Unique Composition of the Preprotein Translocase of the Outer
Mitochondrial Membrane from Plants*
Lothar
Jänsch
,
Volker
Kruft§,
Udo K.
Schmitz
, and
Hans-Peter
Braun
¶
From the
Institut für Angewandte Genetik,
Universität Hannover, Herrenhäuser Strasse 2, 30419 Hannover and § Applied Biosystems, Paul-Ehrlich-Straße 17,
63225 Langen, Germany
 |
ABSTRACT |
Transport of most nuclear encoded mitochondrial
proteins into mitochondria is mediated by heteropolymeric translocases
in the membranes of the organelles. The translocase of the
outer mitochondrial membrane (TOM) was
characterized in fungi, and it was shown that TOM from yeast comprises
nine different subunits. This publication is the first report on the
preparation of the TOM complex from plant mitochondria. The protein
complex from potato was purified by (a) blue native
polyacrylamide gel electrophoresis and (b) by
immunoaffinity chromatography. On blue native gels, the potato TOM
complex runs close to cytochrome c oxidase at 230 kDa and
hence only comprises about half of the size of fungal TOM complexes.
Analysis of the TOM complex from potato by SDS-polyacrylamide gel
electrophoresis allows separation of seven different subunits of 70, 36, 23, 9, 8, 7, and 6 kDa. The 23-kDa protein is identical to the
previously characterized potato TOM20 receptor, as shown by in
vitro assembly of this protein into the 230-kDa complex, by
immunoblotting and by direct protein sequencing. Partial amino acid
sequence data of the other subunits allowed us to identify sequence
similarity between the 36-kDa protein and fungal TOM40. Sequence
analysis of cDNAs encoding the 7-kDa protein revealed significant
sequence homology of this protein to TOM7 from yeast. However, potato
TOM7 has a N-terminal extension, which is very rich in basic amino
acids. Counterparts to the TOM22 and TOM37 proteins from yeast seem to
be absent in the potato TOM complex, whereas an additional low
molecular mass subunit occurs. Functional implications of these
findings are discussed.
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INTRODUCTION |
Most mitochondrial proteins are nuclear encoded, synthesized on
cytoplasmic ribosomes, and posttranslationally transported into the
organelles (1, 2). The transport of proteins into mitochondria relies
on two prerequisites: (i) targeting information of the proteins
destined for mitochondria, which in most cases is encoded by N-terminal
extensions called presequences, and (ii) a protein import apparatus
within the mitochondrial membranes that recognizes the targeting
information and translocates proteins from the cytoplasm to their
subcellular destination. Central components of the protein import
apparatus are two polymeric protein complexes called the
"translocase of the outer mitochondrial
membrane"
(TOM)1 and the
"translocase of the inner mitochondrial
membrane" (TIM) (3). During protein transport, the
translocases are believed to interact dynamically as most nuclear
encoded mitochondrial proteins must cross both mitochondrial
membranes.
The TOM complex was isolated from outer mitochondrial membranes from
Neurospora crassa and yeast (4-6). The yeast translocase consists of nine subunits designated TOM72, TOM70, TOM40, TOM37, TOM22,
TOM20, TOM7, TOM6, and TOM5 according to their calculated molecular
masses (for nomenclature, see Ref. 7). Five of these proteins (TOM72,
TOM70, TOM37, TOM22, and TOM20) are involved in the recognition of
nuclear encoded mitochondrial proteins on the surface of the organelles
forming two heterodimeric receptors (TOM20-TOM22 and TOM37-TOM70/TOM72)
(8-10). TOM40 is the main component of the pore for the translocation
of proteins across the outer mitochondrial membrane (4, 11). It is
associated with the three low molecular weight subunits: TOM7, TOM6,
and TOM5 (12-14). The TOM complex from N. crassa has a
similar subunit composition; TOM complexes from other organisms have
not been characterized so far.
In a structural sense, the TOM complex is a rather dynamic association
of polypeptide components. The receptors are partially present within
TOM complexes but also occur independently within the outer
mitochondrial membrane. Consequently, it is a difficult task to purify
this protein complex. The TOM complexes from yeast and
Neurospora were prepared by immunoprecipitation using
antibodies directed against individual subunits of the translocase
(4-6). The overall molecular mass of the TOM complex from
Neurospora was estimated by gel filtration and lies in the
range of 500 kDa (4).
Little is known about the transport of proteins into plant
mitochondria. Some components of the protein import apparatus could be
defined, namely the mitochondrial processing peptidase, which excises
the presequences of nuclear encoded mitochondrial proteins upon their
import into the organelles. In plants only is this enzyme
membrane-bound and an integral part of the cytochrome c reductase complex of the respiratory chain (15-17). Recently, an additional processing activity in the soluble fraction of plant mitochondria was described, which might be distinct from the
membrane-bound mitochondrial processing peptidase (18). Furthermore,
the mitochondrial heat stress proteins HSP70, HSP60, and HSP10 were
characterized, which are involved in the terminal steps of protein
transport into mitochondria (19-21). Finally, one component of the TOM
complex, TOM20 from potato, has been characterized at the molecular
level (22). TOM20 from potato is a 23-kDa protein of the outer
mitochondrial membrane that exhibits significant sequence identity to
the TOM20 proteins from fungi and mammals. Antibodies directed against
potato TOM20 specifically inhibit protein transport into mitochondria, as demonstrated by in vitro import experiments (22).
Here, we describe the purification of the TOM complex from potato by
native gel electrophoresis and by immunoaffinity chromatography. The
protein complex has a molecular mass of 230 kDa and comprises seven
different subunits that are characterized by direct protein sequencing
and by sequencing corresponding clones encoding a 7-kDa polypeptide.
TOM20 forms part of this complex, as shown by in vitro
assembly of this protein into the 230-kDa complex, by direct sequence
determination and by immunoblotting. The TOM complex from potato has a
unique subunit composition.
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EXPERIMENTAL PROCEDURES |
Purification of the Outer Mitochondrial Membrane from
Potato--
Mitochondria from potato tuber were prepared on Percoll
step gradients as described by Braun et al. (23) with the
following modifications: (i) 0.1 mM PMSF was added to all
buffers, and (ii) after the gradient centrifugation the organelles were
washed and suspended in a buffer containing 0.4 M mannitol,
1 mM EGTA, 0.1% bovine serum albumin, 0.1 mM
PMSF, and 10 mM KH2PO4, pH 7.2. The isolated mitochondria were resuspended in 7.5 ml of swelling buffer (5 mM KiPO4, 1 mM PMSF, pH
7.2)/g of organelles for 8 min at 4 °C. Subsequently, the same
volume of swelling buffer was added and mitochondria were ruptured in a
Potter homogenizer. Separation of outer mitochondrial membranes (OM)
and mitoplasts was achieved by sucrose step gradient centrifugation, as
reported by Heins et al. (24). One gram of potato
mitochondria (100 mg of mitochondrial protein) yielded about 1 mg of OM
protein. The OMs were stored in a buffer containing 10 mM
EDTA, 0.4 mM PMSF, 10% glycerol, 100 mM
MOPS-KOH, pH 7.2, at a protein concentration of 5 mg/ml at
80 °C.
Purification of the Potato TOM Complex by Blue Native
Polyacrylamide Gel Electrophoresis (BN-PAGE) and
Electroelution--
Starting material for the purification of the
potato TOM complex by blue native polyacrylamide gel electrophoresis
were 40-µl OM aliquots (200 µg of OM protein) per lane of the gels.
The aliquots were diluted by 100 µl of double-distilled
H2O; the OMs were sedimented by ultracentrifugation at
100,000 × g for 45 min and resuspended in 75 µl of
ACA buffer (750 mM aminocaproic acid, 0.5 mM
EDTA, 50 mM Bis-Tris, pH 7.0). Membrane proteins were
solubilized by addition of 75 µl of ice-cold digitonin solution (10%
digitonin, 750 mM aminocaproic acid, 0.5 mM
EDTA, 50 mM Bis-Tris, pH 7.0), and insoluble material was
removed by ultracentrifugation at 100,000 × g for 20 min. Supernatants were supplemented with 15 µl of Coomassie solution
(750 mM aminocaproic acid, 5% Coomassie Blue) and directly loaded onto the gel. BN-PAGE was carried out as described previously (25, 26). The potato TOM complex forms a band at about 230 kDa, which
is visible without staining. The band was cut out, and the protein
complex was electroeluted in a buffer containing 25 mM
Tricine, 7.5 mM Bis-Tris, pH 7.0, 0.1 mM PMSF
using the electroeluter from CBS Scientific Co. (Del Mar, CA).
Determination of the Molecular Weight of the Potato TOM
Complex--
For molecular weight determination of the potato TOM
complex, 7 mg of mitochondria (0.7 mg of mitochondrial protein) from potato were resuspended in 75 µl of ACA buffer (see above) and supplemented with either 25 µl of a 10% digitonin solution or 15 µl of a 10% laurylmaltoside solution. The solubilized protein complexes in both fractions were separated by BN-PAGE as described above and subsequently blotted onto nitrocellulose membranes as outlined by Jänsch et al. (26). The respiratory
protein complexes were directly visible due to bound Coomassie Blue,
whereas the potato TOM complex was identified by immunostaining. The
size of the potato TOM complex was determined by comparison to the sizes of the well characterized respiratory protein complexes from
potato.
Immunoaffinity Purification of the Potato TOM
Complex--
Antibodies directed against TOM20 from potato (22) were
covalently bound to CNBr-activated Sepharose (Amersham Pharmacia Biotech, Sweden) according to the supplier's instructions. Potato outer mitochondrial membranes prepared from 0.8 g of isolated organelles were sedimented by ultracentrifugation at 70,000 × g for 90 min and resuspended in 375 µl of coupling buffer
(20 mM Tris acetate, 10 mM NaCl, pH 7.0).
Membrane proteins were solubilized by addition of 375 µl of ice-cold
digitonin solution (10% digitonin, 20 mM Tris acetate, 10 mM NaCl, pH 7.0), and insoluble material was removed by
ultracentrifugation at 100,000 × g for 20 min. About 2 ml of the anti-TOM20-Sepharose were equilibrated in a column with
coupling buffer. After applying the solubilized membrane proteins to
the column, it was washed with coupling buffer and bound protein was
eluted with 5 ml of elution buffer (100 mM glycine, pH
2.5). Eluted proteins were neutralized by addition of 400 µl of 1 M Tris-HCl, pH 8.0. The proteins were precipitated by the Roti-Collect system (Roth GmbH, Karlsruhe, Germany), supplemented with
loading buffer and directly analyzed by Tricine-SDS-PAGE (27).
In Organello Assembly Assay--
The TOM20 protein from potato
was synthesized in vitro by the coupled
transcription/translation system from Stratagene (Heidelberg, Germany)
in the presence of [35S]methionine. Mitochondria were
prepared on Percoll step gradients as described above. For an assembly
assay, 600 µl of mitochondria (10 mg of protein/ml) were combined
with 2.4 ml of assembly buffer (0.25 M mannitol, 80 mM KCl, 1 mM K2HPO4, 1 mM ATP, 1 mM malate, 2 mM NADH, 1 mM dithiothreitol, and 20 mM HEPES, pH 7.5) and
37.5 µl of radiolabeled TOM20 protein in transcription/translation lysate. The assembly mixture was incubated for 20 min at 25 °C. Mitochondria were reisolated by centrifugation for 10 min at
13,000 × g, resuspended in 600 µl of resuspension
buffer (0.4 M mannitol, 1 mM EGTA, 0.1% bovine
serum albumin, 0.1 mM PMSF, and 10 mM
KH2PO4, pH 7.2) and supplemented with 3.4 ml of
untreated potato mitochondria (10 mg of protein/ml) in the same buffer.
Subsequently, outer mitochondrial membranes were prepared as described
above and proteins were separated by two-dimensional
BN/SDS-polyacrylamide gel electrophoresis. Gels were treated with
Amplify (Amersham Pharmacia Biotech, Braunschweig, Germany) and exposed
on x-ray films.
Identification of Subunits of the Potato TOM Complex by Direct
Sequence Determination and Immunoblotting--
The subunits of the
potato TOM complex were separated either by SDS-PAGE of the
electroeluted protein complex or directly by electrophoresis of a BN
gel stripe in a second gel dimension in the presence of SDS.
Tricine-SDS-PAGE according to Schägger and von Jagow (27) gives
best resolution because the TOM complex contains several very small
subunits with similar molecular masses. The separated TOM subunits were
blotted onto polyvinylidene difluoride membranes, and TOM20 was
identified by immunostaining using the Vectastain ABC kit (Vector
Laboratories, Burlingame, CA). Bands corresponding to other TOM
subunits were cut out from the blots and analyzed directly on a
Procise-HT protein sequencer (model ABI 494A, Applied Biosystems,
Foster City, CA). N-terminally blocked proteins were digested with
endoprotease LysC, and the generated peptides were separated by high
performance liquid chromatography and analyzed by direct protein
sequencing.
Isolation of Clones Encoding the Potato TOM7 Subunit--
Clones
encoding TOM7 from potato were isolated by probing a
ZAP-II
cDNA library with a mixture of 23-nucleotide oligomers that were
made degeneratively to an amino acid sequence close to the N terminus
of the protein (Lys-Gly-Lys-Asn-Thr-Lys-Phe).
 |
RESULTS |
Purification of the Potato TOM Complex--
Compared with the
protein complexes of the respiratory chain, the TOM complex must be
considered to be a dynamic supramolecular structure. Consequently,
classical biochemical approaches to isolate the TOM complex has only
had limited success. Recently, BN-PAGE was shown to be an alternative
approach to characterize the translocase of the outer mitochondrial
membrane from yeast (28). BN-PAGE was first used by Schägger and
von Jagow (25) to isolate the respiratory protein complexes from beef
and yeast and was later adapted to characterize the respiratory and
photosynthetic protein complexes from plant organelles (26, 29).
Prerequisite for the isolation of the potato TOM complex was a fraction
containing highly pure outer mitochondrial membranes, which was
generated as described in the experimental procedures. Tricine-SDS-PAGE
of potato outer mitochondrial membranes allows to separate about 30 different proteins (Fig. 1C).
The two most dominant bands represent two isoforms of the pore-forming
protein "porin," which run at 30 kDa on Tricine SDS gels (34-36
kDa on glycine SDS gels according to Laemmli; Ref. 30) and which have been characterized previously (24). BN-PAGE of digitonin-lysed outer
mitochondrial membranes from potato revealed the presence of one
dominant protein complex, which, under the conditions applied, migrates
in the central part of the gel (Fig. 1A). Furthermore some
faint bands with higher electrophoretic mobility are visible on the BN
gel. To characterize the subunit compositions of the separated protein
complexes, a strip of the blue native gel was transferred horizontally
onto a second gel dimension and electrophoresed in the presence of SDS.
On Tricine SDS gels, the dominant protein complex could be resolved
into seven subunits with apparent molecular masses of 70, 36, 23, 9, 8, 7, and 6 kDa (Fig. 1B). If analyzed by the glycine SDS-PAGE
system, the apparent molecular mass of the 36-kDa protein lies at 39 kDa. The subunit composition resembles the composition of the TOM
complex from fungi, which was characterized previously. The protein
complexes with higher electrophoretic mobility on the BN gel turned out
to be aggregates of varying numbers of porin proteins (Fig.
1B, proteins number 8).

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Fig. 1.
Purification of the potato TOM complex by
BN-PAGE. Potato OM proteins were solubilized by digitonin and
subsequently analyzed by BN-PAGE as described under "Experimental
Procedures." A, Coomassie-stained gel of potato OM
proteins after BN-PAGE. B, Coomassie-stained gel of potato
OM proteins after two-dimensional resolution by BN-PAGE (first
dimension) and Tricine-SDS-PAGE (second dimension). C,
Coomassie-stained gel of potato OM proteins after solubilization with
SDS and one-dimensional Tricine-SDS-PAGE. The arrows
indicate proteins that were subjected to direct protein sequencing (see
Table I). The molecular masses of standard proteins are given on the
left in kDa. TOM, translocase of the outer
mitochondrial membrane from potato.
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Determination of the Molecular Weight of the Potato TOM
Complex--
Blue native polyacrylamide gel electrophoresis is a
suitable method for the size determination of protein complexes
solubilized by laurylmaltoside (31). To determine the apparent
molecular weight of the putative potato TOM complex, isolated
mitochondria from potato were treated with laurylmaltoside and directly
analyzed by BN-PAGE (Fig. 2A).
The separated protein complexes were transferred onto nitrocellulose
membranes and stained by immunoblotting. The respiratory protein
complexes of the inner mitochondrial membrane, which have well defined
apparent molecular sizes, could be identified. An antibody directed
against the potato TOM20 protein specifically reacted with a protein
complex migrating close to the cytochrome c oxidase complex
at about 230 kDa. Electrophoresis of the same sample on a second gel
dimension in the presence of SDS revealed that the immunoreactive
protein complex had an identical protein composition as described above
for the putative TOM complex from potato after digitonin solubilization
(data not shown). Determination of the molecular mass of the potato TOM
complex after solubilization of mitochondria with digitonin gave
similar results (Fig. 2B). The separation of the respiratory
protein complexes is slightly different if compared with the separation
of laurylmaltoside lysed mitochondria. However, the TOM complex again
migrates close to the cytochrome c oxidase at 230 kDa.

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Fig. 2.
Determination of the molecular weight of the
potato TOM complex. Mitochondrial protein complexes were
solubilized by laurylmaltoside (A) or digitonin
(B) as described under "Experimental Procedures,"
separated by BN-PAGE, and blotted onto nitrocellulose membranes.
Lanes 1 and 3, blot strips after staining with
Coomassie Blue; lanes 2 and 4, blot strips after
immunostaining using an antibody directed against potato TOM20. The
designations on the left indicate the identity of some
respiratory protein complexes and their molecular weights as determined
previously (26).
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The Identification of TOM20 as Part of the Potato TOM
Complex--
So far, the TOM20 protein from potato is the only
component of the preprotein translocase from plants that has been
characterized at the molecular and physiological level (22). To prove
that the TOM20 protein forms part of the putative potato TOM complex, antibodies directed against pTOM20 were used to immunoaffinity-purify the proteins of a fraction containing outer mitochondrial membranes from potato. The antibody was covalently bound to CNBr-activated Sepharose and immunoaffinity chromatography was carried out using a
small column. Bound protein was eluted with 100 mM glycine
at pH 2.5 as described under "Experimental Procedures" and analyzed on Tricine-SDS-PAGE (Fig. 3). The eluted
fraction contains predominant proteins of 36, 30, 23, 9, 8, 7, and 6 kDa (Fig. 3, lane 2). The 23-kDa protein was immunologically
identified to be TOM20 (Fig. 3, lane 3), and the 30-kDa
protein turned out to be the most abundant protein of the mitochondrial
outer membrane, porin (data not shown). The remaining proteins have
identical molecular masses as the subunits of the potato TOM complex
after purification by BN-PAGE. Hence, TOM20 seems to form part of the
TOM complex from potato.

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Fig. 3.
Purification of the potato TOM complex by
immunoaffinity chromatography using antibodies directed against
pTOM20. Protein fractions were separated by Tricine-SDS-PAGE.
Lane 1, silver-stained gel of separated OM proteins from
potato. Lane 2, silver-stained gel of digitonin-lysed OM
proteins after the immunoaffinity purification step. Lane 3,
immunostained Western blot of the affinity-purified OM proteins using
the pTOM20 antibody. The molecular masses of standard proteins are
given on the left in kDa.
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In a second approach to monitor the presence of TOM20 in the 230-kDa
protein complex of the outer mitochondrial membrane from potato,
in vitro assembly of the TOM20 protein into the 230-kDa complex was tested. Radiolabeled TOM20 from potato was incubated with
isolated mitochondria as described under "Experimental Procedures." Subsequently, the outer mitochondrial membrane was prepared and the
proteins of this fraction were analyzed by two-dimensional BN/SDS-PAGE
and autoradiography. Indeed, the 23-kDa TOM20 protein from potato
assembles into the 230-kDa protein complex as documented in Fig.
4. We conclude that the 230-kDa protein
complex in the outer membrane from potato mitochondria corresponds to
the preprotein translocase designated "TOM" in fungi.

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Fig. 4.
In vitro assembly of potato TOM20 into
the potato TOM complex. pTOM20 was synthesized in the presence of
[35S]methionine and incubated with freshly isolated
potato mitochondria as described under "Experimental Procedures."
Subsequently, the potato TOM complex was isolated by two-dimensional
BN/SDS-PAGE. A, Coomassie-stained gel of the purified TOM
complex; B, autoradiography of the same gel. The
designations on the top and on the left indicate
the sizes of standard proteins in kDa.
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Identification of Subunits of the Potato TOM Complex by Direct
Sequence Determination--
In an attempt to characterize the
components of the potato TOM complex, the proteins of the outer
mitochondrial membrane from potato were separated by two-dimensional
BN/SDS-PAGE and blotted onto filter membranes. The subunits of the TOM
complex were visualized by staining with Ponceau S, cut out, and
subjected to direct protein sequencing. Subunits blocked for direct
protein sequence determination were digested with endoprotease LysC to
generate peptides, which were separated by high performance liquid
chromatography and also analyzed by direct protein sequence
determination. Table I summarizes the
obtained amino acid data for the 36-, 23-, 9-, 8-, 7-, and 6-kDa
subunits of the potato TOM complex. As expected, the peptide sequence
for the 23-kDa subunit is identical to an internal amino acid stretch
of potato TOM20. The peptide sequences for the 36-kDa protein exhibit
some sequence similarity to the published TOM40 sequences from fungi.
Sequence conservation was highest between peptide P1 and the fungal
TOM40 proteins (Fig. 5). The partial sequence data generated for the 9-, 8-, 7-, and 6-kDa proteins of the
potato TOM complex did not exhibit any significant sequence similarity
to components of the TOM complex from other organisms.

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Fig. 5.
The 36-kDa subunit of the TOM complex from
potato is homologous to TOM40 from fungi. An internal sequence (P1
in Table I) of the 36-kDa subunit from potato was aligned with TOM40
sequences from Saccharomyces cerevisiae (P23644),
Neurospora crassa (P24391), and Schizosaccharomyces
pombe (AB004538). Amino acids conserved between the potato
sequence and one or several fungal sequences are underlaid in
black, amino acids that are similar in potato and one or
several fungal sequences are underlaid in gray. The
numbers to the left of the three fungal sequences
indicate the position of the first amino acid within the protein
sequences, as deduced from the open reading frames of the corresponding
genes.
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Isolation of Clones Encoding the TOM7 Protein from Potato--
In
order to obtain complete sequences for the 9-, 8-, 7-, and 6-kDa
proteins of the potato TOM complex, degenerate oligonucleotides designed from amino acid stretches of each protein were used to screen
a cDNA library for potato tuber and to isolate corresponding clones. Screening with an oligonucleotide deduced from the N-terminal sequence of the 7-kDa protein of the potato TOM complex led to the
isolation of two clones designated pTOM221 and pTOM222. Both clones
contained inserts of 653 base pairs with identical sequences (Fig.
6). The inserts include an open reading
frame of 216 base pairs encoding a protein of 72 amino acids with a
calculated molecular mass of 7716 Da. The N-terminal amino acid
sequence of the deduced protein is identical to the sequence directly
determined by cyclic Edman degradation of the 7-kDa protein of the
potato TOM complex, with the exception of the initiator methionine,
which is absent in the mature protein. Comparison between the complete
amino acid sequence of the potato 7-kDa protein and the sequence
entries of protein data bases revealed striking similarities to the
sequence of the TOM7 protein from yeast (Fig.
7). The overall sequence identity lies at
25% and sequence similarity at 50%. Interestingly, the sequence
conservation is rather low in the N-terminal half of the protein but
high in the C-terminal half. We conclude that the 7-kDa protein of the
potato TOM complex is the plant counterpart to TOM7 from yeast. Further
analyses with nucleotide sequence data bases led to the discovery of
some unidentified plant sequences, which encode proteins with high
homology to the sequence of potato TOM7, including the expressed
sequence tag D22755 from rice and L35838 from cabbage (Fig. 7). On the
basis of amino acid sequences for TOM7 proteins from five different
organisms, a consensus sequence for TOM7 could be defined, which should
be useful for analysis of the function of TOM7 by site-directed
mutagenesis.

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Fig. 6.
Nucleotide sequence and deduced amino acid
sequence of the insert of clone pTOM221. The sequence data have
been submitted to the EMBL sequence data banks and are available under
the accession number Y16228.
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Fig. 7.
The potato 7-kDa protein is homologous to
yeast TOM7. Sequence comparison of the 7-kDa protein of the
mitochondrial outer membrane from potato with TOM7 from yeast (S63002)
and with amino acid sequences deduced from unidentified expressed
sequence tags from the plants Oryza sativa (rice, D22755)
and Brassica napus (cabbage, L35838) and with an
unidentified open reading frame of a genomic region of A. thaliana (AB005233). Identical amino acids are underlaid in
black if conserved in at least three organisms. Similar
amino acids are underlaid in gray.
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DISCUSSION |
Solubilization of outer mitochondrial membranes from potato with
digitonin and separation of the solubilized proteins by blue native
polyacrylamide gel electrophoresis allows the purification of a complex
of 230 kDa that consists of seven different subunits of 70, 36, 23, 9, 8, 7, and 6 kDa. The subunit composition resembles the one reported for
fungal TOM complexes. TOM20 from potato, a well defined receptor for
the transport of nuclear encoded mitochondrial proteins into
mitochondria, forms part of the 230-kDa complex as shown by in
vitro assembly of this protein into the 230-kDa complex, by direct
protein sequencing and by immunoblotting. Hence, the 230-kDa complex
represents the potato TOM complex. Blue native polyacrylamide gel
electrophoresis was employed previously to analyze the TOM complex from
yeast (14, 28). The procedure allows separation of two subcomplexes of
the yeast TOM complex of 400 and 120 kDa. The larger subcomplex
contains TOM40, TOM20, TOM5, and presumably TOM22, TOM7, and TOM6,
whereas the smaller subcomplex contains TOM70 and possibly TOM37, as
shown by immunoblotting. In contrast, the potato translocase seems to
be stable during solubilization and native gel electrophoresis as no
subcomplexes or singular subunits of the TOM complex are visible on the
two-dimensional gels (Fig. 1).
The TOM complexes from yeast and Neurospora were isolated by
immunoprecipitations using antibodies against individual subunits of
the TOM complex (4, 6). However, immunoprecipitation often does not
lead to biochemically pure proteins or protein complexes because the
antibodies can be present in the final fractions and because
cross-reactions can occur. In contrast, antibodies are not needed
during blue native gel electrophoresis and the capacity of this
procedure to resolve proteins is high. BN-PAGE seems to be a powerful
tool for the isolation of the potato TOM complex. The 230-kDa protein
complex can be electroeluted from BN gels and used for further
investigations. In fact, it was shown that the protein complexes of the
respiratory chain from beef and potato are physiologically active after
electroelution from BN gels (25, 32). This also may be valid for the
potato TOM complex after purification by BN-PAGE and
electroelution.
Sequence analyses of the subunits of the potato TOM complex allowed us
to relate its subunits to the components of the TOM complex from yeast.
The 23-kDa protein is homologous to fungal TOM20 and the 7-kDa protein
to TOM7 from yeast. The 36-kDa protein from potato exhibits sequence
similarity to fungal TOM40. It was previously shown that antibodies
directed against TOM40 from Neurospora cross-react with a
42-kDa protein of the outer membrane of Vicia faba
mitochondria and that antibodies against this 42-kDa protein inhibit
protein import into mitochondria from V. faba in in
vitro import experiments (33). Most likely, the 36-kDa protein
from potato and the 42-kDa protein from V. faba are
homologues. Furthermore, we speculate that the 70-kDa protein of the
TOM complex from potato is the counterpart of TOM70 from fungi and that
two of the three unidentified small proteins represent the TOM5 and
TOM6 proteins.
Based on these conclusions, the potato TOM complex differs in several
respects from the translocase from fungi: (i) the TOM complex from
potato is only about half the size of the fungal TOM complex, (ii) the
TOM complex from potato comprises four instead of three low molecular
mass subunits, and (iii) two of the four receptor proteins defined for
yeast (TOM22 and TOM37) seem to be absent in potato. However, it cannot
be excluded that the TOM complex from potato contains additional
subunits in vivo that are lost during solubilization of the
outer mitochondrial membrane with digitonin or during native gel
electrophoresis. On the other hand, there are several arguments
indicating that the TOM complex from potato was isolated in complete
form; (a) different concentrations of digitonin during
solubilization always led to the isolation of a protein complex with
the same subunit composition (data not shown), (b)
solubilization of outer mitochondrial membranes from potato with
laurylmaltoside also gave identical results, and (c) two
different purification procedures for the potato TOM complex (BN-PAGE
and immunoaffinity chromatography) led to the isolation of a protein
complex with identical subunit composition. Furthermore, the TOM37
protein has so far only been identified in yeast but not in
Neurospora. Hence, the occurrence of two heterodimeric receptors (one of which is composed of the TOM20 and TOM22 proteins, the other of the TOM37 and TOM70/TOM72 proteins; see Refs. 8-10) might
be an unique feature of yeast mitochondria. Possibly protein transport
into plant mitochondria is based on the presence of two monomeric
receptors of 23 kDa (TOM20) and 70 kDa (TOM70?). However, further
experiments are needed to precisely define the structure and function
of the plant TOM complex, including isolation of the translocase from
other organisms. Preliminary characterization of the TOM complex from
Arabidopsis thaliana also reveals the presence of four low
molecular mass subunits.2
The small proteins of the TOM complex are best characterized in yeast
(13, 14, 34). All of them are associated with TOM40 forming part of the
"general insertion pore" for the transfer of nuclear encoded
mitochondrial proteins into mitochondria. TOM5 functionally links the
receptors and TOM40, whereas TOM6 and TOM7 have antagonistic functions
in the modulation of the assembly and dissociation of the TOM
machinery: TOM6 supports the binding of the receptors with TOM40,
whereas TOM7 destabilizes the same interactions. (If TOM7 from potato
has a similar function, its presence within the 230-kDa TOM complex
together with TOM20 and TOM70 would add a further argument supporting
the absence of heterodimeric receptors in plants in vivo.)
The mature TOM7 proteins from potato and yeast both lack the N-terminal
methionine and have slightly different calculated molecular masses of
7.6 kDa (potato) and 6.7 kDa (yeast). The size difference is due to an
N-terminal extension of the potato protein, which is very rich in basic
amino acids (Fig. 7). TOM7 from potato is more hydrophilic than the
corresponding yeast protein. Most striking is the presence of 11 lysines in the potato protein (yeast: 2 lysines). Similarly to TOM7
from yeast, the potato protein lacks a putative hydrophobic
membrane-spanning helix and most likely is bound to the outer
mitochondrial membrane by protein-protein interactions. Identification
of some additional TOM7 sequences from plants allowed to define a
consensus sequence for TOM7 proteins. There are two highly conserved
amino acid stretches within the C-terminal half of TOM7 proteins
(-H-Y-G-(W/F)-I-P-(F/L)-V- and -(N/Q)-L-L-S-P-(L/V)-). Site-directed
mutagenesis may allow elucidation of the function of these sequence
motifs in protein translocation across the outer mitochondrial
membrane.
Due to its dynamic structure, the translocase of the outer
mitochondrial membrane is difficult to characterize.
Immunopurifications and blue native gel electrophoresis were employed
to characterize both the TOM complex from fungi and the TOM complex
from potato. The purified TOM complexes have several characteristics in
common, but also differ in some respects. So far, it cannot be excluded that the differences are due to isolation artifacts. On the other hand,
the structure and function of the mitochondrial protein import
apparatus from different organisms is known to vary to some extent. One
example is the mitochondrial processing peptidase, which is a soluble
enzyme in fungi and mammals but forms an integral part of the
cytochrome c reductase complex in plants. It should be
interesting to analyze the structure and function of the TOM complex
from some further organisms.
 |
FOOTNOTES |
*
This work was supported by the Deutsche
Forschungsgemeinschaft and by the Fonds der Chemischen Industrie.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) Y16228.
¶
To whom correspondence should be addressed. Tel.:
49-511-7622674; Fax: 49-511-7623608; E-mail:
braun{at}mpimg-berlin-dahlem.mpg.de.
1
The abbreviations used are: TOM,
translocase of the outer mitochondrial
membrane; TIM translocase of the
inner mitochondrial membrane; PMSF,
phenylmethylsulfonyl fluoride; OM, outer mitochondrial membrane; MOPS,
4-morpholinepropanesulfonic acid; Bis-Tris,
bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; BN, blue native;
PAGE, polyacrylamide gel electrophoresis; Tricine, N-tris(hy- droxymethyl)methylglycine.
2
L. Jänsch, U. K. Schmitz, and H.-P.
Braun, manuscript in preparation.
 |
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