondria isolated from wild-type,
tim23 mutant, tim21 ? , and tim50 mutant cells were treated with 500 μ g/ml PK for 20 min at 4 or 16 ° C. After stopping the reaction with 1 mM PMSF, the
mitochondria were reisolated and proteins were analyzed by SDS-PAGE and immunoblotting with antibodies against Tim54 (with a domain exposed to
the IMS), Tom20 (with a domain exposed to the cytosol), and Tim23. Total amounts of Tim23 and Tim23*, a protease-resistant fragment, are set to 100%.
(D) Mitochondria isolated from indicated cells expressing Tim23FLAG with BPA at position 41 after UV irradiation (UV+) or mock treatment (UV ? ) were
analyzed by SDS-PAGE and immunoblotting with the anti-FLAG antibody (left) or were subjected to immunoprecipitation with the anti-FLAG antibody, and
Tom22 was detected by anti-Tom22 antibodies (right). (E) C-terminally (His) 10 -tagged Tom22 with BPA at position 132, 134, or 136 was expressed in cells
with either wild-type (W) or C-terminally FLAG-tagged (F) Tim50 and purifi ed with Ni-NTA resin after UV irradiation (UV+) or mock treatment (UV ? ). The
purifi ed proteins were analyzed by SDS-PAGE and immunoblotting with anti-Tom22 antibodies. The weak bands above the cross-linked products (denoted
with a closed triangle) are nonspecifi c bands. (F) Cell extracts were prepared from TIM50FLAG/TOM22-BPA-X (X = 132, 134, and 136) and were sub-
jected to immunoprecipitation with the antibody against the FLAG epitope tag attached to the C terminus of Tim50. The purifi ed proteins were analyzed
by SDS-PAGE and immunoblotting with anti-Tim50 antibodies (left) and anti-Tom22 antibodies (right). (G) Tom22-(His) 10 with BPA at position 132 or 136
was expressed in tim23-71 , tim50-279,282,286 , or their corresponding wild-type cells and purifi ed with Ni-NTA resin after UV irradiation (UV+) or mock
treatment (UV ? ). Cross-linked products between Tom22 and Tim50 were detected by immunoblotting with anti-Tom22 antibodies and quantifi ed with
normalization by the total amount of authentic Tom22 plus Tom22-(His) 10 . Amounts of the cross-linked products in corresponding wild-type cells were set to
100%. Error bars represent SDs from three independent experiments.
JCB • VOLUME 184 • NUMBER 1 • 2009 136
Tim23 – Tim50 interactions in the IMS
facilitate the late step of protein
translocation across the inner membrane
After presequence transfer from the TOM40 complex to
Tim50, the precursor protein guided by the presequence has to
enter the TIM23 channel. Gating of the TIM23 channel was
proposed to involve Tim50-mediated Tim23 dimerization
( Meinecke et al., 2006 ), and formation of the Tim23 dimer can
be monitored by cross-linking of Tim23 ( Bauer et al., 1996 ;
Alder et al., 2008a ) or by affi nity copurifi cation of Tim23
( Meinecke et al., 2006 ). However unexpectedly, although we
confi rmed that disuccinimidylglutarate generated a Tim23
dimer as a 50-kD cross-linked product ( Fig. 6 A ), the amount
of the cross-linked product did not change with the mutations
of L64S, L71S, L71,78S, or L64,71,78S in Tim23 ( Fig. 6 A ).
We also found that imported Tim23, which was confi rmed by
blue native – PAGE to be correctly assembled into the TIM23
complex (not depicted), was coimmunoprecipitated with pre-
existing FLAG-tagged Tim23 ( Fig. 6 B ), yet the amounts of
imported and coimmunoprecipitated Tim23 did not change
with the L279,282,286S mutation in Tim50 ( Fig. 6 B ). Dis-
rupted or weakened interactions between Tim23 and Tim50 in
the IMS did not lead to reduced ? ? ( Fig. 1 D and Fig. 3 D ) or
impaired permeability barrier of the inner membrane, as well.
These results suggest that the Tim23 – Tim50 interactions in the
IMS do not affect the dimer formation of Tim23, highlighting
the apparently different effects of the Tim50 IMS domain be-
tween intact mitochondria and the reconstituted Tim23 system
( Meinecke et al., 2006 ).
The Tim23 – Tim50 interactions affect the matrix import
and inner membrane sorting differently, the matrix import being
more sensitive to the deteriorated Tim23 – Tim50 interactions than
the inner membrane sorting ( Fig. 2, Fig. 3 E , and Fig. S1). These
results suggest the possibility that, because matrix import requires
multiple turnover of mtHsp70 as an import motor, motor func-
tions of mtHsp70 in the matrix may be affected by the defects in
Tim23 – Tim50 interactions in the IMS. To assess the motor func-
tion of mtHsp70, we took advantage of the fact that the DHFR
domain stabilized by its ligand methotrexate cannot go across the
outer membrane, but instead forms a two-membrane – spanning
intermediate that is closely apposed to the outer membrane by
the motor function of mtHsp70. Such close apposition of the
DHFR domain to the outer membrane is refl ected in its resistance
against externally added protease ( Schwarz et al., 1993 ; Voisine
et al., 1999 ). The presequence-cleaved mature form of the metho-
trexate-bound intermediate of pb 2 (220) ? 19-DHFR was PK resis-
tant in wild-type mitochondria whereas it became PK sensitive in
tim23-64 and tim23-78 mitochondria ( Fig. 6 C ). It should be
noted that motor functions of mtHsp70 in tim23-71 mitochondria
was so defective that two-membrane – spanning intermediate could
not be generated. Therefore the Tim23 mutations with defective
Tim23 – Tim50 interactions in the IMS apparently impair the
motor function of mtHsp70 in the matrix.
To gain more insight into the impaired motor functions of
mtHsp70 caused by deteriorate Tim23 – Tim50 interactions, we
analyzed the effects of overexpression of mtHsp70 on the tim23
or tim50 mutations. Overexpression of mtHsp70 from a multicopy
Tim23 ? 50 and protein A – Tim23 did not show signifi cant growth
defects at any temperature (Fig. S4, B and C). However, when
combined with deteriorated Tim23 – Tim50 interactions, the
N-terminal 50-residue deletion of Tim23 in tim23-64,71,78 ? 50
and tim23-71,78 ? 50 strains and protein A attachment to Tim23
in ProteinA - tim23-71 and ProteinA - tim23-71,78 strains re-
sulted in slower cell growth at elevated temperature than in cells
expressing wild-type, full-length Tim23 (Fig. S4, B and C). Al-
though tim23-64 or tim23-78 strains do not show obvious growth
defects, even in combination with the N-terminal 50-residue de-
letion ( Fig. 1 C and Fig. S4 B), attachment of protein A to ex-
pressed Tim23 with a mutation of L64S or L78S now exhibits
synthetic growth defects (Fig. S4 C, bottom). These results sug-
gest that the Tim23 – Tim50 interactions facilitate the dynamic
interactions of the N terminus of Tim23 with the outer mem-
brane and/or TOM40 complex, which is important for vital cell
growth, rather than mere stabilization of the interaction of Tim23
with the outer membrane.
We then probed possible transient interactions, if any, be-
tween the TIM23 and TOM40 complex, which would facilitate
recruitment of the TIM23 complex to the TOM40 complex at
contact sites, by site-specifi c photo-cross-linking in vivo ( Chin
et al., 2003 ). First, the TIM23FLAG gene with an amber codon
for residue 41 of Tim23 was expressed in the yeast strain that
contains an orthogonal pair of amber suppressor tRNA and its
cognate aminoacyl-tRNA synthetase specifi c for BPA and UV
irradiated. FLAG-tagged Tim23 and its cross-linked products
were affi nity purifi ed and analyzed by SDS-PAGE. Tim23 with
BPA at residue 41 generated a 50-kD cross-linked product,
whose cross-linked partner was identifi ed as Tom22 by immuno-
blotting with anti-Tom22 antibodies ( Fig. 5 D ) and by immuno-
precipitation with anti-Tom22 antibodies followed by detection
with the anti-FLAG antibody (not depicted). Because the 50-kD
cross-linked product was not observed in mitochondria with
Tom22 lacking the IMS domain ( Fig. 5 D , tom22 ? C ), residue
41 of Tim23 interacts with the IMS domain of Tom22. Second,
when the TOM22HIS10 gene with an amber codon for resi-
due 132, 134, or 136 in the IMS domain of Tom22 was ex-
pressed in the same system, UV irradiation led to generation of
90-kD cross-linked products ( Fig. 5 E , lanes 3, 7, and 11). These
cross-linked products were found to involve Tim50 because
they were shifted to 100 kD by attachment of the FLAG tag to
Tim50 ( Fig. 5 E , lanes 4, 8, and 12) and were detected by anti-
Tom22 antibodies after affi nity purifi cation with the antibody
against the FLAG epitope tag attached to the C terminus of
Tim50 ( Fig. 5 F , right). Thus, Tim23 and Tim50 are the fi rst
TIM23 components that can be cross-linked to the intact TOM40
complex even in the absence of translocating precursor proteins.
Although the amounts of the cross-linked products between
Tim23 and Tom22 were not affected by the L71S mutation in
the Tim23 IMS domain ( Fig. 5 D ), those of the cross-linked prod-
ucts between Tim50 and Tom22 moderately increased with the
mutation L71S in Tim23 or L279,282,286S in Tim50 ( Fig. 5 G ).
Therefore, proper Tim23 – Tim50 interactions facilitate dissocia-
tion of Tim50 from Tom22, likely resulting in effi cient transfer
of the presequence from the trans site of the TOM40 complex
137 TIM23 – TIM50 IN MITOCHONDRIAL PROTEIN IMPORT • Tamura et al.
fi rst step, the presequences of both matrix-targeted and inner
membrane – sorted precursor proteins are transferred from the
trans site of the TOM40 complex to Tim50 of the TIM23 com-
plex ( Fig. 7 , 1 → 2). In the second step, the presequence enters
the TIM23 channel in a ? ? -dependent manner ( Fig. 7 , 2 → 3).
This step also requires initial trapping of the presequence by the
ATP-dependent motor mtHsp70 if translocation of the pre-
sequence through the TIM23 channel requires unfolding of the
tightly folded passenger domain outside the mitochondria. In
the third step, translocation of matrix-targeted proteins across
the inner membrane further requires the motor function of
mtHsp70 (and MMC proteins), which undergoes an ATP-dependent
reaction cycle of holding on and off the incoming unfolded seg-
ment of precursor polypeptides at the outlet of the TIM23 chan-
nel ( Fig. 7 , 3 → 4-1), whereas insertion of inner membrane – sorted
proteins into the inner membrane is independent of the function
of mtHsp70 ( Fig. 7 , 3 → 4-2).
Here we found that the N-terminal region of Tim23 and
the IMS domain of Tim50 directly interact with Tom22 in the
absence of substrate precursor proteins. The Tim23 – Tom22 and
Tim50 – Tom22 interactions may lead to effi cient coupling of
protein translocation through the TOM40 complex and TIM23
plasmid caused synthetic growth defects with tim23-64,71,78
or tim50-279,282,286 mutants ( Fig. 6 D , right compared with
left panels for the controls). This fi nding is in contrast to the
cases of overexpression of other components of the TIM23
complex and MMC proteins; overexpression of Tim50 and
Tim17 partly suppress the growth defects of the tim23-64,71,78
mutant at elevated temperature ( Fig. 6 E ). These results suggest
that the Tim23 – Tim50 interactions in the IMS may thus facili-
tate turnover of mtHsp70 as an import motor; mtHsp70 over-
expression will shift the equilibrium of mtHsp70 from the free
state toward the TIM23 complex – bound state, thereby leading
to synthetic growth defects with Tim23 – Tim50 interaction mu-
tants. Interestingly, we found synthetic growth defects of the
coiled-coil mutants of Tim23 or Tim50 with deletion of the
PAM17 gene ( Fig. 6 F ). This suggests that Pam17 also facili-
tates the Tim23 – Tim50 – mediated activation of the motor func-
tions of mtHsp70.
Protein translocation across or insertion into the inner mem-
brane can be dissected into three distinct steps ( Fig. 7 ). In the
Figure 6. Roles of Tim23 – Tim50 interactions
in the IMS in the late step of protein transloca-
tion across the inner membrane. (A) Mitochon-
dria isolated from wild-type control (WT), in
which Tim23 is supplied from the plasmid, and
tim23 mutant cells were incubated in import
buffer containing 0.05% BSA and 75 mM di-
succinimidylglutarate on ice for 30 min. After
quenching the reaction with 50 mM Tris-HCl,
pH 7.4, Tim23 dimer formation was analyzed
by SDS-PAGE followed by immunoblotting with
anti-Tim23 antibodies. The amount of the Tim23
dimer in wild-type mitochondria was set to
100%. (B) Radiolabeled Tim23 was imported
into wild-type mitochondria (WT) with or with-
out Tim23FLAG and into tim50-279,282,286
mitochondria (T) with Tim23FLAG for 20 min
at 25 ° C and PK treated. The mitochondria was
solubilized with 1.0% digitonin and subjected
to coimmunoprecipitation with the anti-FLAG
antibody. Proteins were analyzed by SDS-PAGE
followed by radioimaging and immunoblotting
with indicated antibodies before and after
import. (C) Radiolabeled pb 2 (220) ? 19-DHFR
was incubated with mitochondria for 20 min
at 30 ° C in the presence of 10 mM methotrex-
ate and 100 mM NADPH. The mitochondria
were treated with 20 mg/ml PK for 20 min on
ice. PK treatment was stopped by addition of
1 mM PMSF, and the mitochondria were re-
isolated by centrifugation. Proteins were ana-
lyzed by SDS-PAGE and radioimaging (top).
The amounts of the PK-resistant processed
intermediate (i) were quantifi ed (bottom). The
amounts of the processed intermediate without
PK treatment are set to 100%. p, precursor
form. (D) Serial dilutions of wild-type, tim23 ,
and tim50 mutant strains carrying yeast 2 μ
plasmid, pYO326-TIM23, and pYO325-SSC1
were plated on SD ( ? Ura, ? Trp, ? Leu; left)
and SD ( ? Trp, ? Leu, and +1 mg/ml 5 ? -fl uoroorotic acid; right) and grown for 4 d at 23 ° C. (E) Serial dilutions of tim23-64,71,78 carrying yeast 2 μ plasmid
(pYO326) encoding indicated genes were plated on YPD plates and grown for 2 d at 30 or 35 ° C. (F) Serial dilutions of tim23 and tim50 mutant strains
lacking the PAM17 gene with plasmid, pRS316-Tim23 Δ 50, and pRS316-Tim50, respectively, were plated on a SCD ( ? Trp and +FOA) plate. Error bars
represent SDs from three independent experiments.
JCB • VOLUME 184 • NUMBER 1 • 2009 138
L279,282,286S > A352S/F355S. The normal Tim23 – Tim50
interactions in the IMS appear to be important for the fi rst step of
the protein translocation because they facilitate effi cient transfer
of translocating proteins from the TOM40 complex to the TIM23
complex through transient interactions between the presequence
and Tim50 ( Fig. 5 A ). Defective import of not only matrix-
targeted proteins but also inner membrane – sorted proteins were
observed for mitochondria containing the L71S mutation, but
not the L64S or L78S mutation ( Fig. 2 ); this suggests that the
effi cient protein transfer from the TOM40 to the TIM23 complex
are impaired only when Tim23 – Tim50 interactions are signifi -
cantly disrupted. Because defects in Tim23 – Tim50 interactions
lead to the increased interaction between Tom22 and Tim50
( Fig. 5 G ), Tom22 and presequences may compete with each other
in interactions with Tim50. Proper Tim23 – Tim50 interactions
may well optimize the Tim50 – Tom22 interactions so that Tim50
can stay close to Tom22 yet the presequence can effi ciently sub-
stitute Tom22 to occupy Tim50 as well ( Fig. 7 , 1 → 2).
It was unexpected that changes in the Tim23 – Tim50 inter-
actions in the IMS did not affect the Tim23 dimer formation,
which was proposed to refl ect the channel closure, but instead
complex. Although a similar function was suggested for Tim21
as well, interactions between Tim21 and Tom22 were found
only after solubilization of mitochondrial membranes ( Chacinska
et al., 2005 ; Mokranjac et al., 2005 ) or only for high concentra-
tions of purifi ed recombinant proteins ( Albrecht et al., 2006 ),
but not in intact mitochondria ( Chacinska et al., 2005 ; Mokranjac
et al., 2005 ). We found here that the N-terminal interaction of
Tim23 with the outer membrane ( Fig. 5 C ) or the contact be-
tween Tom22 and Tim23 ( Fig. 5 D ) was not altered by the
absence of Tim21 and that depletion of Tim21 did not show
synthetic growth defects with the tim23 or tim50 mutants (Fig. S4,
D and E). Besides, the amount of the cross-linked product of the
translocation intermediate at the TOM40 complex with Tim50
was not affected by depletion of Tim21 or by wash with 150
mM KCl (unpublished data), which abolishes interactions be-
tween Tim21 and Tom22 ( Albrecht et al., 2006 ). Therefore,
Tim21 does not appear to play a primary role in linking the
TOM40 and TIM23 complexes.
Defects in the Tim23 – Tim50 interactions caused by muta-
tions in the Tim23 IMS domain are in the order of L71S > L64S
> > L78S and those in the Tim50 IMS domain in the order of
Figure 7. A model of protein translocation across and insertion into the inner membrane via the TIM23 complex. Note that the interactions among some
of the components are dynamic rather than static.
139 TIM23 – TIM50 IN MITOCHONDRIAL PROTEIN IMPORT • Tamura et al.
gests that the TIM23 complex is in the two distinct states with
or without activation for the successive action of the import
motor mtHsp70 ( Fig. 7 ). These two states of the TIM23 com-
plex, the TIM23 complexes for motor activation (TIM23 A ) and
for motor resting (TIM23 R ), may contain mtHsp70 and MMC
proteins, but differ in their abilities to allow mtHsp70 to per-
form multiple rounds of holding on and off the incoming un-
folded segments of translocating proteins. Conversion of the
TIM23 R complex to the TIM23 A complex is caused by the
Tim23 – Tim50 interactions in the IMS, likely responding to
the presence of precursor proteins in transit through the Tim23
channel, and is facilitated by Pam17. In other words, the IMS
domains of Tim23 and Tim50 could monitor the presence of
substrate polypeptide chains delivered from the TOM40 com-
plex and transmit this information to the matrix side of the
TIM23 complex, perhaps through Pam17, for on-demand acti-
vation of the motor functions without unnecessary idling. Evi-
dently, future studies need to reveal the structural basis of the
difference and of a switching mechanism between the TIM23 R
and TIM23 A states.
Materials and methods
Plasmids and yeast strains
Yeast strains, plasmids, and primers used in this study are described
in Table S1 (available at http://www.jcb.org/cgi/content/full/jcb
.200808068/DC1). Gene disruption of the TIM23 or TIM21 gene and
FLAG tagging of the TIM23 gene were performed by homologous recom-
bination using the PCR-mediated gene cassette. Introduction of the point
mutations and amber codon into the TIM23 , TIM50 , or TOM22 gene were
performed by the overlap extension method using two pairs of primers or
Cells were grown in YPD (1% yeast extract, 2% polypeptone, and 2% glu-
cose), SD (0.67% yeast nitrogen base without amino acids and 2% glu-
cose), SCD (0.67% yeast nitrogen base without amino acids, 0.5%
casamino acid, and 2% glucose), or lactate (0.3% yeast extract, 0.1%
glucose, 0.05% CaCl 2 -H 2 O, 0.05% NaCl, 0.06% MgCl 2 -6H 2 0, 0.1%
KH 2 PO 4 , and 2% lactic acid, pH 5.6) media with appropriate supple-
ments. Cells that have the kanMX4 gene were selected on YPD + G418
(1% yeast extract, 2% polypeptone, 2% glucose, and 500 μ g/ml G418
sulfate). For plasmid shuffl ing to eliminate the URA3 -containing plasmid,
cells were grown on SCD ( ? Trp and +1 mg/ml 5 ? -fl uoroorotic acid). For
expression of BPA-containing Tim23FLAG, cells were grown in SD ( ? Trp,
? Leu, and +1 mM BPA). Cells were grown in SD ( ? Trp, ? Leu, and +1 mM
BPA) for expression of Tim23FLAG with BPA and in SGal ( ? Trp, ? Leu, and
+1 mM BPA) or SGal ( ? Trp, ? Leu, ? Ade, and +1 mM BPA) for expression
of Tom22 with BPA.
Mitochondria were isolated from W303-1A, tim21 ? , and tim23 mutant
strains grown in lactate medium at 30 ° C and tim50 mutant strains grown
in lactate medium at 23 ° C. Radiolabeled precursor proteins were synthe-
sized with rabbit reticulocyte lysate by coupled transcription/translation in
the presence of [ 35 S]methionine. Mitochondria isolated from W303-1A,
tim21 ? , and tim23 and tim50 mutant cells were incubated with radio-
labeled precursor proteins in import buffer (250 mM sucrose, 10 mM MOPS-
KOH, pH 7.2, 80 mM KCl, 2 mM KPi, 2 mM methionine, 5 mM dithiothreitol,
5 mM MgCl 2 , 2 mM ATP, 2 mM NADH, and 1% BSA) at 30 or 25 ° C. The
import reaction was stopped by addition of 10 μ g/ml valinomycin. Prote-
ase treatment was performed by incubating the mitochondria with 100
μ g/ml PK for 20 min on ice, which was inactivated by subsequent addition
of 1 mM PMSF. The mitochondria were isolated by centrifugation, and pro-
teins were analyzed by SDS-PAGE and radioimaging with a Storm 860
image analyzer (GE Healthcare) or Pharos FX Plus Molecular Imager (Bio-
Rad Laboratories). Two-step import of pSu9-DHFR was performed essen-
tially as described in Kanamori et al. (1999) . In brief, radiolabeled
affected the third step of the translocation of matrix-targeted
proteins across the inner membrane. Defective motor functions
of mtHsp70 usually cause retarded import of not only matrix-
targeted proteins but also the inner membrane – sorted protein
such as pb 2 (220)-DHFR, but not pb 2 (167)-DHFR ( Voos et al.,
1993 ). This is because pb 2 (220)-DHFR, not pb 2 (167)-DHFR,
contains a tightly folded heme-binding domain (residues 81 –
181) downstream of the presequence (residues 1 – 80) and re-
quires mtHsp70 for the second step or initial translocation of
the N terminus of the presequence across the inner membrane
( Glick et al., 1993 ). However, the effects of mutations L64S and
L78S on the import rates of pb 2 (220)-DHFR and pb 2 (167)-
DHFR do not markedly differ ( Fig. 2 ), suggesting that the
Tim23 – Tim50 interactions do not affect binding of mtHsp70 in
the second step of the TIM23 complex-mediated translocation.
Rather, the Tim23 – Tim50 interactions increase effi ciency of
mtHsp70 in the reaction cycle of binding to and release from the
translocating protein in the third step of the inner membrane
translocation. In other words, the Tim23 – Tim50 interactions in
the IMS facilitate turnover of mtHsp70 at the outlet of the
TIM23 channel in the matrix ( Fig. 7 , 4-1). This interpretation
was supported by the observation that overexpression of
mtHsp70, which would hamper the mtHsp70 turnover, nega-
tively affects the cell growth of tim23 and tim50 mutants ( Fig. 6
D ). It is to be noted that depletion of Tim50 leads to defects in
the fi rst and third steps of the TIM23 complex-mediated trans-
location ( Geissler et al., 2002 ) like the tim23 mutants with L64S
or L78S mutation rather than L71S mutation. Besides, the
tim17-5 ( Chacinska et al., 2005 ) and tim23-76 mutant ( van der
Laan et al., 2007 ) were reported, which showed similar pheno-
types, i.e., defects in only the third step of the TIM23 complex-
mediated translocation ( Chacinska et al., 2005 ). However, the
defects in the motor function of mtHsp70 observed here are not
caused by the lack of MMC proteins (Tim14 and Tim16) in the
TIM23 complex because tim23-64,71,78 and tim50-279,282,286
mitochondria contain normal amounts of Tim14 and Tim16
( Fig. 4, C and E ). Although we cannot rule out the possibility
that the observed defects in motor functions are indirect conse-
quences of the growth defects, a more likely explanation is that
tertiary and/or quaternary structural changes of the TIM23 com-
plex caused by defective Tim23 – Tim50 interactions impaired
the motor functions.
Chacinska et al. (2005) proposed that the TIM23 com-
plex is in the equilibrium between the matrix translocation
complex containing mtHsp70 and MMC proteins, but not
Tim21, and the inner membrane – sorting complex containing
Tim21 but not mtHsp70 or MMC proteins. However, this
model was recently challenged by the observations that Tim21
was coimmunoprecipitated with MMC proteins ( Tamura et al.,
2006 ; Popov- Č eleketi ć et al., 2008 ). The previous observation
that Tim21 was mutually exclusive with MMC proteins as a
constituent of the TIM23 complex ( Chacinska et al., 2005 )
may be explained by the fi ndings that Tim21 and MMC pro-
teins occupy only a fraction of the TIM23 core complex
(Tim23 and Tim17) (unpublished data) and/or may be because
of the artifact of using the protein A fusion protein of Tim21
( Popov- Č eleketi ć et al., 2008 ). The present study instead sug-
JCB • VOLUME 184 • NUMBER 1 • 2009 140
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pSu9-DHFR was incubated with mitochondria pretreated with 10 μ M CCCP
for 15 min at 30 ° C. Samples were diluted 11-fold with ice-cold SM buffer
(250 mM sucrose and 10 mM MOPS-KOH, pH 7.2) containing different
concentrations of KCl and incubated for 5 min on ice. The mitochondria
were reisolated and used for chase experiments or analyzed by SDS-PAGE
and radioimaging. For chase reactions, mitochondria were resuspended
with chase buffer (250 mM sucrose, 10 mM MOPS-KOH, pH 7.2, 10 mM
KCl, 5 mM MgCl 2 , 10 mM DDT, 2% bovine serum albumin, 2 mM ATP, 2
mM KPi, 5 mM sodium malate, and 2 mM NADH) and incubated for indi-
cated times at 25 ° C.
In vivo cross-linking
The plasmids for in vivo BPA cross-linking were provided by P.G. Schultz
(The Genomics Institute of the Novartis Research Foundation, San Diego,
CA). GAL-TIM23/TIM23FLAG-BPA41 and its derivative cells were grown
in SGal ( ? Trp and ? Leu) to the saturated phase. To express Tim23FLAG
containing BPA and to suppress expression of endogenous Tim23, cells
were transferred to SD ( ? Trp, ? Leu, +1 mM BPA) and grown to the log
phase. TOM22HIS10-BPA-X and its derivative cells were grown in SD
( ? Trp, ? Leu, and ? Ade) to the saturated phase. For overexpression of
Tom22-(His) 10 containing BPA, cells were transferred to SGal ( ? Trp,
? Leu, ? Ade, and +1 mM BPA) and grown to the log phase. BPA
(BACHEM) was added to appropriate media heated at ? 60 ° C from 1 M
of stock solution in 1 M NaOH. During cultivations, cells were kept in the
dark to prevent BPA from cross-linking reactions. Yeast cells were har-
vested, resuspended in water to 1.0 OD 600 cells/ml, and divided into
halves. One half was UV irradiated for 10 min at room temperature at a
distance of 5 cm from a 365-nm UV lamp (22,000 μ W/cm 2 ; B-100AP;
UVP) and the other half was kept on ice.
Constructions of plasmids and yeast strains and yeast cell growth conditions
are described in the online supplemental material. A binding assay using
immobilized recombinant Tim50-IMS protein was performed as described
previously ( Geissler et al., 2002 ). Site-specifi c in vitro photo-cross-linking
and immunoprecipitation were performed as described previously ( Kanamori
et al., 1999 ; Tamura et al., 2006 ). Immunoblotting was quantifi ed with a
Storm 860 image analyzer or Pharos FX Plus Molecular Imager.
Online supplemental material
Fig. S1 shows the quantifi cation of the import rates of various mitochon-
drial proteins into the tim23 or tim50 mutant mitochondria with defective
Tim23 – Tim50 interactions. Fig. S2 shows the results of glycerol density
gradient centrifugation for the TIM23 complex structures of the tim23 mu-
tant mitochondria with defective Tim23 – Tim50 interactions. Fig. S3 shows
the assignments of the cross-linked partners for Tim23 generated by site-
specifi c photo-cross-linking. Fig. S4 shows that Tim23 – Tim50 interactions
facilitate dynamic assembly of the N terminus of Tim23 into the outer
membrane. Table S1 shows the mutation points of the tim23 mutants and
sequences of the primers to construct various yeast mutant strains used in
this study. Online supplemental material is available at http://www.jcb
We thank Dr. Peter G. Schultz for the plasmids for in vivo BPA cross-linking. We
thank Dr. K. Nakatsukasa and members of the Endo laboratory for discussions
We acknowledge support of this work by Grants-in Aid for Scientifi c
Research of the Ministry of Education, Culture, Sports, Science and Technol-
ogy and a grant from the Japan Science and Technology Agency. Y. Tamura
was a research fellow and Y. Harada and K. Yamano are research fellows of
the Japan Society for the Promotion of Science.
Submitted: 13 August 2008
Accepted: 9 December 2008
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