From the Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada
Received for publication, August 8, 2002, and in revised form, October 15, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The TOM complex
(Translocase of the Outer mitochondrial
Membrane) is responsible for the recognition of
mitochondrial preproteins synthesized in the cytosol and for their
translocation across or into the outer mitochondrial membrane. Tom40 is
the major component of the TOM complex and forms the translocation
pore. We have created a tom40 mutant of Neurospora
crassa and have demonstrated that the gene is essential for the
viability of the organism. Mitochondria with reduced levels of Tom40
were deficient for import of mitochondrial preproteins and contained
reduced levels of the TOM complex components Tom22 and Tom6, suggesting
that the import and/or stability of these proteins is dependent on the
presence of Tom40. Mutant Tom40 preproteins were analyzed for their
ability to be assembled into the TOM complex. In vitro
import assays revealed that conserved regions near the N terminus
(residues 51-60) and the C terminus (residues 321-323) of the
349-amino acid protein were required for assembly beyond a 250-kDa
intermediate form. Mutant strains expressing Tom40 with residues 51-60
deleted were viable but exhibited growth defects. Slow growing mutants
expressing Tom40, where residues 321-323 were changed to Ala residues,
were isolated but showed TOM complex defects, whereas strains in which
residues 321-323 were deleted could not be isolated. Analysis of the
assembly of mutant Tom40 precursors in vitro supported a
previous model in which Tom40 precursors progress from the 250-kDa
intermediate to a 100-kDa form and then assemble into the 400-kDa TOM
complex. Surprisingly, when wild type mitochondria containing Tom40
precursors arrested at the 250-kDa intermediate were treated with
sodium carbonate, further assembly of intermediates into the TOM
complex occurred, suggesting that disruption of protein-protein
interactions may facilitate assembly. Import of wild type Tom40
precursor into mitochondria containing a mutant Tom40 lacking residues
40-48 revealed an alternate assembly pathway and demonstrated that the N-terminal region of pre-existing Tom40 molecules in the TOM complex plays a role in the assembly of incoming Tom40 molecules.
Most mitochondrial proteins are nuclear gene products that must be
synthesized on cytosolic ribosomes, imported into mitochondria, and
sorted to the correct mitochondrial subcompartment. These processes
require the concerted action of complex protein translocases located in
the outer and inner mitochondrial membranes (1-3). The process of
importing mitochondrial preproteins into the organelle is initiated by
the TOM complex (Translocase of the Outer
mitochondrial Membrane),1 which
recognizes mitochondrial preproteins in the cytosol. The TOM complex
facilitates the direct insertion of preproteins targeted to the outer
membrane as well as the passage of preproteins destined for the inner
mitochondrial compartments across the outer membrane. Further import
and sorting of these preproteins requires the action of the TIM
complexes (Translocases of the Inner
mitochondrial Membrane).
The Neurospora crassa TOM holo complex contains seven
different proteins: Tom70, Tom40, Tom22, Tom20, Tom7, Tom6 (4, 5), and
Tom5.2 Analogous proteins are
found in the Saccharomyces cerevisiae TOM complex, which
contains two additional components: Tom71 and Tom37 (6, 7). The large
cytosolic domains of Tom20, Tom22, and Tom70 act as receptors for
mitochondrial precursor proteins. The more tightly associated subunits
of the complex (Tom40, Tom22, Tom7, Tom6, and Tom5) form the TOM core
complex, which is essentially equivalent to the general insertion pore
(5, 8, 9). The general insertion pore serves as the major entry point
for all precursors entering mitochondria (10-15).
Tom40 has been shown to be an essential protein in S. cerevisiae (16). The protein can be cross-linked to precursor
proteins as they pass through the translocation pore (17, 18) and is the major component of the TOM complex pore in both S. cerevisiae and N. crassa (4, 19, 20). Based on
cross-linking studies to presequences, Tom40 may also form the major
portion of the precursor binding trans site on the
intermembrane space side of the outer membrane and contribute to the
cis binding site on the cytosolic side of the membrane (21).
Purified TOM complex and pores containing only Tom40 have similar
structural and electrophysiological properties (19, 22). The TOM
complex pore has been found to be about 20-26 Å in diameter by both
electron microscopic analysis and size exclusion studies (4, 5, 19, 22,
23).
In the TOM complex, Tom40 exists as an oligomer with dimers as the
basic structure (5, 8, 24, 25). However, cross-linking studies have
shown that, as precursors translocate, both the oligomer and the dimer
undergo changes that affect the spatial interactions between different
Tom40 molecules and between Tom40 and other TOM complex components
(24). Predictions of Tom40 structure have suggested that the N and C
termini extend into the intermembrane space. This is consistent with
experimental evidence showing that both termini can be removed by added
protease but only when the mitochondrial membrane is opened to allow
the protease access to the intermembrane space (20). The remainder of
the structure has been predicted to exist as a The integration and assembly of Tom40 itself into the mitochondrial
outer membrane requires the TOM complex and is only accomplished efficiently if the protein exists in a partially folded state (29).
Assembly of Tom40 into the TOM complex is thought to occur via
translocation intermediates (29-31). In the first step of
translocation, the Tom40 precursor binds at the outer surface of the
TOM complex as a monomer. This monomer is imported through the outer
membrane and assembled on the intermembrane space side of the membrane into an intermediate of 250 kDa that also contains pre-existing molecules of Tom40 and Tom5. There are two views for the mechanism by
which Tom40 integrates into the outer membrane. In one model, integration occurs when the precursor that is associated with other
components in the 250-kDa intermediate progresses to a 100-kDa intermediate that most likely contains Tom40 as a dimer of the newly
imported subunit and a pre-existing molecule. This intermediate undergoes further assembly and becomes associated with other Tom proteins to give the fully assembled TOM complex (30). In a second
model, Tom40 was thought to insert into the membrane directly from the
250-kDa form into the 400-kDa fully assembled complex (29). A conserved
set of amino acid residues near the N terminus of Tom40 is required for
assembly and stability of Tom40 within the TOM complex, but is not
involved in targeting of newly synthesized Tom40 to mitochondria
(32).
To date, there has been only one report of cells lacking Tom40.
S. cerevisiae cells depleted of the protein (then named
Isp42p) were shown to accumulate mitochondrial precursors in the
cytosol and ascospores lacking a functional copy of the gene were
inviable (16). To examine and more fully characterize the effects of Tom40 depletion in another organism, we have generated a null allele of
the gene in N. crassa that is maintained in a sheltered heterokaryon. We have also further investigated the assembly pathway of
Tom40 into the TOM complex.
Growth of N. crassa and Strains Used--
Growth and handling of
N. crassa strains was carried out as described previously
(28). Strains used in this study are listed below in Table I.
Race tubes were constructed as described previously (33).
Creation of Sheltered RIP Mutants--
Repeat induced point
mutation (RIP) occurs in N. crassa when one of the nuclei
involved in a sexual cross carries a duplicated DNA sequence. RIP
results in the generation of GC to AT transitions in both copies of the
duplication (34). Because Tom40 was predicted to be an essential
protein in N. crassa, the procedure of sheltered RIP was
used to mutate the tom40 gene. Sheltered RIP insures that nuclei containing non-functioning alleles are present in a heterokaryon that also contains nuclei with a wild type copy of the gene. The rationale and strains utilized for sheltered RIP are described in
detail elsewhere (35, 36). Briefly, molecular restriction fragment
length polymorphism mapping studies (37, 38) using a
tom40-containing cosmid as a probe revealed that the
tom40 gene was located on linkage group V of N. crassa. To create a duplication of tom40, spheroplasts
of the HostV strain for sheltered RIP were transformed with a plasmid
(pRIP-4) carrying hygromycin resistance (39) and 1.8 kb of
PCR-generated tom40 genomic sequence. Transformants were
selected on hygromycin, and strains containing duplications were
identified by Southern analysis. One strain, 40Dupl, was crossed to the
MateV strain. Any ascospores from this cross that were capable of
growth on minimal medium were determined to be heterokaryons. One
nucleus of the heterokaryon was wild type with respect to Tom40
function, whereas the other could contain either RIPed or wild
type tom40 alleles. The desired heterokaryotic strain is
shown in Fig. 1.
Several ascospore isolates from the sheltered RIP cross were screened
for the predicted characteristics of
tom40RIP-sheltered heterokaryons. When
heterokaryons with the genotype shown in Fig. 1 were grown in media
containing lysine, leucine, and cycloheximide (concentrations ranging
from 30 to 50 µg/ml) the RIPed nucleus was forced to predominate the
heterokaryon to provide resistance to the antibiotic. As a result, the
growth rate was severely reduced, because Tom40 could not be supplied by the nucleus providing cycloheximide resistance. One strain (RIP40het), which grew slowly under these conditions, was analyzed further. Southern analysis (not shown) indicated that, in addition to
the gene at the endogenous tom40 locus, the ectopically
integrated RIP substrate was also present in the strain. Using specific
primers, both RIPed versions of the gene were amplified by PCR. The
gene at the endogenous locus was cloned and sequenced entirely. A total of 92 RIP mutations, including a RIP generated stop codon at residue 35, were identified. About 270 base pairs of the ectopic
tom40 sequence were determined directly from specific PCR
products, and 9 RIP mutations were observed, including one that created a stop codon at residue 30. For both RIPed alleles, the stop codons occurred prior to the first predicted membrane-spanning domain (26, 27)
of the protein, and we consider these to be effectively null alleles.
We have briefly described the use of this strain for developing mutants
expressing exclusively variants of Tom40 in a previous report (32).
Transformation of N. crassa--
DNA was transformed into
N. crassa using spheroplasts as described previously (40,
41) or by electroporation of conidia using modifications of a
previously described technique (42, 43). For electroporation, conidia
(1 week old) were harvested, washed three times with 1 M
sorbitol, and resuspended in 1 M sorbitol at a
concentration of 2 to 2.5 × 109 conidia/ml.
Linearized plasmid DNA (5 µg in a final volume of 5 µl) was mixed
with 40 µl of conidia, placed in a pre-chilled electroporation
cuvette, and incubated for 5 min on ice. A Gene Pulser (Bio-Rad,
Hercules, CA) was used with settings of 2.1 kV, 475 Creation of Strains Expressing Mutant Variants of Tom40--
The
method for development of strains expressing only mutant versions of
Tom40 was described previously (32). Briefly, mutant alleles of
tom40 were constructed by site-directed mutagenesis of
single-stranded DNA derived from a Bluescript plasmid derivative containing a genomic version of N. crassa tom40 and a
bleomycin resistance gene (44). Plasmids confirmed to carry the desired mutations were transformed into the
tom40RIP-sheltered heterokaryon (RIP40het). Plasmids
used to develop strains expressing altered Tom40s in this study
contained deletions in the region between residues 50 and 60 of the
N. crassa Tom40 protein. Plasmids pRD, pRDTLL, and p297 lack
the residues, RD, RDTLL, or all of residues 51-60, respectively.
Another plasmid, p16.6, encoded a Tom40 variant where residues KLG at
position 321-323 were changed to alanine residues. Transformants were
selected on media containing cycloheximide (50 µg/ml), lysine, and
leucine (to select for transformants of the nucleus carrying the
tom40RIP alleles), as well as bleomycin (1.5 µg/ml) and caffeine (0.5 mg/ml, to enhance the action of bleomycin),
purified through one round of single-colony isolation on the same
selective medium and tested for nutritional requirements. Plasmids
giving rise to homokaryons requiring lysine and leucine contain mutant
alleles capable of restoring Tom40 function to a level sufficient for viability. The presence of mutant alleles in the transformants was
confirmed by sequencing tom40-specific PCR products
generated from genomic DNA.
Creation of Tom40 Variants for in Vitro Import Studies--
A
tom40 cDNA was cloned into plasmid pGEM7Zf(+). Mutant
alleles were created by site-directed mutagenesis and used for in vitro transcription and translation to produce mutant Tom40
precursor proteins for import into isolated mitochondria.
Import of Radiolabeled Proteins into Isolated
Mitochondria--
For in vitro import studies, the
isolation of mitochondria (45), import of Tom40 precursors (32), and
import of other mitochondrial precursor proteins (46) were as
described. Import was analyzed by SDS-PAGE or blue native gel
electrophoresis (BNGE) and gels were viewed by autoradiography.
For some experiments, carbonate extraction was performed to determine
if imported precursor proteins were inserted into membranes. Mitochondria were suspended in 0.1 M sodium carbonate (pH
11) for 30 min at 0 °C. The mixture was centrifuged at 20,000 rpm in
a TLA55 rotor (Beckman Instruments, Palo Alto, CA) for 30 min at
2 °C, and the pellets were processed for BNGE.
Blue Native Gel Electrophoresis--
Mitochondria (50-100 µg)
were solubilized in 50 µl of buffer containing detergent (either 1%
digitonin or 1% dodecyl maltoside in 20 mM Tris-Cl, pH
7.4; 0.1 mM EDTA; 50 mM NaCl; 1% glycerol; 1 mM phenylmethylsulfonyl fluoride). After gentle mixing at
4 °C for 15 min and a clarifying spin (30 min, 14,000 × g), 5 µl of sample buffer (5% Coomassie Brilliant Blue
G-250 in 100 mM Bis-Tris, 500 mM 6-aminocaproic
acid, pH 7.0) was added, and the mixture was analyzed on a 6-13%
gradient blue native gel (47, 48).
Electron Microscopy--
One milliliter of cell suspension from
liquid cultures was fixed in 1.5% KMnO4 for 30 min at room
temperature followed by several washes with distilled water. The sample
was then suspended in 0.05 M sodium cacodylate buffer
containing 2% glutaraldehyde and 15% sucrose. The cells were pelleted
by brief centrifugation at room temperature in a clinical centrifuge
and were resuspended in the same buffer. After a 30-min incubation on
ice, cells were post-fixed in 1% (w/v) OsO4 and 1.5%
(w/v) K2Cr2O7 for 90 min on ice.
Samples were then post-stained in 1% (w/v) uranyl acetate overnight at
room temperature. The steps of dehydration, embedding, and sectioning
were performed by the Microscopy Unit, Department of Biological
Sciences, University of Alberta. Sections were examined in a
transmission electron microscope.
Other Techniques--
The standard techniques of agarose gel
electrophoresis, Southern and Northern blotting of agarose gels,
preparation of radioactive probes, transformation of Escherichia
coli, isolation of bacterial plasmid DNA, and the PCR using a
mixture of Taq and Vent polymerase (New England
BioLabs, Beverly, MA) to minimize replication errors, were all
performed as described (49). The following procedures were employed
using the supplier's recommendations or previously described
procedures: isolation of total RNA with the Qiagen RNeasy plant mini
kit (Qiagen Inc., Santa Clarita, CA), separation of mitochondrial
proteins by polyacrylamide gel electrophoresis (50), Western blotting
(51), Western blot detection using LumiGLO chemiluminescent substrate
(Kirkegaard and Perry Laboratories, Gaithersburg, MD), genomic DNA
extraction (52), protein determination with the Coomassie dye binding
assay (Bio-Rad, Hercules, CA), manual DNA sequencing using
thermosequenase (Amersham Biosciences, Cleveland, OH), and automated
sequencing using a DyeNamic sequencing kit (Amersham Biosciences) with
a Model 373 stretch sequencer separation system (Applied Biosystems,
Foster City, CA), and site-directed mutagenesis using the Muta-Gene
system (Bio-Rad). Radioactive precursor proteins for import were
generated by coupled in vitro transcription and translation
with the Promega (Madison, WI) TNT reticulocyte lysate
system in the presence of [35S]methionine (ICN, Costa
Mesa, CA).
Isolation of N. crassa tom40 Null Mutants--
Because Tom40 is
the major component of the mitochondrial outer membrane translocation
pore (22) and is an essential gene in yeast (16), we used
the procedure of sheltered RIP to obtain N. crassa tom40
null mutants. The product of the procedure was a heterokaryotic strain
(designated RIP40het) in which the tom40 gene in one nucleus
is inactivated by RIP, whereas the other nucleus retains a wild type
copy of the gene (Fig. 1). Sequence
analysis revealed that the tom40RIP-bearing nucleus
contains only null alleles of the gene (see "Materials and
Methods"). To determine if tom40 is an essential gene in
N. crassa, conidiaspores produced by RIP40het were streaked
onto medium containing all the nutritional requirements of both nuclei in the heterokaryon (Fig. 1 and Table I).
N. crassa conidiaspores are usually multinucleate and the
heterokaryon should produce three separate types of conidiaspores,
assuming random segregation of nuclei into conidia: homokaryons for the
lysine-leucine-requiring tom40RIP nucleus,
homokaryons for the inositol-requiring sheltering nucleus, and
heterokaryons containing both nuclei. If tom40 is essential, no lysine-leucine-requiring conidia should be viable. Testing of
nutritional requirements of 181 individual colonies isolated from these
plates revealed that 120 were heterokaryons, 61 were inositol-requiring
homokaryons, and none were lysine-leucine-requiring homokaryons. Thus,
the tom40RIP nucleus was inviable. To confirm that
the effects of RIP were specific to the tom40 gene, the
sheltered heterokaryon was transformed with a bleomycin resistance
plasmid containing a wild type copy of tom40. When selected
on media containing lysine, leucine, cycloheximide, and bleomycin,
viable lysine-leucine-requiring homokaryotic strains were recovered.
Considered together, these data show that tom40 is an
essential gene in N. crassa.
Characteristics of tom40-deficient Cells--
The two nuclei in
RIP40het differ with respect to auxotrophic and antibiotic resistance
markers, which make it possible to force the
tom40RIP nucleus to predominate the heterokaryon by
growth in medium containing lysine, leucine, and cycloheximide (Fig.
1). Under these conditions, the growth rate of RIP40het was clearly
reduced (Fig. 2A), because the
cycloheximide resistance-conferring tom40RIP nucleus
was unable to supply sufficient levels of Tom40 when it predominated in
the heterokaryon. Analysis of mitochondrial proteins in RIP40het grown
under these conditions showed the predicted reduction in Tom40 (Fig.
2B). BNGE of the TOM complex from Tom40-deficient mitochondria revealed no differences in size or stability (Fig. 2C), demonstrating that the complex forms normally in the
mutant but is simply reduced in amount.
Mitochondria deficient in Tom40 were also examined with respect to the
levels of other mitochondrial proteins, including other members of the
TOM complex. The level of the TOM holo-complex receptor proteins Tom70
and Tom20, as well as the mitochondrial proteins Hsp70 and porin were
unaffected by the reduction in Tom40 levels. However, the amounts of
the TOM core complex proteins, Tom22 and Tom6, were severely reduced
(Fig. 2B). To rule out the possibility that the Tom40
deficiency might signal a down-regulation of transcription of the
tom22 and tom6 genes, we analyzed Northern blots
for the presence of tom40, tom22, and
tom6 mRNAs. The level of tom40 transcript is
severely reduced in RIP40het cells grown under conditions where the
tom40RIP nucleus predominates the culture (Fig.
3). This was expected, because genes that
have undergone severe RIP produce little or no transcript (53, 54). We
were unable to detect a transcript for tom6 in any control
or mutant strain (not shown), suggesting that few transcripts are
produced and/or that they are short-lived. However, transcripts of
tom22 were present in normal amounts in Tom40-deficient
cells (Fig. 3) so that the decreased levels of this protein, and by
analogy Tom6, were not due to decreased levels of transcription. Thus,
the data suggest that either the import and/or stability of Tom22 and
Tom6 in the membrane is/are dependent on assembly into the core complex
with Tom40. It should be noted that the reduction in these two
components was not due to a generalized decrease in import capacity
that would be expected in Tom40-deficient mitochondria, because other
mitochondrial proteins were present at normal levels (Fig.
2B). It is likely that the reduced growth rate of the cells
reflects the rate at which mitochondrial proteins can be imported so
that their steady-state levels do not differ significantly from wild
type cells. This interpretation is supported by the observation that
import of mitochondrial precursors into isolated Tom40-deficient
mitochondria was reduced (Fig. 4).
To determine the structure of Tom40-deficient mitochondria, cells
depleted of the protein by growth in cycloheximide were examined by
electron microscopy. Mitochondria with reduced levels of Tom40 were
smaller than controls and contained virtually no cristae (Fig.
5).
A Region Near the N Terminus of Tom40 Affects Assembly and
Stability of the TOM Complex--
Little is known about the domains
and amino acid residues of Tom40 that are necessary for it to
accomplish its functions or for its own assembly into the TOM complex.
Alignment of the protein from several organisms reveals the existence
of several conserved residues (Fig. 6).
We had previously shown that a conserved region near the N terminus
(residues 40-48) was important for assembly of N. crassa
Tom40 into the TOM complex (32). The region is followed by another set
of residues (51-60) that are very well conserved between yeast and
N. crassa and reasonably well conserved between fungi and
animals (Fig. 6). To determine if these residues were also important
for the assembly of Tom40, we developed cDNA versions of the
gene-encoding variants lacking the RD residues (
Genomic versions of all three mutant variants were found to rescue the
tom40RIP nucleus of strain RIP40het. Strains
containing the A Conserved C-terminal Region of Tom40 Is Also Required for
Assembly--
To examine the effects of mutations outside the
N-terminal region on Tom40 assembly, we chose a block of residues in
the most C-terminal region of similarity between all the species shown in Fig. 6. Mutant versions of N. crassa tom 40 cDNA in
which the residues KLG at position 321-323 were either deleted
( Nature of TOM Complex Assembly Intermediates--
We consistently
observed a high molecular weight band on our blue native gels of Tom40
import experiments that had not been discussed in previous reports. We
estimated the band to be ~500 kDa in size. The band did not appear in
substantial amounts when import was stalled at the 250-kDa stage (Fig.
7B and 8B). To assess the relevance of the
500-kDa band we performed import experiments where Tom40 precursor was
added for only a 4-min pulse at 25 °C. The kinetics of precursor
assembly were then followed over a period of 240 min at 25 °C. The
experiment showed that the amount of the 500-kDa form did not change
appreciably over the course of the experiment (Fig.
9). Thus, the 500-kDa band most likely
represents Tom40 precursor in a non-productive state. This experiment
also showed the precursor-to-product relationship between the 100- and
250-kDa intermediates and the fully assembled 400-kDa form, because the
amount of radioactive Tom40 precursor in the intermediates gradually
decreases as more radioactivity accumulates in the 400-kDa form (Fig.
9). When the levels of radioactivity in the intermediates were
quantified for each time point, we could not demonstrate that the
appearance of Tom40 precursor in the 250-kDa form precedes its
appearance in the 100-kDa intermediate as previously reported (30). In
our experiments the Tom40 precursor appeared in both intermediates
simultaneously. The rate of disappearance of the precursor from each
intermediate, as Tom40 assembled to the 400-kDa form, was also
virtually identical. Attempts to delay the appearance of the
intermediates by lowering the temperature of import to 10 or 15 °C,
and shortening the pulse time to 1 min, reduced the rate of assembly
but revealed no differences in precursor product relationships (not
shown). However, results obtained during studies of import and assembly
of mutant Tom40 precursors did support the hypothesis that the 250-kDa
intermediate is formed prior to the 100-kDa form (see below).
Carbonate Treatment Enhances the Assembly of Wild Type Tom40
Precursor into the TOM Complex--
Based on the results of sodium
carbonate extraction experiments it was previously suggested that a
newly imported Tom40 precursor in the 250-kDa intermediate is
peripherally associated with the mitochondrial outer membrane in the
intermembrane space. On the other hand, when the precursor progresses
to the 100-kDa intermediate and the 400-kDa assembled form, it becomes
an integral membrane protein (30). We attempted to confirm the
carbonate extractability of Tom40 precursor in the 250-kDa intermediate
and did observe that the amount of the intermediate form was reduced
following extraction of imports performed at 25 °C. However, the
interpretation of these data was not straightforward, because we also
observed that newly imported Tom40, which accumulates at intermediate
steps when import is performed at 0 °C, was assembled into the
400-kDa form of the TOM complex as a result of carbonate treatment.
This is shown in Fig. 10 where the
expected accumulation of Tom40 precursor in the 250-kDa form during
import at 0 °C was seen in the untreated samples and very little of
the fully assembled 400-kDa complex was present, even after 60 min of
import (Fig. 10A, Tom40wt lanes). This contrasts
with results obtained when import was performed at 25 °C, when most
of the newly imported precursor was present in the fully assembled form
(Fig 10A, Tom40wt lanes). However, after
mitochondria were subjected to carbonate extraction, much of the
precursor imported at 0 °C was present in the fully assembled complex (Fig. 10B, Tom40wt lanes). The trivial
explanation that further assembly to the 400-kDa form occurs as a
result of the extra time at 0 °C during incubation with sodium
carbonate cannot account for the data, because the untreated samples
(panel A) were handled in an identical fashion except for
the addition of carbonate. These data demonstrate that assembly of
precursors into the 400-kDa form is stimulated by the addition of
carbonate. Thus, we cannot confirm the carbonate extractability of
Tom40 precursor from the 250-kDa intermediate, because the possibility that Tom40 in the intermediate may have assembled into the 400-kDa form
during the carbonate treatment cannot be excluded. It was possible that
the newly imported Tom40 in the 400-kDa complex formed during carbonate
treatment did not contain correctly assembled Tom40. To test this
possibility we treated samples with proteinase K, which gives rise to
characteristic 26- and 12-kDa cleavage products when the protein is
correctly assembled (29, 32). These fragments can be clearly seen in
samples not treated with sodium carbonate (Fig. 10C). The
carbonate-treated samples also show evidence of these bands, although
both are reduced in amount and the 26-kDa fragment is present as a
series of shortened fragments (Fig. 10C). This is most
likely because Tom40 in the membrane sheets created by the action of
carbonate is fully exposed to the added proteinase and undergoes more
degradation than Tom40 in intact mitochondria. We conclude that the
material in the 400-kDa form that arose by the action of carbonate
represents correctly assembled Tom40.
Mutant Tom40 Precursors in the 250-kDa Assembly Intermediates Are
Extractable by Carbonate--
The precursor of the
Further confirmation that Tom40 precursor in the 250-kDa form is
extractable with carbonate was obtained in similar experiments using
the precursor form of the The Assembly Characteristics of Mutant Tom40 Variants Provide
Evidence That Formation of the 250-kDa Intermediate Precedes Formation
of the 100-kDa Intermediate--
Our results on the assembly of the
An Alternative Assembly Pathway for Tom40 in Mitochondria
Containing Mutant TOM Complex--
It was of interest to determine how
effectively Tom40 precursors would be imported/assembled into
mitochondria with TOM complex containing only mutant versions of Tom40.
Wild type Tom40 precursor proteins were imported into mitochondria
isolated from a strain developed by rescue of the null nucleus of
RIP40het with the Tom40 was previously shown to be essential for the viability of
S. cerevisiae cells and reduced levels of Tom40 resulted in the accumulation of mitochondrial precursors in the cytosol (16). We
have confirmed these findings by showing that tom40 is an
essential gene in N. crassa and that mitochondria containing
reduced levels of Tom40 are deficient in their capacity to import
precursor proteins in vitro. We have extended the original
findings by showing that mitochondria with lowered levels of Tom40 are
smaller than normal and are devoid of cristae. It is probable that
growth and accumulation of mitochondria are limited by the deficiency
of Tom40 and that the reduction in growth rate of cells with depleted
levels of the protein is related to the reduced capacity to accumulate
essential factors in the organelle. Similar mitochondria are observed
in slow growing cells deficient in other important components of the
TOM complex such as Tom20 and Tom22 (12, 46). Mitochondria deficient in
Tom40 also have reduced levels of the TOM core complex components Tom22
and Tom6. These deficiencies are most likely due to the inability to
import and/or assemble the proteins in the absence of Tom40. The
possibility that reduced levels of Tom40 might signal reduced
transcription of these genes was eliminated by demonstrating that the
mRNA for Tom22 is present at normal levels.
We have previously shown that a well conserved region near the N
terminus of Tom40 results in TOM complex assembly defects (32). The
previously described mutant, lacking residues 40-48 of the N. crassa Tom40 protein, was less efficient at assembling into the
complex than wild type Tom40 precursors. Here we show that an adjacent
region has similar effects. The The lack of assembly to any high molecular weight intermediate of the
It has been suggested that Tom40 precursor appears in the 250-kDa
intermediate prior to the 100-kDa form on the assembly pathway (30). We
were unable to confirm this in time course experiments using wild type
Tom40 precursor, because both forms appeared and decreased
concomitantly. When the assembly of mutant Tom40 molecules was
examined, many variants gave rise to both the 250- and the 100-kDa
intermediates. Because both intermediates can be seen during the import
of wild type and various mutant forms of the precursor, it appears that
neither represents a strictly rate-limiting step on the assembly
pathway. Nonetheless, the existence of the 250-kDa form with no
evidence of the 100-kDa intermediate in the Analysis of the import and assembly of wild type Tom40 precursors into
mitochondria containing only the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-barrel, similar to
bacterial porins (26, 27), with 14 anti-parallel
-strands spanning the mitochondrial outer membrane. A high level of
-sheet was seen in
Tom40 expressed in bacteria and refolded from exclusion bodies (19),
but spectral analysis of Tom40 purified directly from mitochondria
revealed less
-sheet and more
-helix than predicted (22).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, 25 microfarads. Immediately after the pulse (time constant, 11-12 ms), 1 ml of ice-cold 1 M sorbitol was added and the conidia were
allowed to recover for 1 h at 30 °C. Aliquots (10-100 µl) of
the mixture were added to top agar containing appropriate antibiotics for selection of transformants, and the mixture was poured onto plates
containing the same medium.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
[in a new window]
Fig. 1.
Sheltered heterokaryon for the
tom40RIP mutant. The box
symbolizes the heterokaryotic RIP40het strain used in this study,
containing two distinct nuclei whose genotypes are enclosed by
circles. Nucleus 1 contains no functional copies
of tom40; nucleus 2 contains a wild type allele
of the gene. Other markers necessary for the manipulation of the
heterokaryon are indicated. The mutant allele of the cyh-2
gene provides resistance to cycloheximide. The heterokaryon was
produced as described under "Materials and Methods."
Strains used in this study
View larger version (23K):
[in a new window]
Fig. 2.
Characteristics of Tom40-deficient
cells. A, the control strain (40Dupl) was grown in the
presence of lysine and leucine (control, diamonds) and
RIP40het was grown in minimal medium (RIP40het, triangles)
to compare their growth rates under conditions where complementation of
Tom40 function occurs in the sheltered heterokaryon. To assess the
growth rate of Tom40-deficient cells, the RIP40het strain was grown in
medium containing lysine, leucine, and cycloheximide at 50 µg/ml
(RIP40het + CHI, squares), which forces the
tom40RIP nucleus to predominate the culture. The
cycloheximide resistant control strain was grown under similar
conditions (control + CHI, crosses). B,
mitochondria were isolated from the indicated strains grown under the
conditions described in A. Mitochondrial proteins were
separated by SDS-PAGE, blotted to nitrocellulose, and immunodecorated
with antisera directed against the indicated proteins (mt,
mitochondrial). C, mitochondria from the control strain
(40Dupl) and RIP40het grown in the presence of lysine, leucine, and
cycloheximide were solubilized in either digitonin (DIG) or
dodecyl maltoside (DDM) and analyzed by BNGE. The gel was
blotted to PVDF membrane and immunodecorated with antiserum against
Tom40. The positions of molecular weight markers are indicated on the
left.
View larger version (61K):
[in a new window]
Fig. 3.
The mRNA for Tom22 is not deficient in
cells lacking Tom40. Control cells (40Dupl) and RIP40het cells
were grown as described for Fig. 2A. RNA was isolated from
the cultures and electrophoresed on formaldehyde-agarose gels. The gel
was stained with ethidium bromide, photographed, and blotted to
nitrocellulose. The blot was cut in half and hybridized with
32P-labeled DNA specific for either tom40 or
tom22. Top portion: ethidium bromide-stained gel.
Bottom portion: autoradiogram of the blots following
hybridization with the probe indicated at the bottom of the
figure. The positions of molecular weight markers (kb) are shown on the
left.
View larger version (35K):
[in a new window]
Fig. 4.
Tom40-deficient mitochondria have reduced
ability to import mitochondrial precursor proteins. Mitochondria
were isolated from the control strain (40Dupl) and RIP40het following
growth in lysine, leucine, and cycloheximide (30 µg/ml) so that
mitochondria in RIP40het contained reduced levels of Tom40. Import of
radiolabeled precursors (F1 , the
-subunit of
the F1-ATPase; MPP, the mitochondrial processing
peptidase; and AAC, the ATP/ADP carrier protein) was
performed at 20 °C for the times shown. Following a post-import
treatment with proteinase K to remove non-imported precursors,
mitochondria were re-isolated and subjected to SDS-PAGE. The gels were
blotted to nitrocellulose and exposed to x-ray film. The lysate lane
contained 33% of the input lysate used in each import reaction. The
precursor (p) and mature (m) forms of MPP and
F1
are indicated. One sample from each strain was
treated with trypsin prior to import (Pre-trypsin) to
demonstrate that import was receptor-dependent.
View larger version (185K):
[in a new window]
Fig. 5.
Appearance of Tom40-deficient
mitochondria. The cycloheximide-resistant control strain (40Dupl)
and RIP40het were grown under the conditions described in the legend to
Fig. 2A. At the indicated times, mycelium was harvested and
processed for electron microscopy as described under "Materials and
Methods."
RD), the RDTLL
residues (
RDTLL), and the ten residues from position 51-60
(
51-60) (Fig. 7A). These
were transcribed and translated in vitro to generate Tom40
precursor proteins for import into isolated mitochondria. Wild type
Tom40 precursor has been shown to accumulate in a 250-kDa intermediate
when imported into isolated mitochondria at 0 °C (29, 32). The most
recent model of Tom40 assembly suggests that the precursor further
assembles into the 400-kDa TOM complex via a 100-kDa intermediate
during import at 25 °C (30). All three deletion variants had a
reduced capacity to be imported into the fully assembled TOM complex at 25 °C and tended to accumulate in the high molecular mass
250-kDa assembly intermediate (Fig. 7B). A fraction of the
RD and
RDTLL precursors did reach the assembled state, suggesting
a relatively mild affect on assembly. However, the
51-60 variant
was more severely affected, and none of this variant became fully
assembled after 20 min of import at 25 °C. There was a similar
gradient of effect on the formation of the 100-kDa intermediate, which was not present as a discrete band at either temperature when the
51-60 variant was imported.
View larger version (101K):
[in a new window]
Fig. 6.
Alignment of Tom40 proteins. The Tom40
proteins of N. crassa (Nc), S. cerevisiae (Sc), Schizosaccharomyces pombe
(Sp), Mus musculus (Mm),
Caenorhabditis elegans (Ce), and Drosophila
melanogaster (Dm) are shown. The number of residues in
each protein is indicated to the right. Black
shading indicates amino acid identity in at least four of the six
species shown. Gray shading indicates amino acids of the
same family in at least four of the six species.
View larger version (82K):
[in a new window]
Fig. 7.
Assembly of Tom40 N-terminal variants into
the TOM complex. A, a region of the alignment from Fig.
6 is shown to illustrate the location and residues affected in the
Tom40 variants RD,
RDTLL, and
51-60. Deleted residues are
indicated by dashes. For the
51-60 mutant, residue 50 was also changed from a Glu to an Ala (lowercase a).
Organisms in the comparison are as defined in the legend to Fig. 6.
B, radiolabeled precursors of a wild type Tom40
(Tom40wt) and the indicated variants were incubated at
either 0° or 25 °C with wild type (74A) mitochondria
for 20 min. Mitochondria were re-isolated and solubilized in buffer
containing 1% digitonin. The samples were electrophoresed on blue
native gels, blotted to PVDF membrane, and analyzed by autoradiography.
The positions of the 400-kDa TOM complex and the 100- and 250-kDa
intermediates are indicated. M, Tom40 monomer. C,
growth phenotype of
51-60 strains. Conidia from a
51-60 strain
(open squares) and a control strain (40Dupl, closed
circles) were inoculated into flasks containing liquid medium,
grown with shaking at 15 °C, and harvested at the indicated times.
D, BNGE analysis of N-terminal deletion strains.
Mitochondria were isolated from a control (40Dupl) and strains
expressing the Tom40 variants
RD,
RDTLL, or
51-60. The
organelles were solubilized with either digitonin (DIG) or
dodecyl maltoside (DDM) and examined by BNGE. The gel was
blotted to PVDF membrane and decorated with antiserum to Tom40. The
position of molecular weight markers is indicated on the
left. E, SDS-PAGE analysis. Mitochondria from
control strain (40Dupl) and a
RDTLL mutant strain were dissolved
with Laemmli gel cracking buffer and subjected to SDS-PAGE. The gel was
blotted to nitrocellulose and immunodecorated with antiserum to Tom40
and Tom22.
RD and
RDTLL forms of Tom40 exhibit only slightly
reduced growth rates and an inability to climb the walls of growth
flasks (not shown). Despite the finding that virtually none of the
51-60 variant assembled into the TOM complex in vitro,
the rate of assembly in vivo must be sufficient to allow
viability, because strains expressing only this version of Tom40 were
obtained. However, growth defects in the
51-60 strains were evident
and were enhanced at 15 °C where the deletion strains grew at
roughly half the rate of controls (Fig. 7C). The
51-60
strains also produced fewer conidiaspores than the strains containing
the milder variants (not shown). Thus, the level of assembly of these
Tom40 variants that was observed in vitro correlates with
the severity of phenotype observed in vivo. In addition,
when analyzed by BNGE, the TOM complex in each of the deletion strains
appears to be more fragile than in controls when mitochondria are
solubilized with dodecyl maltoside and break down into a series of
smaller complexes (Fig. 7D). A striking deficiency of Tom40
also appears in the lanes containing mitochondria from the
51-60
strain. When mitochondria from this strain were solubilized in SDS, no
difference in the amount of Tom40 was observed relative to controls
(Fig. 7E). Therefore, the deficiency of Tom40 observed
following BNGE may have been due to aggregation of the complex from
this strain, resulting in an inability to enter the gel.
321-3) or changed to Ala residues (321AAA) were constructed (Fig.
8A). The variants were
transcribed and translated in vitro to produce mutant
precursor proteins for use in import studies. The import and assembly
of the 321AAA precursor was similar to the wild type precursor,
although assembly to the final 400-kDa complex was somewhat less
efficient (Fig. 8B). On the other hand, the
321-3 variant did not progress to the 250-kDa intermediate during import at
0 °C and did not reach the fully assembled complex at either temperature. Thus, the deletion of the KLG residues dramatically affects the ability of Tom40 precursors to assemble into the TOM complex, whereas changing the residues to alanines has little effect.
In keeping with the severe assembly defects observed in vitro, we were unable to rescue the tom40RIP
nucleus with constructs containing the
321-3 construct. However, using the 321AAA construct, strains with growth defects were isolated (Fig. 8C). BNGE analysis of mitochondria isolated from
321AAA strains revealed striking defects in TOM complex stability (Fig. 8D). When solubilized in the mild detergent digitonin, a
substantial fraction of Tom40 was not present in the 400-kDa fully
assembled form and migrated at a position that would correspond roughly to a Tom40 dimer. From these data it is impossible to determine if the
smaller form of the complex forms during solubilization and BNGE or if
it exists in vivo. Solubilization of mitochondria containing
the 321AAA Tom40 variant in dodecyl maltoside results in
Tom40 being released as a monomer. We have previously shown that the
C-terminal extension in fungal Tom40 proteins (residues 330-349 of the
N. crassa protein, Fig. 6) is not required for assembly (32)
or function of the TOM
complex.3 TOM complex
containing the variant lacking this fungal C-terminal extension
(residues 330-349, Fig. 6) was virtually indistinguishable from
controls in terms of TOM complex stability on blue native gels (Fig.
8D). Thus, instability of the complex is not caused by
general perturbations of the protein at the C terminus. The role of
residues 321-323 in the function and stability of the TOM complex was
further demonstrated by the fact that mitochondria containing the
321AAA variant imported mitochondrial precursors less efficiently than
control mitochondria (Fig. 8E).
View larger version (79K):
[in a new window]
Fig. 8.
Assembly of the
321-3 and 321AAA variants of Tom40.
A, a region of the alignment from Fig. 6 is shown to
illustrate the location and residues affected in the Tom40 variants
321AAA and
321-3. Deleted residues are indicated by
dashes, and amino acid changes are shown as lowercase
letters. Organisms in the comparison are defined in the legend to
Fig. 6. B, radiolabeled precursors of wild type Tom40
(Tom40wt) and variants
321-3 and 321AAA were imported
into wild type (74A) mitochondria for 20 min at either 0°
or 25 °C. Mitochondria were re-isolated and solubilized in 1%
digitonin. The samples were electrophoresed on blue native gels,
blotted to PVDF membrane, and analyzed by autoradiography. The
positions of the 400-kDa TOM complex and the 100- and 250-kDa
intermediates are indicated. M, Tom40 monomer. C,
growth of the 321AAA mutant. A control strain (40Dupl, square
symbols) and a 321AAA mutant strain (circles) were
inoculated in race tubes and incubated at either 22 °C (filled
symbols) or 15 °C (open symbols). The extent of
mycelial elongation was measured daily. D, BNGE analysis of
C-terminal mutant strains. Mitochondria were isolated from a control
(40Dupl) and strains expressing the Tom40 variants 321AAA or a
C-terminal deletion of Tom40 lacking residues 330-349
(
C-term). Mitochondria were solubilized with either
digitonin (DIG) or dodecyl maltoside (DDM) and
examined by BNGE. The gel was blotted to PVDF membrane and decorated
with antiserum to Tom40. The position of molecular weight markers is
indicated on the left. E, import of precursors
into 321AAA mitochondria. Import into mitochondria isolated from a
control strain (40Dupl), and a 321AAA strain was performed as described
in the legend to Fig. 4 using the precursors of MPP, the mitochondrial
processing peptidase, and Su-9 dihydrofolate reductase, a fusion of the
N-terminal 69 amino acids of N. crassa ATPase subunit 9 to
the coding sequence of mouse dihydrofolate reductase (57).
View larger version (73K):
[in a new window]
Fig. 9.
Kinetics of TOM complex assembly. Import
of radiolabeled wild type Tom40 precursor was allowed to proceed into
wild type (74A) mitochondria for 4 min at 25 °C.
Mitochondria were re-isolated at 4 °C and resuspended in fresh
import mix containing no additional Tom40 precursor. Import was allowed
to continue at 25 °C, and aliquots were removed at the times
indicated. Mitochondria were re-isolated from the aliquots and
processed for BNGE. The gel was blotted to PVDF membrane and analyzed
by autoradiography. The sizes and position of bands are indicated on
the left.
View larger version (42K):
[in a new window]
Fig. 10.
Sodium carbonate treatment
results in assembly of Tom40 precursors into the TOM complex. For
A and B, radiolabeled precursors of wild type
Tom40 and variant 51-60, were imported into wild type
(74A) mitochondria at 0° and 25 °C for the times
indicated. The sample was divided equally, and mitochondria were
pelleted. One tube from each import was held on ice while the
mitochondria in the other were resuspended in 0.1 M sodium
carbonate. After 30 min on ice, the membrane fraction from the
carbonate-treated samples and the untreated mitochondrial samples were
pelleted at 2 °C. Both pellets were suspended in 1% digitonin and
processed for BNGE. A, BNGE of untreated samples;
B, BNGE of carbonate-treated samples; C, import
of radiolabeled wild type Tom40 precursor into wild type
(74A) mitochondria was done for 20 min at either 0° or
25 °C. For each temperature the experiments were performed in
quadruplicate, and mitochondria were pelleted. In the first sample, the
pellets were solubilized in 1% digitonin and prepared for BNGE. In the
second sample, the mitochondria were treated with sodium carbonate. The
resulting membrane fraction was solubilized in 1% digitonin and
processed for BNGE. In the third sample, the pellets were suspended in
import buffer, and treated with proteinase K (0.1 µg/µl) for 15 min. Mitochondria were re-isolated and processed for SDS-PAGE. In the
fourth sample, mitochondria were subjected to sodium carbonate
extraction, and the membrane fraction was pelleted, resuspended in
import buffer, and treated with proteinase K as for the third sample.
The membranes were re-isolated and processed for SDS-PAGE. Samples not
treated with proteinase K and analyzed by BNGE are shown on the
left. Samples treated with proteinase K and examined by
SDS-PAGE are on the right. Gels were blotted to PVDF
membrane (BNGE) or nitrocellulose (SDS-PAGE) and analyzed by
autoradiography. For all panels, the positions and size of bands are
indicated on the left. M, Tom40 monomer.
51-60 Tom40
variant accumulated at the 250-kDa intermediate stage during import at
both 0° and 25 °C (Fig. 7B). We wished to determine if
the variant protein arrested at this stage would also assemble into the
400-kDa complex as the result of carbonate treatment. As shown in Fig.
10A (
51-60 lanes), the mutant form accumulates at the
250-kDa stage even after 60 min of import at 25 °C. Unlike the wild
type precursor at this stage, the variant was not efficiently converted
to the assembled form in the presence of sodium carbonate (Fig.
10B), and a substantial amount of the protein in the
intermediate was removed (compare panels A and B,
51-60 lanes). Thus, a variant precursor that is
inefficient at progressing past the 250-kDa stage is largely
carbonate-extractable. A small fraction of the
51-60 variant does
remain in the 250-kDa intermediate following carbonate treatment
suggesting that at least some of the newly imported
51-60 Tom40
molecules at the 250-kDa stage are inserted into the membrane. Another
small amount may be converted to the 100-kDa form (Fig. 9B,
compare the smear in the
51-60 lanes near 100 kDa in panel A to the discrete bands in
panel B). The 100-kDa form appears to have a faster
electrophoretic mobility when the variant Tom40 precursor forms part of
this intermediate.
321-3 Tom40. This variant is also
entirely extractable from the 250-kDa intermediate with carbonate and
is not converted to the assembled form (Fig.
11, A and B). Even long exposures of the blots in Fig. 11 to x-ray film or a phosphorimaging screen did not reveal any of the 250-kDa intermediate or the 400-kDa assembled form in carbonate-extracted samples. There is
also no indication that this variant of Tom40 reaches the 100-kDa
intermediate stage. It appears that the
321-3 form of Tom40 is
incapable of progressing past the carbonate-extractable 250-kDa
intermediate stage of the assembly pathway. Thus, assembly mutants that
accumulate at the 250-kDa intermediate stage provide good evidence that
the Tom40 precursor has only a peripheral association with the membrane
at this stage.
View larger version (41K):
[in a new window]
Fig. 11.
The 321-3
precursor is fully extracted from the 250-kDa intermediate by sodium
carbonate. Radiolabeled wild type and
321-3 Tom40 precursors
were imported into wild type (74A) mitochondria and analyzed
as described for panels A and B of Fig. 10. The
positions and size of bands are indicated on the left.
M, Tom40 monomer.
321-3 and
51-60 mutant variants of Tom40 showed that these
forms of the protein accumulate at the 250-kDa intermediate stage of
the pathway. Although a smear in the region from Tom40 monomer up
to the 100-kDa form is often seen during the assembly of these mutants
(Figs. 7, 8, 10, and 11), no clear band corresponding to the 100-kDa
form seen in the import of wild type precursor is observed. These
observations support the notion that formation of the 250-kDa form
precedes the formation of the 100-kDa intermediate.
40-48 variant of Tom40 described previously (32).
The TOM complex in these mitochondria contains only the mutant version
of the protein. Surprisingly, wild type Tom40 precursors accumulated in
a larger form of about 450 kDa when imported into mitochondria of the
mutant at 0 °C (Fig.
12A). This band was not
identical to the unproductive band at 500 kDa that was discussed
earlier, as shown by comparing the control and
40-48
lanes in Fig. 12A. A small amount of precursor did
exist in the usual 250-kDa intermediate, but the 100-kDa form was not
observed (Fig. 12A). When import was performed for 20 min at
25 °C, assembly to the 400-kDa form was quite efficient, but the
usual intermediates were almost entirely absent. To more fully address
this finding, we examined conversion of the wild type precursor to the
assembled form over a time course of 0-120 min of import into the
40-48 variant mitochondria. A small amount of the 250-kDa
intermediate was observed, but a discrete band for the 100-kDa
intermediate was not evident at any time point (Fig. 12B).
The conversion of precursor molecules in the 450-kDa intermediate to
the 400-kDa form occurred in a pattern that is consistent with a
precursor to product relationship (Fig. 12B). Carbonate
extraction showed that Tom40 precursor was removed from the higher
molecular weight intermediate, but not the assembled form (Fig.
12B). As with import into wild type mitochondria, at least
some of the Tom40 precursor was converted into the assembled 400-kDa
form as the result of carbonate treatment. Digestion with proteinase K
demonstrated that Tom40 was not assembled in its final conformation in
the high molecular weight intermediate that was the most abundant form
present at 0 min but was in the correct conformation in the 400-kDa
form at subsequent time points as judged by the formation of the 26- and 12-kDa fragments (Fig. 12C). These results indicate that
the assembly pathway of Tom40 precursor was influenced by the altered
N-terminal intermembrane space domain of Tom40 molecules in the TOM
complex of the
40-48 mutant mitochondria.
View larger version (34K):
[in a new window]
Fig. 12.
Wild type Tom40 precursor assembles into the
TOM complex via an alternate pathway in mitochondria containing a
mutant form of Tom40. A, radiolabeled wild type Tom40
precursor was imported for 20 min at either 0 or 25 °C into
mitochondria isolated from either control (74A) cells or
cells expressing only the 40-48 mutant version of Tom40.
Mitochondria were re-isolated and processed for BNGE. The gel was
blotted to PVDF membrane and analyzed by autoradiography. B,
samples were processed as in the legend for Fig. 9, except that
radiolabeled wild type Tom40 precursor was imported into mitochondria
isolated from cells expressing only the
40-48 mutant Tom40 protein.
Duplicates of the 0- and 10-min samples were subjected to sodium
carbonate extraction following import and re-isolation of mitochondria
(+carb). C, samples were developed as in
B, but after import and re-isolation, mitochondria were
treated with proteinase K and processed for SDS-PAGE as described in
the legend to Fig. 10. The positions and size of bands are indicated on
the left of all panels. M, Tom40 monomer.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
51-60 mutant form of Tom40 is
unable to progress past the 250-kDa intermediate stage to the fully
assembled TOM complex even after 60 min of import at 25 °C. The
deletion of residues 51-60 does not totally abrogate assembly in
vivo, because strains expressing only this form of the protein are
viable, although they display growth defects and alterations in TOM
complex stability. Thus, the region encompassed by both the
40-48
and
51-60 mutations plays an important role for achieving the
interactions necessary for progression past the 250-kDa intermediate on
the assembly pathway.
321-3 variant at 0 °C was striking. At this temperature the
altered protein may not be in an import-competent conformation. At
25 °C, the precursor can be imported to the 250-kDa intermediate but
cannot proceed to the membrane integration step of the assembly pathway. The severity of the assembly defects in vitro are
supported by the observation that a tom40 gene encoding a
321-3 variant cannot rescue the tom40 null nucleus. It
has been shown that Tom40 must be in a partially folded state for
effective integration into the membrane (29) and the
321-3 variant
may be unable to achieve the correct conformation for integration.
However, it seems unlikely that there are gross overall changes in
conformation at 25 °C, because the protein assembles past the
initial recognition stage and reaches the 250-kDa intermediate in
vitro. The KLG residues might provide a specific signal for
integration into the membrane or assembly with another TOM complex
component, but this seems unlikely, because changing the residues to
alanines has little effect on in vitro assembly and a
tom40 gene encoding the 321AAA variant can restore
viability, although the resulting strains have growth and TOM complex
defects. Finally, it is conceivable that the KLG residues form part of
a membrane-spanning domain. In this case, a reasonable explanation for
the drastically different behavior of the deletion and substitution
variants could be that replacement with alanine residues still allows
the region to span the membrane, whereas loss of the residues does not
allow membrane spanning and prevents integration. Such a
membrane-spanning region might also be important for channel formation.
Given its position near the C terminus and the striking fragility of
the TOM complex from the 321AAA strain, it is conceivable that the
region could also be involved in maintaining interactions between TOM
complex subunits. The presence of the Tom40 dimer-sized complex in
digitonin-treated samples is reminiscent of the subcomplexes of Tom40
dimers seen in Tom22-deficient yeast cells (55). Thus, the 321AAA Tom40 mutant might have weakened interactions with Tom22. The breakdown of
the complex in 321AAA mitochondria to Tom40 monomers following solubilization with dodecyl maltoside suggests that the region could
also contribute to the formation and maintenance of Tom40 dimers.
51-60 and
321-3
variants favors the notion that the Tom40 precursor appears in the
250-kDa form first and then progresses to the 100-kDa form. It has also
been proposed that the Tom40 precursor in the 250-kDa intermediate is
associated with the outer membrane on the intermembrane space side and
is extractable with sodium carbonate (30). Our data from carbonate
extraction of mitochondria following import of wild type Tom40 into
wild type mitochondria do not allow conformation of this aspect of the
model due to the unexpected finding that carbonate enhances assembly of
Tom40 precursors in intermediates into the final 400-kDa form. However,
the observation that precursors of the
51-60 and
321-3 variants
of Tom40 in the 250-kDa intermediate were extractable by carbonate does
support the notion that the Tom40 precursor is only peripherally
associated with the membrane at this stage. One explanation for the
action of sodium carbonate on wild type precursors might be that
protein-protein interactions hold the molecule at an intermediate stage
of assembly, and these are disrupted in the presence of carbonate. It
is also conceivable that interactions between Tom40 molecules in
existing TOM complexes may be weakened, facilitating replacement of
existing subunits with incoming molecules.
40-48 mutant form of Tom40
revealed differences from the normal assembly pathway. Very little of
the precursor was seen in the 250-kDa intermediate, and virtually none
was detectable in the 100-kDa form. Instead, the precursor was found in
a 450-kDa form, which appeared to assemble directly into the 400-kDa
form. The simplest interpretation for the larger form is that it
represents a precursor molecule associated with an existing TOM
complex. Most of the precursor at this stage was extractable with
sodium carbonate so that its initial association with the components in
the high molecular weight form must precede integration into the
membrane. Because there is no formation of the 100-kDa form, the
precursor may integrate directly into the existing TOM complex, perhaps
by displacing a pre-existing subunit. This interpretation suggests that
amino acid residues 40-48 of Tom40 molecules in the TOM complex may
interact with incoming subunits. Although it may be surprising that an
alternate pathway exists, it is also possible that a small amount of
assembly occurs by this pathway under normal conditions but is not
easily detected. Although the N terminus of Tom40 is not required for
stable interactions between Tom40 molecules (56), our results suggest
that the region is important for assembly of the protein into the TOM
complex, because alterations in the N terminus of both Tom40 precursors and Tom40 molecules in the TOM complex have an affect on the assembly pathway.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Bonnie Crowther and Lara Corrigan for technical assistance and to Doron Rapaport for comments on the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the Canadian Institutes of Health Research (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.
Supported by scholarships from the Natural Sciences and
Engineering Research Council of Canada and the Alberta Heritage
Foundation for Medical Research.
§ To whom correspondence should be addressed. Tel.: 780-492-5375; Fax: 780-492-9234; E-mail: frank.nargang@ualberta.ca.
Published, JBC Papers in Press, October 23, 2002, DOI 10.1074/jbc.M208083200
2 S. Nussberger and W. Neupert, personal communication.
3 R. D. Taylor and F. E. Nargang, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TOM, translocase of the outer mitochondrial membrane; TIM, translocases of the inner mitochondrial membrane; RIP, repeat induced point mutation; RIPed, inactivated by repeat induced point mutation; BNGE, blue native gel electrophoresis; PVDF, polyvinylidene difluoride.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Pfanner, N., and Geissler, A. (2001) Nat. Rev. Mol. Cell. Biol. 2, 339-349[CrossRef][Medline] [Order article via Infotrieve] |
2. | Paschen, S., and Neupert, W. (2001) Life 52, 101-112[Medline] [Order article via Infotrieve] |
3. |
Rehling, P.,
Wiedemann, N.,
Pfanner, N.,
and Truscott, K. N.
(2001)
Crit. Rev. Biochem. Mol. Biol.
36,
291-336 |
4. | Künkele, K.-P., Heins, S., Dembowski, M., Nargang, F. E., Benz, R., Thieffry, M., Walz, J., Lill, R., Nussberger, S., and Neupert, W. (1998) Cell 93, 1009-1019[Medline] [Order article via Infotrieve] |
5. |
Ahting, U.,
Thun, C.,
Hegerl, R.,
Typke, D.,
Nargang, F.,
Neupert, W.,
and Nussberger, S.
(1999)
J. Cell Biol.
147,
959-968 |
6. | Neupert, W. (1997) Annu. Rev. Biochem. 66, 863-917[CrossRef][Medline] [Order article via Infotrieve] |
7. | Pfanner, N., Craig, E. A., and Hönlinger, A. (1997) Annu. Rev. Cell Dev. Biol. 13, 25-51[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Dekker, P. J.,
Ryan, M. T.,
Brix, J.,
Muller, H.,
Honlinger, A.,
and Pfanner, N.
(1998)
Mol. Cell. Biol.
18,
6515-6524 |
9. |
Meisinger, C.,
Ryan, M. T.,
Hill, K.,
Model, K.,
Lim, J. H.,
Sickmann, A.,
Müller, H.,
Meyer, H. E.,
Wagner, R.,
and Pfanner, N.
(2001)
Mol. Cell. Biol.
21,
2337-2348 |
10. | Kiebler, M., Keil, P., Schneider, H., van der Klei, I., Pfanner, N., and Neupert, W. (1993) Cell 74, 483-492[Medline] [Order article via Infotrieve] |
11. | Hönlinger, A., Kübrich, M., Moczko, M., Gärtner, F., Mallet, L., Bussereau, F., Eckerskorn, C., Lottspeich, F., Dietmeier, K., Jacquet, M., and Pfanner, N. (1995) Mol. Cell. Biol. 15, 3382-3389[Abstract] |
12. | Nargang, F. E., Künkele, K.-P., Mayer, A., Ritzel, R. G., Neupert, W., and Lill, R. (1995) EMBO J. 14, 1099-1108[Abstract] |
13. | Gratzer, S., Lithgow, T., Bauer, R. E., Lamping, E., Paltauf, F., Kohlwein, S. D., Haucke, V., Junne, T., Schatz, G., and Horst, M. (1995) J. Cell Biol. 129, 25-34[Abstract] |
14. |
Komiya, T.,
Rospert, S.,
Schatz, G.,
and Mihara, K.
(1997)
EMBO J.
16,
4267-4275 |
15. |
Kurz, M.,
Martin, H.,
Rassow, J.,
Pfanner, N.,
and Ryan, M. T.
(1999)
Mol. Biol. Cell
10,
2461-2474 |
16. | Baker, K. P., Schaniel, A., Vestweber, D., and Schatz, G. (1990) Nature 348, 605-609[CrossRef][Medline] [Order article via Infotrieve] |
17. | Vestweber, D., Brunner, K. P., Baker, A., and Schatz, G. (1989) Nature 341, 205-209[CrossRef][Medline] [Order article via Infotrieve] |
18. | Kiebler, M., Pfaller, R., Söllner, T., Griffiths, G., Horstmann, H., Pfanner, N., and Neupert, W. (1990) Nature 348, 610-616[CrossRef][Medline] [Order article via Infotrieve] |
19. | Hill, K., Model, K., Ryan, M. T., Dietmeier, K., Martin, F., Wagner, R., and Pfanner, N. (1998) Nature 395, 516-521[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Künkele, K.-P.,
Juin, P.,
Pompa, C.,
Nargang, F. E.,
Henry, J.-P.,
Neupert, W.,
Lill, R.,
and Thieffry, M.
(1998)
J. Biol. Chem.
273,
31032-31039 |
21. |
Rapaport, D.,
Neupert, W.,
and Lill, R.
(1997)
J. Biol. Chem.
272,
18725-18731 |
22. |
Ahting, U.,
Thieffry, M.,
Engelhardt, H.,
Hegerl, R.,
Neupert, W.,
and Nussberger, S.
(2001)
J. Cell Biol.
153,
1151-1160 |
23. |
Schwartz, M. P.,
and Matouschek, A.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
13086-13090 |
24. | Rapaport, D., Kunkele, K. P., Dembowski, M., Ahting, U., Nargang, F. E., Neupert, W., and Lill, R. (1998) Mol. Cell. Biol. 9, 5256-5262 |
25. | Model, K., Prinz, T., Ruiz, T., Radermacher, M., Krimmer, T., Kühlbrandt, W., Pfanner, N., and Meisinger, C. (2002) J. Mol. Biol. 316, 657-666[CrossRef][Medline] [Order article via Infotrieve] |
26. | Court, D. A., Lill, R., and Neupert, W. (1995) Can. J. Bot. 73 (Suppl. 1), S193-S197 |
27. | Mannella, C. A., Neuwald, A. F., and Lawrence, C. E. (1996) J. Bioenerg. Biomembr. 28, 163-169[Medline] [Order article via Infotrieve] |
28. | Davis, R. H., and De Serres, F. J. (1970) Methods Enzymol. 17, 79-143 |
29. |
Rapaport, D.,
and Neupert, W.
(1999)
J. Cell Biol.
146,
321-331 |
30. | Model, K., Meisinger, C., Prinz, T., Wiedemann, N., Truscott, K. N., Pfanner, N., and Ryan, M. T. (2001) Nat. Struct. Biol. 8, 361-370[CrossRef][Medline] [Order article via Infotrieve] |
31. | Rapaport, D. (2002) Trends Biochem. Sci. 26, 191-197 |
32. |
Rapaport, D.,
Taylor, R.,
Käser, M.,
Langer, T.,
Neupert, W.,
and Nargang, F. E.
(2001)
Mol. Biol. Cell
12,
1189-1198 |
33. | White, B., and Woodward, D. (1995) Fungal Genet. Newsl. 42, 79 |
34. | Selker, E. U. (1990) Ann. Rev. Genet. 24, 579-613[CrossRef][Medline] [Order article via Infotrieve] |
35. | Metzenberg, R. L., and Grotelueschen, J. S. (1992) Fungal Genet. Newsl. 39, 37-49 |
36. |
Harkness, T. A. A.,
Metzenberg, R. L.,
Schneider, H.,
Lill, R.,
Neupert, W.,
and Nargang, F. E.
(1994)
Genetics
136,
107-118 |
37. | Metzenberg, R., Stevens, J., Selker, E., and Morzycka-Wroblewska, E. (1984) Neurospora Newsl. 31, 35-39 |
38. | Metzenberg, R., Stevens, J., Selker, E., and Morzycka-Wroblewska, E. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 2067-2071[Abstract] |
39. | Staben, C., Jensen, B., Singer, M., Pollock, J., and Schechtman, M. (1989) Fungal Genet. Newsl. 36, 79-81 |
40. | Schweizer, M., Case, M. E., Dykstra, C. C., Giles, N. H., and Kushner, S. R. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 5086-5090[Abstract] |
41. | Akins, R. A., and Lambowitz, A. M. (1985) Mol. Cell. Biol. 5, 2272-2278[Medline] [Order article via Infotrieve] |
42. | Margolin, B. S., Freitag, M., and Selker, E. U. (1997) Fungal Genet. Newsl. 44, 34-36 |
43. | Margolin, B. S., Freitag, M., and Selker, E. U. (2000) Fungal Genet. Newsl. 47, 112 |
44. | Austin, B., Hall, R. M., and Tyler, B. M. (1990) Gene (Amst.) 93, 157-162[CrossRef][Medline] [Order article via Infotrieve] |
45. | Mayer, A., Lill, R., and Neupert, W. (1993) J. Cell Biol. 121, 1233-1243[Abstract] |
46. | Harkness, T. A. A., Nargang, F. E., Van der Klei, I., Neupert, W., and Lill, R. (1994) J. Cell Biol. 124, 637-648[Abstract] |
47. | Schägger, H., and von Jagow, G. (1991) Anal. Biochem. 199, 223-231[Medline] [Order article via Infotrieve] |
48. | Schägger, H., Cramer, W. A., and von Jagow, G. (1994) Anal. Biochem. 217, 220-230[CrossRef][Medline] [Order article via Infotrieve] |
49. | Ausubel, R. A., Brent, R., Kingston, R. E., Moore, D. D., and Seidman, J. G. (1992) Current Protocols in Molecular Biology , Greene and Wiley Interscience, New York |
50. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
51. | Good, A. G., and Crosby, W. L. (1989) Plant Physiol. 90, 1305-1309 |
52. | Wendland, J., Lengeler, K., and Kothe, E. (1996) Fungal Genet. Newsl. 43, 54-55 |
53. |
Rountree, M. R.,
and Selker, E. U.
(1997)
Genes Dev.
11,
2383-2395 |
54. | Grad, L., Descheneau, A., Neupert, W., Lill, R., and Nargang, F. (1999) Curr. Genet. 36, 137-146[CrossRef][Medline] [Order article via Infotrieve] |
55. | van Wilpe, S., Ryan, M. T., Hill, K., Maarse, A. C., Meisinger, C., Brix, J., Dekker, P. J., Moczko, M., Wagner, R., Meijer, M., Guiard, B., Honlinger, A., and Pfanner, N. (1999) Nature 401, 485-489[CrossRef][Medline] [Order article via Infotrieve] |
56. | Gordon, D. M., Wang, J., Amutha, B., and Pain, D. (2001) Biochem. J. 356, 207-215[CrossRef][Medline] [Order article via Infotrieve] |
57. | Pfanner, N., Tropschug, M., and Neupert, W. (1987) Cell 49, 815-823[Medline] [Order article via Infotrieve] |