From the Institut für Physiologische Chemie der
Universität München, 80336 München, Germany,
§ Department of Biological Sciences, University of Alberta,
Edmonton, Alberta T6G 2E9, Canada, and ¶ Department of
Biochemistry, The Hebrew University, Hadassah Medical School, Jerusalem
91120, Israel
Received for publication, October 23, 2000, and in revised form, February 27, 2001
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ABSTRACT |
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Translocation of preproteins across the
mitochondrial outer membrane is mediated by the translocase of the
outer mitochondrial membrane (TOM) complex. We report the
molecular identification of Tom6 and Tom7, two small subunits of the
TOM core complex in the fungus Neurospora crassa.
Cross-linking experiments showed that both proteins were found to be in
direct contact with the major component of the pore, Tom40. In
addition, Tom6 was observed to interact with Tom22 in a manner that
depends on the presence of preproteins in transit. Precursors of both
proteins are able to insert into the outer membrane in
vitro and are assembled into authentic TOM complexes. The
insertion pathway of these proteins shares a common binding site with
the general import pathway as the assembly of both Tom6 and Tom7 was
competed by a matrix-destined precursor protein. This assembly was
dependent on the integrity of receptor components of the TOM machinery
and is highly specific as in vitro-synthesized yeast Tom6
was not assembled into N. crassa TOM complex. The targeting
and assembly information within the Tom6 sequence was found to be
located in the transmembrane segment and a flanking segment toward the
N-terminal, cytosolic side. A hybrid protein composed of the C-terminal
domain of yeast Tom6 and the cytosolic domain of N. crassa
Tom6 was targeted to the mitochondria but was not taken up into TOM
complexes. Thus, both segments are required for assembly into the TOM
complex. A model for the topogenesis of the small Tom subunits is discussed.
The import of proteins into mitochondria is mediated by
multisubunit translocases in the outer
(TOM1 complex) and inner (TIM
complexes) mitochondrial membranes (for reviews see Refs. 1-4). The
TOM complex contains components that expose domains to the cytosol and
function as preprotein receptors. The major receptor is Tom20, which is
involved, together with Tom22, in the translocation of most precursors
(5-7). Another receptor that forms a binding site for a more
restricted set of preproteins, most notably the mitochondrial carrier
family, is Tom70 (8, 9). These receptors are loosely attached to the other components of the TOM machinery that form the core complex (10,
11). The subunits of the core complex (Tom40, Tom22, Tom7, Tom6, and
Tom5) are embedded in the outer membrane and form the translocation
pore (10, 11). Tom40 represents the major component of the
translocation pore (12, 13), whereas Tom22 and Tom5 probably transfer
preproteins from the receptors to the pore (14, 15). The yeast Tom6 and
Tom7 were suggested to modulate the stability of the association of the
Tom components (16, 17).
Tom6 is a small protein that was first identified in yeast as a high
copy number suppressor of a temperature-sensitive mutant of Tom40 (18).
Tom6 was proposed to support the cooperation between the receptors, in
particular Tom22, and the general insertion pore (10, 15, 16). The
protein contains one putative transmembrane domain close to its C
terminus and is oriented with its N terminus in the cytosol. Thus, it
belongs to the class of membrane proteins with a C-terminal anchor. It
has been suggested that the insertion of the protein into the outer
membrane is independent of surface receptors or the function of Tom40
(19).
Tom7 in yeast consists of 59 amino acid residues. It's topology in the
mitochondrial outer membrane is unknown (17). A lack of Tom7 was
reported to stabilize the interaction of the receptors Tom20 and Tom22
with the pore element, Tom40. These findings suggested that Tom7 plays
a role opposite to that of Tom6 by exerting a destabilizing effect on
the association of Tom components (17). At present, Tom6 and Tom7 have
been investigated only in yeast, though homologues from other organisms
have been identified (4). Information on the molecular environment of
Tom6 and Tom7 in the TOM complex would be of special interest for
understanding the molecular function of the TOM machinery.
As Tom6 is a tail-anchored protein, it can serve as a model protein for
the study of membrane insertion and assembly into functional complexes
for this group of membrane proteins. Currently, the information on how
tail-anchored proteins are targeted to mitochondria and inserted into
the mitochondrial outer membrane is very limited. In this report we
describe the cloning of TOM6 and TOM7 from
the fungus Neurospora crassa and an analysis of the
processes by which they are inserted into the mitochondrial outer
membrane and assembled into the TOM core complex. We also investigated
the molecular environment of the two proteins in their assembled state
and found them to be in direct contact with Tom40, whereas Tom6 was
also found to interact with Tom22 in a manner that depends on the
presence of preproteins in transit.
Cloning of N. crassa Tom6 and Tom7--
The TOM complex was
isolated according to Künkele et al. (20), and its
protein components were separated by urea-SDS polyacrylamide gel
electrophoresis and blotted onto polyvinylidene difluoride membrane (20). The bands corresponding to Tom6 and Tom7 were isolated,
and their N-terminal sequences were determined by Edman degradation.
For Tom6, 25 amino acid residues were determined, and degenerate
primers were synthesized for use in PCR reactions utilizing a cDNA
library from N. crassa as the template. By this method a
70-base pair DNA fragment was amplified that encodes 23 of these 25 amino acid residues. A larger PCR product containing most of the coding
sequence and the 3'end of the gene was produced from a pool of cloned
cDNAs using primers derived from the sequence of the 70-base pair
fragment and the T3 promotor region of the cDNA-containing vector.
This PCR product was labeled with Digoxigenin (Roche Molecular
Biochemicals) and used to probe both a cDNA library and a cosmid
library. One cDNA was completely sequenced and found to encode a
protein of 60 amino acid residues. For determination of the genomic
sequence a cosmid containing the TOM6 gene was isolated, and
the TOM6-containing part was sequenced.
For Tom7, 28 residues were determined from the N terminus of the
protein. A PCR-based strategy, similar to the one used for Tom6,
revealed the entire sequence of both the cDNA and genomic versions
of the gene.
Biochemical Procedures--
Isolation of mitochondria and outer
membrane vesicles (OMV) from N. crassa was performed as
described (21). The fusion protein, pSu9(1-69)-DHFR, was purified by
nickel-nitrilotriacetic acid affinity chromatography from cell extracts
of Escherichia coli strain BL21 carrying the
pQE60-pSu9(1-69)-DHFR-His6 overexpression vector.
Antibodies against N. crassa Tom6 and Tom7 were raised in
rabbits by injecting peptides coupled to keyhole limpet hemocyanin (Pierce), which corresponded to the 12 N-terminal amino acid residues. Blotting to polyvinylidene difluoride or nitrocellulose membranes and
immunodecoration were according to standard procedures, and visualization was by the ECL method (Amersham Pharmacia Biotech).
For cross-linking experiments,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)
was added to isolated OMV in either the absence or presence of various
amounts of pSu9-DHFR. After incubation for 30 min at 25 °C, excess
cross-linker was quenched by the addition of glycine, pH 8.0, to 80 mM, and the reactions were kept for 10 min at 25 °C.
Aliquots were removed before and after addition of the cross-linking reagent.
For co-immunoprecipitation experiments, OMV or mitochondria were
dissolved in buffer containing 1% of digitonin, Import of Preproteins into Isolated
Mitochondria--
Radiolabeled precursor proteins were synthesized in
rabbit reticulocyte lysate in the presence of
[35S]methionine (Amersham Pharmacia Biotech) after
in vitro transcription using SP6 polymerase from pGEM4
vector containing the cDNA of interest. Import reactions were
performed by incubation of radiolabeled preproteins with 30-50 µg of
mitochondria in import buffer (0.25% (w/v) bovine serum albumin, 250 mM sucrose, 80 mM KCl, 5 mM
MgCl2, 2 mM ATP, 10 mM MOPS-KOH, pH
7.2) at specified temperatures. Trypsin pretreatment (200 µg/ml) of
mitochondria was performed on 250 µg of mitochondria in 500 µl of
import buffer for 15 min on ice. Trypsin activity was blocked by
soybean trypsin inhibitor. Proteinase K treatment of samples was
performed by incubation with the protease for 15 min on ice followed by
inhibition by addition of 1 mM phenylmethylsulfonyl fluoride. Import was analyzed by SDS-PAGE or blue native gel
electrophoresis (BNGE), and the gels were viewed by autoradiography or
quantified by phosphorimaging (Fuji BAS 1500).
Blue Native Gel Electrophoresis--
Mitochondria (50 to 100 µg) were lysed in 50 µl of buffer (20 mM Tris-HCl, 0.1 mM EDTA, 50 mM NaCl, 10% glycerol, pH 7.4) containing 1% of digitonin, Triton X-100, or Cloning and Primary Sequence of N. crassa TOM6 and TOM7--
The
N-terminal sequences of isolated Tom6 and Tom7 were determined.
Degenerate primers were constructed according to the sequences obtained, and the genomic DNAs cloned by a PCR-based procedure (see
details under "Experimental Procedures"). Comparison of the amino
acid sequence obtained by N-terminal sequencing to that deduced from
the cDNA revealed the initiator methionine residue to be removed in
Tom6 but not in Tom7. The corresponding genes were sequenced from
PCR-amplified genomic DNA and cosmids containing TOM6 and TOM7,
respectively. The TOM6 gene contains two introns (GenBankTM accession number AF321882). N. crassa TOM6 encodes a protein of 60 residues and has one predicted
transmembrane segment near the C terminus (Fig.
1A). There is little
similarity between N. crassa Tom6 and yeast Tom6 in the
N-terminal region of the protein. However, the C-terminal regions,
including the predicted transmembrane segment, are 44% identical over
the last 25 residues (Fig. 1A).
The TOM7 gene is interrupted by three introns
(GenBankTM accession number AF321883). It encodes a protein
of 53 amino acid residues with a single predicted transmembrane segment
(Fig. 1B). N. crassa Tom7 has 53% identity to
yeast Tom7 (Fig. 1B).
Tom6 and Tom7 Are Integral Components of the TOM Core
Complex--
Antisera raised against peptides corresponding to the
first 12 amino acids of either Tom6 or Tom7 reacted selectively with the corresponding protein and were used to determine the enrichment of
the proteins in purified TOM complex as related to mitochondria or OMV
(Fig. 2A).
To analyze the association of Tom6 and Tom7 with the TOM complex in
N. crassa, BNGE was conducted on OMV solubilized in the mild
detergent digitonin. The conditions during BNGE cause the Tom20 and
Tom70 receptors of both the yeast and N. crassa TOM complexes to dissociate from the remaining components that constitute the TOM core complex (10, 11, 23). Both Tom6 and Tom7 comigrate with
Tom22 and Tom40 demonstrating their firm association with the core
complex (Fig. 2B). The stability of the interaction was further demonstrated by solubilizing OMV with 1% of either
The interactions between the various components of the N. crassa TOM complex were further investigated by
co-immunoprecipitation. OMV were solubilized with either digitonin, a
detergent that is known to keep the TOM holo complex intact (20), or
DDM, which results in the formation of the TOM core complex (11). Tom6 and Tom7 were precipitated with antibodies against Tom22 and Tom40 (Fig. 2, C and D). Hence, both Tom6 and Tom7 are
in close association with the other components of the TOM core complex.
In agreement with previous observations (10, 11), antibodies against
Tom70 precipitated only minor amounts of the other Tom components (Fig. 2, C and D), supporting the notion that Tom70 is
loosely associated with the other components of the TOM machinery. In
contrast to the observation in yeast (10) antibodies against Tom20 did
precipitate with high efficiency the other components of the TOM
complex from mitochondria dissolved in digitonin suggesting that each
complex contains at least one molecule of Tom20. The observation that not all the Tom20 molecules residing in the outer membrane could be
precipitated by antibodies against other components of the TOM complex
suggests the existence of a subpopulation of Tom20 molecules that are
not, or only loosely, attached to the complex. Antibodies against Tom20
could not, however, precipitate the other Tom components after
solubilization of the OMV with Tom6 and Tom7 Are in Direct Contact with Other Members of the TOM
Core Complex--
Tom6 was previously found to be in the vicinity of
Tom40, and their interaction was modified by the formation of specific translocation intermediates of a precursor protein (25). To determine
whether both Tom6 and Tom7 are only in the vicinity of Tom40 or
actually in direct contact with the protein, the cross-linking reagent
EDC was added to outer membrane vesicles, and cross-linking products were analyzed by immunodecoration. Specific cross-linking adducts of Tom40 with both Tom6 and Tom7 could be identified (Fig. 3A). As EDC is a zero-spacer
cross-linking reagent, it can be concluded that Tom40 is in direct
contact with both proteins.
In addition, a cross-linking adduct between Tom6 and Tom22 was
identified (Fig. 3B). The formation of this adduct was
reduced gradually by adding increasing amounts of the precursor,
pSu9(1-69)-DHFR, a chimeric preprotein consisting of the first 69 amino acids of subunit 9 of the mitochondrial F0-ATPase
fused to mouse dihydrofolate reductase, before performing the
cross-linking reaction. The Tom6-Tom22 adduct was also observed using
other cross-linking reagents (not shown). Hence, Tom6 and Tom22 are in
a direct contact, and their interaction is dynamically modulated by
preproteins in transit. A similar interaction between Tom22 and Tom7
was not observed (not shown).
Membrane Insertion and Assembly of Tom6 and Tom7--
To determine
whether in vitro-synthesized precursors of Tom6 and Tom7 can
be imported and assembled into authentic TOM complexes we took
advantage of the characteristic migration of the endogenous Tom6 and
Tom7 on BNGE. Isolated mitochondria were incubated with precursors of
Tom components at various temperatures, solubilized with digitonin, and
their proteins were analyzed by BNGE. A significant amount of the
precursors of Tom6 and Tom7, like those of Tom22 and Tom40, were
assembled in a temperature-dependent manner into the
endogenous pre-existing TOM core complex (Fig.
4A). This assembly was
specific. In a control experiment the imported precursor of porin,
another outer membrane protein, did not migrate with the endogenous TOM
complex (not shown). The lower molecular weight bands in Fig.
4A are probably insertion intermediates or unproductively bound precursors, as shown previously when these low molecular weight
bands were analyzed in detail following import of Tom40 (23). In this
experiment typical precursor-product relationships were not observed
for all Tom components. A significant portion of the precursor
molecules that are absorbed to the surface of the mitochondria during
the incubation at 0 °C dissociate from the mitochondria during
the wash and centrifugation, which are performed before loading the
material on BNGE. Hence, part of the molecules observed after
incubation at 25 °C are products of early intermediates that cannot
be observed with BNGE. Assembly of precursors of Tom components into
the TOM core complex was also observed after solubilization of
mitochondria with another detergent, DDM (Fig. 4B).
The assembly of newly synthesized Tom6 and Tom7 was further tested by
co-immunoprecipitation. Radiolabeled precursors were incubated with
mitochondria, and following the import reactions, mitochondria were
solubilized with digitonin and subjected to immunoprecipitation with
antibodies against various Tom components. Both precursors were
precipitated with antibodies against Tom20, Tom22, and Tom40 (Fig.
4C). Thus, both precursor proteins are imported into the
mitochondrial outer membrane and assembled into TOM complexes.
The requirements for efficient insertion of Tom6 and Tom7 into the
mitochondrial outer membrane were investigated. A previous report
suggested that neither import receptors nor Tom40 were required for
insertion of Tom6 and that other, unknown, proteins were involved (19).
We studied assembly into the endogenous TOM complex as a criterion for
correct insertion and addressed the question of whether Tom6 and Tom7
use the general insertion pore of the TOM complex for insertion. The
protein conducting pore was blocked by accumulating chemical amounts of
a translocation intermediate of the fusion protein pSu9(1-69)-DHFR in
the presence of methotrexate (26, 27). When precursors of Tom6 and Tom7 where imported into these blocked mitochondria a strong reduction in
the level of assembly of both was observed (Fig.
5A). Thus, precursors of a
matrix-destined protein apparently compete with precursors of Tom6 and
Tom7 for sites required for translocation. However, we cannot exclude
the possibility that the reduction in assembly upon blocking the pore
results from induction of a conformational change in the complex, which
masks a distal Tom6 binding site.
Does Tom6 require the receptor components for its proper assembly?
Mitochondria were treated with trypsin resulting in the removal of the
exposed parts of the surface receptors (Fig. 5B, inset). The ability of Tom6 precursor to assemble into the
TOM complex was reduced by this procedure (Fig. 5B). This is
in contrast to previous reports (17, 19) and suggests that the receptor proteins significantly enhance the assembly of Tom6. In summary, our
results support the hypothesis that Tom6 and Tom7 follow an import
pathway involving the TOM complex.
Targeting, Insertion, and Assembly Information within the Sequence
of Tom6--
Tom6, like all outer membrane proteins, does not contain
a cleavable targeting sequence. To determine which portions of the protein contain information for targeting, insertion, and integration we constructed fusion proteins containing Tom6 variants (Fig. 6A). To improve the detection
of the newly synthesized Tom6, we used a fusion protein where a DHFR
domain was present at the N terminus of Tom6. A similar fusion
construct in yeast was shown to be capable of functionally replacing
the native Tom6 protein in vivo (19). Our constructs
included a chimeric protein with the complete Tom6 protein (DHFR-Tom6)
and two mutant variants lacking either residues 1-12 of Tom6
(DHFR-Tom6
The primary sequence of yeast Tom6 has high similarity to the N. crassa protein in the C-terminal 20 amino acid residues (Fig. 1A). To study further the requirements for specific assembly
into the TOM complex, we constructed a hybrid precursor composed of the
C-terminal domain of yeast Tom6 fused to the cytosolic domain of
N. crassa Tom6 (Fig.
7A). We then asked whether
in vitro-synthesized yeast Tom6 and the hybrid construct
would be assembled into the N. crassa complex. Radiolabeled
precursors were imported into mitochondria isolated from N. crassa, and specific assembly was investigated by BNGE. Both yeast
Tom6-containing precursors migrated as low molecular weight bands
indicating that the precursors were bound to mitochondria but not
assembled (Fig. 7B). When the integration of these
precursors into the yeast TOM complex was examined, the opposite
integration behavior was observed (Fig. 7C). Yeast Tom6 was
assembled into the yeast complex, but the N. crassa Tom6 and the hybrid precursor were only bound as non-assembled species. The
results of importing the precursors into N. crassa
mitochondria were verified by co-immunoprecipitation (Fig.
7D). Although the precursor of N. crassa Tom6 was
efficiently precipitated by antibodies against Tom22 and Tom40, only
minor amounts of yeast Tom6 were brought down by these antibodies.
Hence, the Tom6 precursor from each organism contains specific
information that allows it to assemble only into the TOM complex from
the corresponding organism. Furthermore, this information cannot be
localized exclusively to the more variable N-terminal, cytosolic domain
as the hybrid precursor containing the yeast C terminus also did not
assemble into the N. crassa TOM complex.
We have cloned Tom6 and Tom7 from N. crassa and
investigated their insertion into the mitochondrial outer membrane,
integration into the TOM core complex, and their interactions with
other Tom components. These two small subunits, like the other
components of the TOM complex, have significant sequence similarity to
their yeast counterparts. Hence, their function can be predicted to be
similar to the yeast homologues.
The components of the TOM complex, Tom22, Tom70 and Tom40, use the
pre-existing TOM complex for their own insertion (23, 28, 29). In
contrast to previous observations (19), we report that Tom6 also
utilizes the TOM complex for insertion. How can this discrepancy be
explained? Although the temperature-sensitive tom40-3 allele
used in the previous study (19) does not affect the import of Tom6,
other tom40 temperature-sensitive alleles might do so.
Indeed, the tom40-3 allele does not affect the import of
porin, a major outer membrane protein, but two newly characterized alleles, tom40-2 and tom40-4, clearly do so
(30). Moreover, we have used BNGE as a specific assay to directly
monitor the assembly of in vitro imported Tom6 into
pre-existing TOM complexes. The criterion of resistance to alkaline
extraction used in the previous study (19) can be used to distinguish
insertion into membranes but can be misleading when taken as a measure
for correct assembly.
The highest sequence similarity between Tom6 from N. crassa
and yeast resides in the C-terminal domain, which includes the putative
transmembrane segment. Therefore, this region is likely to be important
for the function and/or assembly of Tom6, and this part of yeast Tom6
was reported to be essential for targeting to mitochondria and for
proper assembly (31). Using N. crassa Tom6 our study has
validated these findings. In addition, we have demonstrated that the
C-terminal domain is necessary for initial targeting and membrane
insertion, though is not sufficient for assembly into the TOM complex.
The C-terminal domain apparently contains the information required for
initial recognition of Tom6 precursor by the Tom components. This
initial recognition is not specific with regard to the organism, as all
constructs containing either the yeast or the N. crassa
version of this segment formed an early insertion intermediate with
N. crassa mitochondria. Although we cannot exclude the
possibility that this initial insertion is independent of the TOM
complex, we favor a model where this intermediate is loosely attached
to the TOM complex and dissociates from it under the conditions of
BNGE. The second possibility is supported by the observation that this
early intermediate can be precipitated, albeit inefficiently, by
antibodies against Tom components.
The C-terminal domain is also required for membrane insertion as it
contains the segment required for anchoring the protein in the outer
membrane. Following membrane insertion Tom6 is assembled into
pre-existing TOM complexes. Membrane insertion of Tom6 and its assembly
into functional complexes are not necessarily coupled events. Different
conditions are required for each process, and it may be that in each
step different segments of the Tom6 molecule are involved. The
C-terminal tail-anchor domain is probably required for membrane
insertion, whereas the process of assembly is more specific and
requires additional non-conserved residues in the N-terminal cytosolic
domain. Yeast Tom6 could not assemble into N. crassa TOM
complex and vice versa. As Tom6 was found to be in dynamic contact with
Tom22 and Tom40, these cytosolic residues may be involved in such
interactions. This is highly reminiscent of the situation with Tom22,
another tail-anchored protein, where import and assembly were also
found to be dependent on a short segment of the cytosolic domain (32,
33). The information for assembly of Tom6 into the complex is most
likely located at the N-terminal flanking region of the putative
transmembrane segment. A proteolytic fragment of Tom6 that lacked few
N-terminal residues of its cytosolic domain maintained the ability to
interact with Tom40 (25). Furthermore, deletion of the N-terminal 12 amino acid residues did not affect assembly of the resulting construct, whereas deletion of 36 of 38 residues of the cytosolic domain resulted
in an assembly-incompetent precursor. Like our findings on Tom6,
a Tom22 variant that lacks part of the cytosolic domain was still
delivered to the mitochondria in intact yeast cells but could not
complement the phenotype of Following their assembly, Tom6 and Tom7 were found to be part of the
core structure of the TOM complex. Tom7 is in direct contact with
Tom40, whereas the contact of Tom6 with Tom22 and Tom40 is dynamically
modulated by preproteins in transit. Because chemical cross-linking did
not reveal a direct contact between Tom40 and Tom22 (not shown), these
results provide experimental support to the previously suggested role
of Tom6 as a linking component between Tom40 and Tom22 (10,
25).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-dodecylmaltoside, or Triton X-100 for 30 min at 4 °C. The lysed material was
centrifuged (15 min at 20,000 × g), and the
supernatant was incubated with antibodies coupled to protein
A-Sepharose beads. At the end of the binding reaction the protein
A-Sepharose beads were washed with detergent-containing buffer, and
bound proteins were eluted with sample buffer and subjected to
SDS-PAGE. The gels were blotted and immunodecorated with antibodies
against the various Tom components.
-dodecylmaltoside. After incubation on ice for 30 min and a clarifying spin (20 min, 22,000 × g, 1.5 cm), 5 µl of sample buffer (5%
(w/v) Coomassie Brilliant Blue G-250, 100 mM BisTris, 500 mM 6-aminocaproic acid, pH 7.0) were added for another 5 min on ice, and the mixture was analyzed on a 6-13% gradient blue
native gel (22). The cathode and anode buffers for performing the
electrophoresis were as described (22). Electrophoresis was started at
100 V until the samples were within the stacking gel and continued with
voltage and current limited to 500 V and 15 mA. At the end of the
electrophoresis excess dye was removed, the gel was blotted on a
polyvinylidene difluoride membrane using a semi-dry apparatus, and
immunodecoration was as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Deduced amino acid sequences of N. crassa Tom6 and Tom7. A, deduced amino acid
sequences of N. crassa (N.c.) and S. cerevisiae (S.c.) Tom6. Similar residues are indicated
by one dot, and identical residues are indicated by
two dots. The putative transmembrane segments are
underlined. B, deduced amino acid sequences of
N. crassa (N.c.) and S. cerevisiae
(S.c.) Tom7. Symbols are as in A.
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Fig. 2.
Tom6 and Tom7 are integral parts of the
N. crassa TOM core complex. A,
presence of Tom6 and Tom7 in the isolated TOM complex. Intact
mitochondria (Mit.), OMV, and purified TOM holo complex
(TOM) (50 µg in each lane) were loaded on a
high Tris urea SDS-PAGE gel, blotted, and decorated with antibodies
against Tom6, Tom7, or Tom40. Apparent molecular masses are given on
the left. B, analysis by BNGE of Tom6 and Tom7 in
the TOM complex. OMV (20 µg) were solubilized in a buffer containing
1% of digitonin (Dig.), DDM, or Triton X-100
(Tx100) and analyzed by BNGE. For detection of Tom proteins
antibodies against Tom6, Tom7, Tom22, and Tom40 were used.
C, analysis of Tom6 and Tom7 in the TOM holo complex by
co-immunoprecipitation (IP). OMV (50 µg per
lane) were lysed in a buffer containing 1% digitonin and
added to protein A-coupled Sepharose beads containing prebound
antibodies against the indicated Tom components. Proteins bound to the
beads were subjected to SDS-PAGE, blotted, and immunodecorated with the
antibodies indicated at the left side. Contr., control.
D, analysis of Tom6 and Tom7 in the TOM core complex. OMV
(50 µg per lane) were lysed in a buffer containing 1%
DDM, and clarifying spin was performed (1 h, 100,000 × g, 1.5 cm). Further treatment was as in C.
-dodecylmaltoside or Triton X-100. Yeast Tom6 and Tom7 were reported
to dissociate from the TOM core complex after solubilization with
Triton X-100 (10). In contrast, with N. crassa most of Tom7
and Tom 22 and all of Tom6 and Tom40 migrated as a high molecular mass
complex (Fig. 2B). This latter complex migrates faster than
the complex solubilized with digitonin. This difference can be
explained by variations in the lipid contents of the various complexes.
We have previously reported a much higher phospholipid content in the
complex isolated with digitonin as compared with the complex isolated
with DDM (24).
-dodecylmaltoside (Fig.
2D). These conditions are known to result in the formation of the TOM core complex that lacks both receptor proteins, Tom20 and
Tom70 (11). Taken together, our data indicate that Tom6 and Tom7 are
integral components of the N. crassa TOM core complex.
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Fig. 3.
Tom6 and Tom7 are in a close dynamic contact
with other components of the TOM core complex. A, Tom6
and Tom7 are in contact with Tom40. OMV were incubated in the presence
or absence of the cross-linker EDC for 30 min at 25 °C. Each sample
was split into three aliquots, and proteins were separated by SDS-PAGE
and analyzed by immunostaining with antibodies against Tom40, Tom6, and
Tom7. B, Tom6 is in dynamic contact with Tom22. OMV in the
absence or presence of the indicated amounts of pSu9-DHFR were
incubated with the cross-linker EDC for 30 min at 25 °C.
Each sample was split into two aliquots, and proteins were separated by
SDS-PAGE and analyzed by immunostaining with antibodies against Tom22
and Tom6.
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Fig. 4.
Assembly of precursors of Tom6 and Tom7
synthesized in vitro into authentic TOM
complexes. Radiolabeled precursor proteins of Tom22, Tom7, Tom6
(panel A), DHFR-Tom6 (panel B), and Tom40 were
incubated for 20 min at the indicated temperatures with 50 µg of
mitochondria. The mitochondria were reisolated and solubilized for 30 min in 40 µl of buffer containing either 1% digitonin (A)
or 0.5% DDM (B). After a clarifying spin the samples were
analyzed by BNGE. The endogenous TOM complex was detected using
antibodies against Tom22 and Tom40. C, analysis by
co-immunoprecipitation. Tom6 and Tom7 precursors were incubated with
isolated mitochondria for 20 min at 25 °C. Mitochondria were
reisolated, solubilized with 1% digitonin, and subjected to
immunoprecipitation with the indicated antibodies or with antibodies
from control (Contr.) serum.
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Fig. 5.
Tom6 and Tom7 utilize the TOM complex for
their assembly into functional TOM complexes. A, a
matrix-destined precursor can compete with Tom6 and Tom7. Radiolabeled
precursors of Tom6 and Tom7 were incubated for 20 min at 25 °C in
import buffer (see "Experimental Procedures") containing 0.5 mM NADPH and 1 µM methotrexate with
either mitochondria (control) or with mitochondria preincubated with 12 µM pSu9-DHFR for 20 min on ice. At the end of the import
reactions mitochondria were washed, reisolated, and analyzed by BNGE.
The radioactive bands migrating together with the authentic TOM complex
were quantified and presented as a percent of integration in control
mitochondria. B, cytosolic domains of receptor components
promote insertion of Tom6. Radiolabeled precursor of DHFR-Tom6 was
incubated at 25 °C in import buffer for various time periods with
either intact mitochondria (circles; control) or with
mitochondria pretreated with trypsin (200 µg/ml) for 20 min on ice
(squares). Further treatment and data quantification was as
above. The inset shows Western blot analysis of intact
mitochondria and mitochondria pretreated with trypsin.
12) or residues 1-36 (DHFR-Tom6
36). All constructs
were targeted to mitochondria and inserted into the outer membrane as
shown by their recovery in the membrane pellet following carbonate
extraction (not shown). Co-immunoprecipitation and BNGE were employed
to investigate the assembly of the variant Tom6 precursors into the
endogenous TOM complex. Using BNGE we observed that only the wild
type construct and the DHFR-Tom6
12 variant were assembled into TOM
complexes whereas DHFR-Tom6
36 was found to be attached to
mitochondria but not assembled (Fig. 6B). Assembly of
DHFR-Tom6
36 was further analyzed by coimmunoprecipitation with
antibodies against Tom22 and Tom40. Imported native Tom6 was
efficiently co-immunoprecipitated, but only minor amounts of
DHFR-Tom6
36 were precipitated by these two antibodies (Fig.
6C). These minor amounts of precipitated DHFR-Tom6
36 may
represent translocation intermediates of the precursors that are
attached to, but not assembled into, the TOM complex. Only background
levels were precipitated by antibodies against Tom22 in a control
experiment where OMV were first solubilized with digitonin, and only
then was precursor added (not shown). These results indicate that amino
acid residues 13-36 in the cytosolic domain of Tom6 contain essential
information for either the correct assembly of the protein or for the
overall folding of the protein, which then affects assembly.
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Fig. 6.
A segment in the cytosolic domain of Tom6 is
essential for assembly into the TOM complex. A,
sequences of DHFR fusions with Tom6 variants. The putative
transmembrane segment of Tom6 is underlined. B,
DHFR-Tom6 fusion proteins were incubated with isolated mitochondria for
20 min at the indicated temperatures and further treated as described
in the legend to Fig. 5. For clarity of comparison the part containing
the variant precursors is taken from longer exposure. Assembled TOM
complex and non-assembled precursors (m) are indicated.
C, DHFR-Tom6 and DHFR-Tom6 36 were incubated with isolated
mitochondria at 25 °C for 30 min. At the end of the import reaction
mitochondria were reisolated. One aliquot was directly analyzed by
SDS-PAGE, and the rest was solubilized with Triton-containing buffer
and split into three aliquots, which were subjected to
immunoprecipitation with antibodies against Tom22 or Tom40 or with
antibodies from control serum. The control serum did not precipitate
the fusion proteins (not shown).
View larger version (70K):
[in a new window]
Fig. 7.
Precursors of Tom6 assemble exclusively into
the TOM complex from the corresponding organism. A,
sequences of DHFR-Tom6 fusion constructs. Tom6 from N. crassa and the portion from N.crassa Tom6 in the hybrid
construct are underlined. B, yeast Tom6 and a
hybrid Neurospora-yeast Tom6 are not assembled into authentic N. crassa TOM complexes. DHFR-Tom6 constructs were incubated with
isolated N. crassa mitochondria for 20 min at the indicated
temperatures, and further treatment was as described in the legend to
Fig. 6. Assembled TOM complex and non-assembled precursors
(m) are indicated. C, N. crassa Tom6
and a hybrid Neurospora-yeast Tom6 are not assembled into authentic
yeast TOM complexes. Import and analysis were as in panel B. D, precursors of DHFR-N.crassa-Tom6 or DHFR-yTom6
were incubated with isolated N. crassa mitochondria at
25 °C for 30 min. Further treatment and analysis were as in Fig.
6C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
tom22 yeast mutants (33). A
hybrid precursor consisting of the cytosolic domain from N. crassa Tom6 and the C-terminal domain from yeast Tom6 were not
able to assemble into either complex. Thus, a specific interaction
between these two domains may facilitate assembly into the
corresponding complex.
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ACKNOWLEDGEMENTS |
---|
We thank P. Heckmeyer, S. Neubauer, M. Braun, I. Dietze, and C. Nargang for excellent technical assistance, Dr. E. Wachter for protein sequencing, Dr. H. Prokisch for helpful discussions, U. Ahting for purified TOM complex, and M. Kaeser for the DHFR-yTom6 construct.
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FOOTNOTES |
---|
* This work was supported in part by grants of the Sonderforschungsbereich 184 of the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, and the Medical Research Council of Canada (to F. E. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF321882 and AF321883.
To whom correspondence should be addressed. Tel.:
972-2-6758292; Fax: 972-2-6757379; E-mail:
rapaportd@md.huji.ac.il.
Published, JBC Papers in Press, March 6, 2001, DOI 10.1074/jbc.M009653200
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ABBREVIATIONS |
---|
The abbreviations used are:
TOM, translocase of the outer mitochondrial membrane;
TIM, translocase of
the inner mitochondrial membrane;
BNGE, blue native gel
electrophoresis;
DDM, n-dodecyl -D-maltoside;
EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride;
PCR, polymerase chain reaction;
OMV, outer membrane vesicles;
DHFR, dihydrofolate reductase;
MOPS, 4-morpholinepropanesulfonic acid;
PAGE, polyacrylamide gel electrophoresis;
BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.
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