From the Department of Pediatrics, Wake Forest University School of
Medicine, Winston-Salem, North Carolina 27157-1081
The association between ribosomes and the pore
proteins at the endoplasmic reticulum membrane is important to
co-translational translocation. To determine if a similar association
occurs between the ribosome and mitochondrial membrane protein(s)
during protein import in higher eukaryotes, we examined
ribosome-mitochondria binding. By using spectral measurements, analysis
of mitochondrial associated RNA, and electron microscopy, we
demonstrated that ribosomes stably bind to purified rat liver
mitochondria in vitro. Binding of ribosomes to mitochondria
was markedly reduced by GTP and nearly abolished by the
non-hydrolyzable GTP analogue, guanosine-5'-[thio]-triphosphate (GTP
S), but was only modestly reduced by GDP or ATP and unaffected by CTP. The initial rate of GTP hydrolysis by mitochondria was increased by ribosomes, whereas the rate of ATP hydrolysis by mitochondria was not affected. Ribosomes programmed with mRNA for
92 amino acids of the N terminus of mitochondrial malate dehydrogenase bound to mitochondria, but unlike unprogrammed rat liver ribosomes, neither GTP nor GDP disrupted binding; however, GTP
S did. These data
show that receptors specific for ribosomes are present on the
mitochondrial membrane, and a GTP-dependent process
mediates this binding. The presence of a nascent chain alters these
binding characteristics. These findings support the hypothesis that a co-translational translocation pathway exists for import of proteins into mitochondria.
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INTRODUCTION |
Proteins can cross membranes by either post-translational or
co-translational translocation. Based on in vitro
observations, proteins targeted to the mitochondria are thought to be
completely synthesized in the cytoplasm and cross the mitochondrial
membrane(s) post-translationally (1). However, evidence consistent with a co-translational translocation pathway for mitochondrial protein import has been reported. For example, 1) the surface of mitochondria isolated from cycloheximide-treated yeast cells is observed to be
studded with polysomes (2); 2) the number of bound polysomes is
dependent on the metabolic state of the cells from which the mitochondria are isolated (3); 3) ribosomes are bound to the mitochondria at the contact sites (4); and 4) the mRNA of polysomes that co-isolate with mitochondria is enriched in messages for mitochondrial proteins (5, 6). In addition, both in vitro and in vivo, mitochondrial protein import can be
instantaneously disrupted by cycloheximide-induced translation arrest
indicating that no detectable pool of full-length precursor exists in
the cytosol and that translation and import are tightly coupled (7-9). Furthermore, methotrexate, which inhibits post-translational import of
cytochrome oxidase subunit IV-dihydrofolate reductase by preventing its
unfolding, does not inhibit cytochrome oxidase subunit IV-dihydrofolate reductase import in vivo (9). This indicates that
co-translational translocation may eliminate the need for precursor
proteins to be maintained in a translocation-competent state by
chaperones in the cytoplasm.
Almost all mitochondrial matrix and inner membrane proteins are
synthesized with an N-terminal presequence and then imported into the
mitochondria (10-12). The presequence-containing precursor protein is
presumably held in a translocation-competent conformation in the
cytoplasm by hsp70 chaperonins probably in concert with the
presequence-specific chaperonin, e.g. "targeting factor"
or mitochondrial stimulating factor, and the precursor is recognized and imported by a multisubunit translocation complex in the
mitochondrial membranes (13-18). Since these early events in targeting
and translocation clearly and specifically involve the N-terminal
presequence, it is entirely possible that mitochondrial protein import
can be initiated long before translation is complete.
To examine the hypothesis that, in vivo, proteins import
into the mitochondria by a co-translational translocation pathway, and
to determine whether receptor(s) for the translation machinery at the
outer membrane play a role in targeting an incompletely synthesized
nascent polypeptide chain to the mitochondrial import site, we examined
the interaction of the ribosome with the mitochondria. Specifically,
ribosome binding to mitochondria was shown using three independent
methods, and the ability of ribosome-bound nascent polypeptide chains
to target the ribosome to the mitochondria was determined. Finally,
because recent studies have shown that GTP-binding proteins are not
only involved in initiating protein translocation at the membrane of
the endoplasmic reticulum
(ER),1 but are also involved
in protein import into chloroplast (19), we determined the effect of
ATP and GTP on ribosomes binding to mitochondria and the early events
in protein translocation.
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MATERIALS AND METHODS |
Preparation of Ribosomes and Rough Endoplasmic
Reticulum--
Ribosomes and rough endoplasmic reticulum (RER) were
isolated from the post-mitochondrial supernatant of rat liver
homogenates by a previously described method (20). To remove endogenous GTPase activity, isolated ribosomes were resuspended in 50 mM Tris, pH 7.4, 0.5 M KOAc, 10 mM
MgCl2, and 4 mM dithiothreitol containing 1 unit/µl RNasin (Promega), incubated at room temperature 15 min, and
re-isolated by sedimentation through a 0.5 M sucrose cushion containing 0.5 M KOAc, 50 mM Tris, pH
7.6, 5 mM MgOAc, 4 mM dithiothreitol, 1 unit/µl RNasin. Rabbit reticulocyte ribosomes were isolated as
described (21). The final ribosomal pellets were resuspended in
ribosome binding buffer (RB, 50 mM Tris, pH 7.4, 100 mM KOAc, 10 mM MgCl2, 1 mM dithiothreitol containing 1 unit/µl RNasin) and stored
at
70 °C in small aliquots.
Preparation of High Salt-washed Mitochondria--
Mitochondria
were isolated from Sprague-Dawley rat livers using previously published
methods (22). Mitochondria were further purified to reduce ER
contamination using metrizamide-Percoll density gradient centrifugation
as described previously (23). Final preparations were brought to a
protein concentration between 5 or 15 mg/ml in 10 mM Tris,
pH 7.4, 70 mM mannitol, and 250 mM sucrose
(MIB) and either used immediately or flash-frozen in liquid nitrogen
and stored at
70 °C. Ribosome binding activity and GTP hydrolysis
was essentially the same for fresh and previously frozen mitochondria,
although electron microscopy indicated integrity of the intermembrane
was often disrupted in the previously frozen mitochondria. Mitochondria
were washed using a previously described method for stripping ER
membranes of ribosomes (24). For all experiments described, EDTA, high
salt-washed mitochondria (EKMT) were used unless otherwise
specified.
Enzyme Assays--
Glucose-6-phosphatase activity and
arylesterase activity were measured as described previously (25, 26, respectively). The amount of inorganic phosphate (Pi)
produced from trinucleotide hydrolysis by mitochondria and ribosomes
was determined using a modification of a previously described method
(27). Briefly, 17-µl samples were prepared as described above, and
after incubation and pelleting, the supernatants were brought to 500 µl with water and 100 µl of 7.5 N
H2SO4, 100 µl of 6.6%
(NH4)6Mo7O24·4H2O,
and 80 µl of freshly prepared 0.36 M
FeSO4·7H2O, 0.15 N
H2SO4 were added sequentially. After a 20-min
incubation, the absorbance was measured at 650 or 700 nm. To correct
for light scattering, Pi contamination, and/or nucleotide
degradation, background samples containing ribosomes, mitochondria, or
ribosome-mitochondria complexes but lacking nucleotides, as well as
samples containing only nucleotides, were run in parallel, and their
absorbance was subtracted from the corresponding samples.
Preparation of Ribosome-Nascent Chain Complexes--
Rat
mitochondrial malate dehydrogenase (mMDH) mRNA was synthesized by
in vitro transcription of linearized pGEM-mMDH as described previously (22). Plasmids were cleaved in the mMDH coding region with
FokI, and truncated mMDH mRNAs produced by in
vitro transcription were translated in nuclease-treated rabbit
reticulocyte lysate (Promega) in the presence of
[35S]methionine to produce stable ribosome-nascent chain
complexes carrying peptides of defined length (28). The TNT® T7
luciferase control plasmid was cleaved in the coding region with
Cfr10, and ribosome-nascent chain complexes were produced
using the TNT®-coupled transcription/translation reticulocyte lysate
system as recommended by the supplier (Promega). Ribosome-nascent chain
complexes were isolated as described (21) except the ribosomes were not
high salt- washed and the sucrose cushion contained 100 mM
KOAc instead of 500 mM KOAc.
Ribosome-Mitochondria Binding Assay--
Before each binding
assay, ribosomes were centrifuged at 17,000 × g for 2 min to remove insoluble material. Mitochondria (75-300 µg as
indicated) and ribosomes (2-16 µg as indicated) were mixed in a
total reaction volume of 17 or 25 µl of RB/MIB (1:3). Either ATP,
GTP, GDP, GTP
S, or CTP was added to a final concentration of 1.2 mM as indicated. Samples were incubated for 0-15 min at room temperature, and mitochondria were immediately sedimented at
17,000 × g for 2 min. The mitochondrial pellets were
washed with MIB and resuspended in MIB containing 1 mM
MgCl2, and the absorbance of the supernatant and/or
mitochondrial suspension was measured at 260 and 280 nm for each
sample. In parallel, samples containing only mitochondria and
nucleotide were assayed and set as background for the corresponding
ribosome, mitochondria, and nucleotide sample. When ribosomes carried
[35S]methionine-labeled nascent chains, the relative
amount of ribosome-nascent chain complex binding was also determined by
liquid scintillation counting of the ribosome-mitochondrial pellet.
To verify that absorbance changes of the resuspended mitochondrial
pellets reflected differential ribosomal binding, 400-450 µg of
mitochondria were incubated with approximately 75 µg of ribosomes in
200 µl of RB/MIB (1:3) for 5 min at room temperature. The
ribosome-mitochondria complexes were divided into 30-µl aliquots, sedimented (10,000 × g, 5 min, 4 °C), and
resuspended in 30 µl of RB/MIB (1:3) containing either 0.1 or 5 mM ATP, GTP, GDP, GTP
S, CTP, or no nucleotide. Samples
were again incubated at room temperature for 5 min and sedimented
through an equal volume of 1.0 M sucrose cushion
(10,000 × g, 5 min, 4 °C). Pellets and supernatants
were extracted with phenol/chloroform/isoamyl alcohol (25:24:1) and the
RNA ethanol-precipitated. The resuspended RNA was subjected to both
agarose gel electrophoresis and dot blot analysis, using a
[32P]dCTP-radiolabeled 300-nucleotide cDNA probe that
had been amplified by polymerase chain reaction using cytoplasmic 18 S
rRNA as a template (GenBankTM accession number V01270.
Sense primer, 5'-TACATGCCGACGGGCGCTGACC-3'; antisense primer,
5'-CCTGCTGCCTTCCTTGGATG-3'). The 18 S probe was tested by Northern
blot analysis to confirm that the RNA visualized by ethidium bromide
staining corresponded to cytosolic 18 S rRNA and not mitochondrial
rRNA.
Electron Microscopy--
Samples were prepared for electron
microscopy by adding 100 µl of mitochondria or ribosome-mitochondria
complexes (approximately 7.5 µg of mitochondrial protein/µl) to 1 ml of fixative containing 4% glutaraldehyde, 100 mM
sucrose, 100 mM cacodylate buffer, pH 7.4 (glutaraldehyde/sucrose/cacodylate buffer). Fixed samples were washed
with 100 mM sucrose, 100 mM cacodylate buffer,
pH 7.4, postfixed in 1% OsO4, and dehydrated in graded
ethanols. Samples were embedded in Spurr's resin (29), thin sectioned, and examined using a Philips 400 TEM operating at 80 KeV (Micromed, Wake Forest University School of Medicine). Ten randomly selected fields for mitochondria under each condition were photographed at a
magnification of × 15,200, and the number of ribosomes per field
was counted by hand. To ensure that each field had the same amount of
mitochondrial surface area, point count stereology was used to quantify
the relative surface areas of en face versus cross-sectional
views of mitochondria (30). Between all fields in all conditions, this
ratio was very constant at 0.31 to 0.33, and thus, absolute numbers of
ribosomes are reported for comparison between fields. For each
condition, the fields with the highest and lowest counts of ribosomes
were discarded, and the average and standard deviation of the remaining
eight fields are reported.
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RESULTS |
Ribosomes Bind to Mitochondria--
Rat liver mitochondria
prepared by differential centrifugation have been shown to contain
other organelles including a significant amount of rough endoplasmic
reticulum-derived microsomes (26). In order to examine only the
ribosome-mitochondria interaction, crude mitochondrial preparations
were either subjected to EDTA and high salt treatment (24),
purification by density gradient centrifugation (23), or both. All
preparations were analyzed for activity of the RER-specific enzymes,
glucose-6-phosphatase, and arylesterase. Based on enzyme activity,
either density gradient centrifugation (MPMT) or EDTA/high salt
treatment of mitochondria (EKMT) reduced the amount of RER
contamination by about 75%, whereas density gradient centrifugation
followed by EDTA/high salt treatment (EKMPMT) essentially eliminated
RER contamination (Fig. 1).

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Fig. 1.
Enzyme activity. Glucose-6-phosphatase
(B) and arylesterase (A) activities were
determined on preparations of endoplasmic reticulum, ribosomes, and
mitochondria. Each solid bar represents the mean of at
least three separate experiments. Error bars represent the
standard deviation. MT, mitochondria from differential
centrifugation; MPMT, metrizamide/Percoll-purified
mitochondria; EKMT, EDTA/high potassium salt washed
mitochondria; EKMPMT, EDTA/high-salt,
metrizamide/Percoll-purified mitochondria; RER, rough
endoplasmic reticulum.
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Prior to conducting ribosome-mitochondria binding experiments,
cytosolic rat liver ribosomes were washed in high salt to remove endogenous GTPase activity and were tested for inorganic phosphate production from GTP or ATP. The initial rate of GTP hydrolysis by
ribosomes was reduced approximately 80% by high salt treatment (Table
I). Ribosomes preparations were also
analyzed for RER enzyme markers and were shown to be free of RER
contamination (Fig. 1).
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Table I
Phosphate production
Each value listed represents the mean ± S.D. from at least three
experiments. Each assay included 150 µg of EDTA/high salt-washed
mitochondria, 5 µg of ribosomes, and 1.2 mM nucleotides.
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High salt-washed ribosomes were incubated with EDTA/high salt-washed,
density gradient purified mitochondria (EKMPMT); the ribosome-mitochondria complexes were separated from free ribosomes by
sedimentation, and the A260 of resuspended
complexes and/or the free ribosomes remaining in the supernatant were
measured. The difference in the A260 between
resuspended mitochondria incubated without ribosomes and mitochondria
incubated with ribosomes was taken to represent ribosome binding. As
shown in Fig. 2, ribosomes bind to both
freshly isolated and previously frozen mitochondria. The number of
ribosomes bound per mg of mitochondria is much higher in the previously
frozen samples, presumably because the mitochondrial contents leak out
of the mitochondria upon freeze-thawing. Freshly isolated mitochondria
will bind approximately one ribosome per 1.7 fg of mitochondrial
protein when saturated with ribosomes (Fig. 2).

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Fig. 2.
Binding of ribosomes to mitochondria.
EDTA/high salt- washed mitochondria (150 µg) were incubated with high
salt-washed ribosomes (2-16 µg corresponding to a final
concentration of 19-160 nM) in 25 µl. Identical samples
without ribosomes were incubated in parallel, and the
A260 of each ribosomal concentration was also
measured. After 5 min, the ribosome-mitochondria complexes, or
mitochondria controls, were sedimented, and the supernatants were
brought to 350 µl in 50 mM Tris, pH 7.4, 100 mM KOAc, 10 mM MgCl2, and their
absorbance was measured at 260 nm. For each condition, the
A260 of the mitochondria incubated without
ribosomes was subtracted from the A260 of the
mitochondria incubated with ribosomes. Data were subjected to a
one-site binding analysis which calculated a binding maximum of
9.6 ± 0.9 pmol/mg, r2 = 0.9748, for
freshly isolated mitochondria.
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Because ATP is required for post-translational import of proteins into
the mitochondria and because GTP is required for co-translational translocation of protein into the ER, we hypothesized that nucleotides would alter ribosome binding to mitochondria. Formation of
ribosome-mitochondria complexes was measured as before except samples
were initially incubated with or without ATP, GTP, GDP, GTP
S, or
CTP. A differential increase in the A260 and
A280 resulting in an increased
A260/A280 ratio (Table I)
was observed when resuspended mitochondria had been incubated with
ribosomes compared to when resuspended mitochondria had been incubated
alone. This indicates that the ribonucleic acid-containing ribosomes
associate with mitochondria, and the increase in the
A260 was arbitrarily set to represent 100%
ribosome binding (Fig. 3, lane
MR). Similar increases in the A260 and
A280 were observed when 1.2 mM CTP
was included in binding assay (Fig. 3, lane MRCTP). However,
when 1.2 mM GTP or GTP
S was included, only minimal
increases in absorbance were observed (Fig. 3, lanes MRGTP
and MRGTPS). Inclusion of GDP or ATP in the binding assay resulted in A260 increases that were somewhat
lower than values obtained in the absence of nucleotide but were also
significantly higher than GTP and GTP
S. This indicates that the
association between ribosomes and mitochondria can be blocked by GTP or
GTP
S but is only mildly affected by ATP or GDP. Under the conditions used here, it is unclear if the effect of ATP on binding is direct or a
result of conversion of ATP to GTP from existing GDP. However, the
dramatic effects of GTP or GTP
S on complex formation was statistically different from complex formation under all other conditions tested indicating that GTP plays a dominant role in controlling the ribosome-mitochondria association.

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Fig. 3.
The effect of nucleotides on
ribosome-mitochondria interaction. EDTA/high salt-washed
mitochondria (EKMT) (75 or 150 µg) were incubated with high
salt-washed ribosomes (5 µg) in the absence and presence of 1.2 mM ATP, GTP, GDP, GTP S, or CTP, all in 17 µl.
Identical samples without ribosomes were incubated in parallel. After 5 min, the ribosome-mitochondria complexes, or mitochondria controls,
were sedimented, and pellets were resuspended in 50 mM
Tris, pH 7.4, 100 mM KOAc, 10 mM
MgCl2, and the absorbance was measured at 260 nm. For each
condition, the A260 of the mitochondria
incubated without ribosomes was subtracted from the
A260 of the mitochondria incubated with
ribosomes. This is graphed as the change in A260
for each condition divided by the change in A260
when ribosomes are bound to mitochondria in the absence of nucleotide
and represents the relative amount of ribosomes bound to mitochondria.
MR, mitochondria and ribosomes; MRATP,
mitochondria, ribosomes, and ATP; MRGTP, mitochondria,
ribosomes, and GTP; MRGDP, mitochondria, ribosomes, and GDP;
MRGTPS, mitochondria, ribosomes, and GTP S;
MRCTP, mitochondria, ribosomes, and CTP. The data represent
the average of at least three experiments and were subjected to one-way
analysis of variance. * indicates significant difference
(p < 0.05) from mitochondria and ribosomes.
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To confirm that the increases in the A260 and
the A280 for mitochondria incubated with
ribosomes were due to ribosome-mitochondria binding,
ribosome-mitochondria complexes were pre-formed by incubation of EKMPMT
with high salt-washed ribosomes in the absence of nucleotides. The
pre-formed complexes were isolated by sedimentation, and the pellets
were resuspended in RB/MIB containing either no nucleotide, GTP, GDP,
GTP
S, or ATP at a concentration of either 0.1 or 5 mM.
Complexes were re-isolated by sedimentation, and to assess whether
ribosomes remained bound under each condition, the relative amount of
rRNA in the supernatants and pellets was determined by subjecting the
extracted samples to agarose gel electrophoresis followed by ethidium
bromide staining or by dot blot analysis (Fig.
4). Ribosomal RNA extracted from the
pellet represents the stably bound ribosomes, whereas rRNA in the
supernatant represents ribosomes that were released from the
mitochondria.

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Fig. 4.
GTP destabilizes ribosome-mitochondrial
binding. EDTA/high salt-washed mitochondria (EKMT) (approximately
450 µg) were incubated alone or with ribosomes (75 µg) to form
mitochondria-ribosome complexes. The 30 µl of mitochondria and
mitochondria-ribosome complexes were sedimented and resuspended in
RB/MIB (50 mM Tris, pH 7.4, 100 mM KOAc, 10 mM MgCl2, 10 mM Tris, pH 7.4, 70 mM mannitol, and 250 mM sucrose; 1:3) alone, or
RB/MIB containing GTP, GDP, GTP S, or ATP. After 5 min at room
temperature, mitochondria and complexes were re-sedimented, and the
rRNA was extracted from supernatants and pellets and was subjected to
either 1% agarose gel electrophoresis or dot blot analysis.
A, the rRNAs were visualized by ethidium bromide staining.
Shown are the 28 S and 18 S rRNA remaining with mitochondrial pellets
when no nucleotide was incubated ( NTP) or when 5 mM GTP, GDP, GTP S, or ATP, respectively, was incubated
with pre-formed mitochondria-ribosome complexes for 5 min.
B, supernatants from samples corresponding to those in
A were subjected to RNA dot blot analysis to quantify and
confirm that differences in mitochondrial-associated rRNA were the
result of ribosome release and not degradation. The dot blot was probed
with [32P]dCTP-labeled cDNA for a portion of the
18 S rRNA. Dots were quantified using an AMBIS radiometric
counter, and the cpm for each condition were plotted.
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When no nucleotide was added to the pre-formed complexes, nearly all
rRNA re-sedimented with the mitochondria (Fig. 4, A and B, lane
NTP). Again, this indicates that the ribosomes
remained stably bound to mitochondria in the absence of nucleotide. As expected, pellets obtained from pre-formed ribosome-mitochondria complexes incubated with GTP, GDP, or GTP
S contained less rRNA demonstrating that guanine nucleotides selectively disrupt binding (Fig. 4A, lanes GTP, GDP, and
GTP
S). ATP did not significantly disrupt
binding under these conditions, and the effect of GDP on binding was
considerably less than GTP. These differences were observed even at low
concentration (0.1 mM) of GTP (data not shown). Samples
containing mitochondria alone were treated exactly as the
ribosome-mitochondria complexes and subjected to dot blot analysis in
parallel, but no radioactivity was detectable for these samples. In
agreement with spectral data, GTP and GTP
S have the greatest effect
on ribosome-mitochondrial complex stability. These data show that 1)
the spectroscopic absorbance changes correlate well with changes in
ribosome binding, and 2) the ribosome-mitochondrial interaction is not
the result of nonspecific binding since, when ribosome-mitochondria
complexes are pre-formed, this interaction can be specifically reversed
by GTP or GTP
S.
Finally, to confirm that the spectral data and dot blot analysis
measured ribosome-mitochondria binding and not another process, such as
ribosome aggregation and de-aggregation, ribosome-mitochondria complexes were examined by electron microscopy. Frozen unwashed mitochondria were thawed and washed twice in MIB. An aliquot was removed and fixed in glutaraldehyde/sucrose/cacodylate buffer. The
remaining mitochondria were then EDTA/high salt-washed (EKMT) and an
aliquot of these mitochondria was removed and fixed. Ribosomes were
then added to the rest of the sample, and the sample was incubated for
5 min at room temperature. An aliquot was then removed, and the
ribosome-mitochondria complexes were sedimented, washed in MIB, and
fixed as before. The remaining sample was divided into 2 aliquots, and
GTP was added to one of the aliquots at a final concentration of 1 mM. These two samples were incubated for 5 min, sedimented,
washed in MIB, and then fixed.
As shown in Fig. 5A, ribosomes
are bound to the mitochondrial membrane surface prior to EDTA/high-salt
treatment. EDTA/high salt-washed mitochondrial membranes are devoid of
ribosomes (Fig. 5B), but when ribosomes are added to these
mitochondria, ribosomes again bind to the mitochondrial membrane
surface (Fig. 5A, compare panels B and
C). Finally, when GTP is added to the ribosome-mitochondria complexes, the number of ribosomes associated with the mitochondrial surface is markedly reduced compared with ribosome-mitochondria complexes before addition of GTP (Fig. 5A, compare
panels C and D) or compared with
ribosome-mitochondria complexes incubated without GTP (data not shown).
To quantify these observed differences in ribosome binding, we counted
the number of ribosomes binding to mitochondria from 10 random fields
of all four conditions. Fig. 5B, lanes A and
B, demonstrates that the observed differences in ribosome
binding from Fig. 5A is real and that there is a
statistically significant difference between panels C and
D. Thus, these data show that ribosomes co-isolating with
mitochondria remain bound to the mitochondrial surface even after
freeze-thawing but that these ribosomes can be completely removed by
EDTA/high salt treatment. EDTA/high salt treatment, however, does not
cause mitochondria to lose their ability to bind ribosomes (panel
C). Finally, GTP is very effective at removing ribosomes from the
mitochondrial surface and clearly disrupts the ribosome-mitochondrial
interaction (panel D).

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Fig. 5.
Electron micrographs of mitochondria and
ribosome-mitochondria complexes. Panel A, endogenous
rat liver ribosomes (arrow) remain bound to mitochondria
after freezing and thawing. Panel B, however, mitochondria
are essentially free of ribosomes after EDTA/high-salt treatment
(EKMT). Panel C shows that when total ribosomes, isolated
from rat liver, are added back to EKMT, the ribosomes
(arrow) are again seen on the mitochondrial membranes.
Finally, in panel D, addition of GTP to these complexes
eliminates almost all ribosomes from the mitochondrial surface. The
bar represents 1 µm. B, the average number of
surface-bound ribosomes/220 µm2 and the standard
deviation for each condition represented in lanes A-D were
plotted. With respect to ribosomes binding to EKMT, C and
D are significantly different. M, mitochondria
isolated by differential centrifugation (lane A);
EKM, mitochondria that have been EDTA/high salt-washed
(lane B); R-EKM, ribosomes added back to EKM
(lane C); R-EKM-G, GTP added to R-EKM (lane
D).
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In summary, we have used three different experimental approaches to
independently prove that ribosomes specifically bind to mitochondria.
These data also show that the interaction is affected by GTP and is
reversible, providing strong evidence that this interaction is
physiologically significant.
The Ribosome-Mitochondria Interaction Affects GTP
Hydrolysis--
Because the presence of nucleotide had a marked effect
on ribosome-mitochondria binding, we hypothesized that nucleotide
hydrolysis was associated with this binding. The amount of inorganic
phosphate (Pi) produced on the cytosolic side of isolated
mitochondria was quantified in the absence and presence of ribosomes
and nucleotides (Table I). Following sedimentation of the mitochondria,
essentially no Pi was detected in the supernatants from
EKMT, high salt-washed ribosomes, or ribosomes and EKMT together, when
incubated without a trinucleotide. In addition, very little
Pi was produced when EKMT, ribosomes, or ribosomes and EKMT
together were incubated with GTP
S (Table I). In contrast, Fig.
6 shows the curve for Pi
produced from GTP by EKMPMT, in the absence and presence of ribosomes.
The initial rate of hydrolysis of GTP by mitochondria alone was 20 nmol/min/mg but was much higher at 85 nmol/min/mg for mitochondria and
ribosomes together. Since ribosomes had no GTPase activity,
acceleration of Pi production by mitochondria in the
presence of ribosomes is due to the interaction between mitochondria
and ribosomes increasing the rate of GTP hydrolysis. The rate of
Pi production in the presence of ATP and, therefore, the
rate of ATP hydrolysis by mitochondria in the presence of ribosomes, is
approximately equal to the sum of the individual rates of
Pi production by mitochondria and ribosomes alone
indicating that the ribosome-mitochondria interaction does not affect
the rate of ATP hydrolysis (Table I). Thus, the ribosome-mitochondrial interaction stimulates GTP hydrolysis which suggests this is a specific
receptor-ligand interaction that is controlled by a
GTP-dependent step.

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Fig. 6.
GTP hydrolysis by mitochondria alone or in
the presence of ribosomes. The amount of inorganic phosphate
produced from GTP (1.2 mM) by EDTA/high salt-washed,
metrizamide/Percoll-purified mitochondria (EKMPMT) (Mito,
150 µg, ), ribosomes (Ribo, 10 µg, ), and EKMPMT
and ribosomes incubated together (Mito. + Ribo.,
) in a total volume of 17 µl was measured at 2, 5, 10, and 15 min.
Each point listed represents the mean value from three or more
experiments. The curves indicate the best fit of the data to
a hyperbolic nonlinear regression.
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Interaction of Programmed Ribosomes with Mitochondria--
The
presence of ribosome-binding sites on the mitochondria suggested that
ribosome-mitochondrial binding might be functionally important in
co-translational translocation. At physiologic concentrations of GTP,
however, ribosomes isolated from the post-mitochondrial cytosol did not
stably bind to mitochondria. Since the transit peptide is responsible
for targeting a mitochondrial protein in the post-translational import
pathway, we proposed that a nascent chain containing a mitochondrial
transit peptide would be capable of directing the ribosome to the
mitochondria and stabilize binding in the presence of GTP, whereas
ribosomes carrying non-mitochondrial nascent chains would behave like
unprogrammed ribosomes in the presence of GTP.
Ribosomes protect about 40 of the most recently added C-terminal amino
acids of the growing polypeptide chain from macromolecules of the
cytosol but allow exposure of the N-terminal end of the nascent
polypeptide to the cytosol prior to complete translation (31). A
truncated mRNA lacking a stop codon and containing the coding
region of the N-terminal end of precursor mMDH was designed to minimize
exposure of the mature portion of mMDH while allowing complete exposure
of the transit peptide to the cytosol (32). The mRNA for this
92-amino acid polypeptide was translated using [35S]methionine in rabbit reticulocyte lysate, and the
ribosome-nascent chain complexes were isolated by sedimentation, and
the complexes were assayed for EKMT binding activity in the absence or
presence of di- or trinucleotides. An 86-amino acid nascent luciferase polypeptide was used as a non-mitochondrial, targeted protein control
and was produced similarly to the mMDH precursor.
The results of these experiments are summarized in Fig.
7 and show that when the ribosomes carry
a mitochondrial transit peptide-containing nascent chain, the presence
of GTP does not significantly reduce the amount of ribosome-nascent
chain complexes bound to the mitochondria compared with the amount of
ribosome-nascent chain complexes bound when no nucleotide is present
(Fig. 7A). Similarly, when GDP was included, the number of
ribosome-nascent chain complexes bound to mitochondria was not
significantly different from the number of complexes bound in the
absence of nucleotide. However, the presence of GTP
S greatly reduced
the formation of stable ribosome-mitochondria complexes. These results
differ from our earlier findings using unprogrammed ribosomes that
showed that GTP caused a significant reduction in stable
ribosome-mitochondria complex formation. In addition, the ribosomes
carrying the non-mitochondrial luciferase nascent chains behave like
unprogrammed ribosomes (Fig. 7B) and do not stably bind to
mitochondria in the presence of either GTP or GTP
S. Thus, the
presence of the transit peptide blocks GTP-dependent release of the ribosome from the mitochondria and allows stable formation of a ribosome-nascent chain-mitochondrial complex.

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Fig. 7.
Binding of mMDH ribosome-nascent chain
complexes and luciferase ribosome-nascent chain complexes to
mitochondria. The first 92 amino acids of mMDH and the first 86 amino acids of luciferase gene were translated in the presence of
[35S]methionine. Ribosome-mitochondria complexes
containing mMDH (A) or luciferase (B) were
prepared by incubating isolated ribosome-nascent chain complexes for 5 min at room temperature with 75 or 150 µg of EDTA/high-salt
mitochondria (EKMT) in the presence of the indicated nucleotide (1.2 mM) and a total volume of 17 µl. Ribosome-nascent
chain-mitochondria complexes were sedimented and resuspended in 350 µl of 50 mM Tris, pH 7.4, 100 mM KOAc, 10 mM MgCl2, and the A260
(solid bars) and cpm (open bars) were measured.
M92, ribosome-mMDH 92-mer-mitochondria
complexes; L86 = ribosome-luciferase
86mer-mitochondria complexes. Data were standardized by setting the
value of (mitochondria-ribosome) minus (mitochondria) at 100% bound.
Relative binding in the presence of nucleotide was then calculated as
follows: ((M + N + R) (M + N)) ((M + R) (M)), where M is EKMT,
R is ribosome, and N is nucleotide. A represents the
averaged data from at least three experiments and was subjected to
one-way analysis of variance. * indicates significant difference from
M92 (p < 0.05). B represents
the averaged data of at least two experiments and was subjected to
one-way analysis of variance. * indicates significant different
(p < 0.05) from L86.
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These experiments show that the transit peptide causes selective
stabilization of ribosome-mitochondria binding in the presence of GTP
and suggest a mechanism for selective import in the presence of a
nonselective ribosome receptor; unprogrammed ribosomes and ribosomes
programmed with non-mitochondrial proteins are not stably bound to the
mitochondrial membrane. Additionally, it is clear that in the presence
of ribosomes, the transit peptide alone is not capable of directing the
nascent polypeptide to the mitochondria since the non-hydrolyzable
GTP
S destabilizes this binding. A GTP-dependent process,
therefore, must function in ribosome binding to mitochondria.
 |
DISCUSSION |
In this report we have demonstrated that ribosomes bind
specifically to rat liver mitochondria and that both GTP and the nature of the nascent chain attached to the ribosome affect binding. Specifically, GTP significantly attenuated stable ribosome-mitochondria binding unless mitochondrial transit peptide-containing translation intermediates were complexed with the ribosomes. In addition, a
nonhydrolyzable analog of GTP almost completely abolished the formation
of stable ribosome-mitochondria complexes whether or not ribosomes
carried presequence-containing nascent chains. We also demonstrated
that whereas isolated intact mitochondria hydrolyze GTP, hydrolysis of
GTP was greatly accelerated when GTPase-depleted ribosomes were added
to the reaction.
This report supports, but does not prove, the hypothesis of
co-translational mitochondrial import by establishing a central element
in this model, namely ribosomes selectively and stably bind to
mitochondria. Our data suggest that the interaction of ribosomes with
the mitochondria could use components and proceed by a pathway, similar
to those used to initiate co-translational translocation at the ER
membrane. It is now becoming clear that ribosomes target to the
translocation apparatus, specifically, the sec61p complex at the ER
membrane independent of the signal recognition particle (SRP) (21, 33).
The interaction of the ribosome with both SRP and the translocation
apparatus at the ER membrane involves GTP in both complexes (34). At
the ER membrane, GTP also controls the release of SRP from SRP receptor
and the initiation of delivery of the ribosome-nascent chain complex to the membrane. In short, ribosomes, alone, possess the ability to bind
specifically to the ER translocon, but under physiological conditions,
G-proteins affect the interactions between components of the targeting
apparatus including the SRP-ribosome interaction, the SRP-SRP receptor
interaction, and the ribosome-membrane attachment to accomplish correct
protein trafficking.
A cytosolic mitochondrial precursor-specific chaperonin, mitochondrial
stimulating factor, has been functionally compared with SRP; however,
it is an ATP-dependent protein and has only been shown to
function post-translationally having no known interaction with the
ribosome (13-16). In our experiments, ribosome-mitochondria binding
was only modestly affected by ATP, and the ribosome-mitochondria interaction did not have any effect on the rate of ATP hydrolysis. Whether ATP has a direct effect on binding or simply provides a source
for generating GTP is unclear. What is clear, however, is that GTP
S
almost completely disrupted the ribosome-mitochondria binding showing
that GTP hydrolysis has an important role in controlling this
interaction.
Our results demonstrate that, similar to what occurs at the ER
membrane, ribosomes bind tightly to the mitochondrial membrane surface
and that at least one GTP-binding factor, and the nature of the nascent
chain bound to the ribosome, affect the stability of this interaction.
These analogies to protein targeting to the ER have led us to propose
that higher eukaryotic mitochondria contain ribosome receptors,
possibly similar to the sec61p complex, which are active in initiating
co-translational translocation.
We observed that ribosome-mitochondria binding is actively destabilized
by GTP unless the ribosome carries a transit peptide-containing nascent
chain. That binding is stable in the absence of nucleotide and only
mildly disturbed by GDP indicates that ribosomes interact with
mitochondria when a G-protein of the binding apparatus is free of
nucleotide or loaded with GDP. Since most G-proteins tightly bind GDP,
ribosomes probably bind to mitochondria when a G-protein of the binding
apparatus is GDP-bound. In general, it is the release of GDP by the
action of guanine nucleotide release or exchange protein (GNRP) that
allows G-proteins to bind GTP and the action of a third protein, G
activation protein, that induces hydrolysis of GTP by G-proteins (35).
Because GTP
S destabilizes ribosome-mitochondria binding, we know
that hydrolysis of GTP is not required to destabilize binding. This
implies that it is the action of a GNRP upon ribosome-mitochondria binding that destabilizes the interaction. Since both stimulation of
GNRP activity and G activation protein activity result in accelerated GTP hydrolysis, this is consistent with the observation that the rate
of GTP hydrolysis by mitochondria increases in the presence of
ribosomes. Therefore, we propose that the ribosome-mitochondria interaction stimulates GNRP activity, and that GTP binding to an
unidentified G-protein destabilizes binding unless a transit peptide is
present. We also suggest that a GNRP is located either on the ribosome,
or the mitochondria, and that the G-protein is located on the other so
that GTP binding essentially lowers the affinity of the ribosome for
the mitochondria. The ribosome-mitochondria interaction behaves
similarly to the ribosome-ER interaction except that the action of an
SRP-like component that recognizes the transit peptide occurs at the
mitochondrial membrane.
These data clearly show a direct link between the cytosolic translation
machinery and the mitochondria, as well as a ribosomal influence on
mitochondrial GTP hydrolysis. These findings are strongly supportive of
the hypothesis that in vivo mitochondrial proteins are
imported by co-translational translocation and indicate the potential
importance of GTP in the mitochondrial protein import system (7). The
current view is that precursor proteins are translated in the cytosol,
accompanied by chaperones to the mitochondrial surface, and imported by
a specific, multi-subunit import apparatus. However, the experimental
observations on which this model is based is biased for several
reasons. First, nearly all of the studies defining mitochondrial
protein import have used in vitro systems in which the
precursor proteins are first translated and then the translated product
is added to mitochondria for import. This system works well in defining
the import apparatus, but mechanisms for co-translational import are
completely missed by this approach. Second, most of the work defining
protein import has been accomplished in yeast and
Neurospora. However, there are significant differences in
transmembrane protein transfer at the ER between yeast and higher
eukaryotic cells, showing that yeast is not always representative of
more complex cellular processes such as those found in mammalian mitochondria (36). Finally, it is clear that the interior of a
eukaryotic cell is highly structured so that even soluble cytoplasmic proteins do not diffuse freely but rather are recruited in specific cytoplasmic domains (37). The current assumption that precursor proteins are translated in the cytosol and then reach their target by
chemical diffusion is inconsistent with the current view of a highly
structured cytosol and could potentially be rate-limiting for cells
with high rates of metabolism. While at least some completely synthesized mitochondrial proteins import post-translationally into
isolated mitochondria, the fact that most mitochondrial matrix and
inner membrane proteins contain an N-terminal transit peptide suggests
that recruiting begins long before protein synthesis is complete.
Further work is necessary to establish the relationship between
mitochondria, ribosomes, the nascent polypeptide, and GTP. It is
intriguing, however, that other investigators have found G-proteins in
the membrane and contact sites of mitochondria of higher eukaryotes
(38-40), and it has been recently reported that GTP hydrolysis is
required for post-translational protein import into yeast mitochondria
(41). Their identity and function remain to be evaluated.
We thank Dr. Carroll Cunningham and Dr. Mark
O. Lively for critical reading of this manuscript and Dr. Walter G. Jerome for helpful advice in electron microscopy analysis.